Introduction
Raman spectroscopy offers a number of benefits for testing and characterization. It is rapid and non-destructive, requires only limited sample preparation and allows for sample volumes in the microliter range. In addition, Raman can be used to measure aqueous samples or samples with high moisture content, and allows researchers to capture data from a sample contained in plastic or other materials that are optically transparent to the wavelengths of interest.

Raman is particularly useful for pharmaceutical applications. For example, Raman techniques are used to discern characteristics of pharmaceutical raw materials, including active ingredients, binders, fillers, lubricants and other excipients. Raman is also useful for through-container measurements of pharmaceutical blister packs, pill bottles and vials.

Experimental Conditions
To illustrate the capabilities of our Raman systems we analyzed Paracetamol (acetaminophen) and Carbamazepine, which are pharmaceutical active ingredients, and the excipients alpha and beta lactose. The samples studied consisted of simple organic compounds contained in standard, clear borosilicate scintillation vials. No additional preparation was necessary.

Samples were analyzed using a modular Raman setup comprising our QE65000 Spectrometer, a 785 nm laser with 500 mW output and a fiber optic probe. The spectrometer was set from ~780-940 nm and configured with a 50 µm slit for good optical resolution. High reflectivity optical bench mirrors increased spectrometer sensitivity.

To collect signal, we placed the tip of the probe at the bottom of three glass vials containing the samples. We measured the samples at an integration time of 8 seconds and averaged three spectra.

Results
Our measurements confirmed that this Raman configuration can differentiate various pharmaceutical raw materials based on their spectral fingerprints. Also, the experiment helped demonstrate that, with proper method development and application of chemometric analysis, our Raman setups can be used to obtain semi-quantitative data of active ingredients in a pharmaceutical mixture.

Our experiment also showed that fluorescence occurs in the lactose samples. Fluorescence is a common phenomenon in Raman measurements of some organic compounds and depends on the wavelength of the laser utilized.

Conclusions
The availability of both turnkey and modular Raman systems, complemented by sophisticated chemometric analysis packages and spectral libraries, makes Raman spectroscopy a versatile choice for a host of applications.

Components for Oxygen:
NeoFox Phase Fluorometer
RedEye® Oxygen Patches (Headspace and In-Solution measurement)
Bifurcated Fiber Optic probe

Components for pH:
Jaz Spectrometer
LS-1 Tungsten Light Source
Bifurcated Fiber Optic Probe
Reflective pH Patches

Experimental Procedure:
•    Oxygen Patches were placed inside the Bio-flask to monitor the oxygen in headspace and in solution.
•    pH patch was placed in solution to monitor pH changes during the fermentation process.
•    Fresh red grapes were mashed and the must was left to sit for 2 days.
•    The liquid was added in the bio-flask. Initial measurements were recorded and yeast cells and nutrients were added to start the fermentation process.
•    This process was observed over a period of 60 hours.

Optical Oxygen Sensors:
•    Prominent method for luminescence sensing in which the lifetime of the indicator compound changes in response to the analyte sensed (phase measurement).
•    A fluorescence method measures the partial pressure of dissolved or gaseous oxygen. The fluorescence is generated at the tip of the optical electrode by a light source.
•    When oxygen in the gas or liquid samples diffuses into the thin film coating, it quenches the fluorescence.
•    The degree of quenching correlates to the level of oxygen pressure.

RedEye Oxygen Sensor Patch:
•    RedEye is a revolutionary oxygen sensing product designed specifically to measure oxygen.
•    RedEye consists of a sensor coating formulation trapped in a sol gel matrix, immobilized and protected from the package contents
•    The RedEye patch has no minimum and maximum diameter  sizes, but typically sizes will vary from a few millimeters to several centimeters
•    RedEye can be easily integrated into any system because of its self-adhesive acrylic  patch having the sensor coating formulation.

O2 Sensing Electronics – NeoFox:
NeoFox is an instrument platform for measurement of fluorescence lifetime and phase for oxygen sensing
•    The frequency domain electronics uses a blue LED excitation and an  avalanche photodiode for detection.
•    A bifurcated optical fiber carries excitation light produced by the blue LED to the thin-film coating of the RedEye Oxygen Sensor
•    Fluorescence generated at the surface of the RedEye Patch is collected by the probe and carried by the optical fiber to the detector.

Lifetime Measurement Technique:
The phase shift between the blue LED excitation and emission signal of fluorescence is used to calculate lifetime.

Advantages of Optical Oxygen Sensors:
•    Uses fiber optic cable for non-intrusive measurements
•    Not contaminated by water or other solutions
•    Immune to EMI
•    Non-electrical, non-conductive
•    Operable in temperature and concentration range of various applications
•    Simple calibration
•    Works with colored samples without any color or light interference

Smart pH Technology:
•    pH buffers used for calibration, references taken by software
•    Absorbance curves seen as pH increases
•    Absorbance peak at 620nm, baseline correction at 512 nm

Temperature Compensation:
Compensation for temperature effects is achieved through the van’t Hoff equation:

•    Isothermal correction corrects pH value based on user-input temperature
•    Dynamic temperature correction available for non-isothermal systems with temperature monitoring

Result Analysis and Conclusion:
•    The graph shows the complete measurement of both oxygen and pH over a period of 60 hours.
•    The next graph shows the measurements over the first two hours. The oxygen sensor in solution quickly dropped from air saturation as soon as the yeast cells and nutrients were added. The pH sensor in solution dropped slightly as the oxygen decreased and CO2 is released. Hence the drop in pH value.
•    The following graph indicates the oxygen sensor in the headspace stays at air saturation approximately the first 2.5 hours. Once the oxygen in solution is completely quenched, the yeast cells and nutrients start consuming oxygen from the headspace.
•    Conclusion: Ocean Optics sensor patches are able to monitor both oxygen and pH non-intrusively which can be used in the fermentation industries.

Experimental Conditions:
Several encoder wheels were sent for analysis. The samples are described below.

50% Sandblasted sample (Zn encoder wheel with 50% of one of the surfaces sandblasted)
Sample 1: Zn plated encoder wheel (not sandblasted)
Sample 2: ABS or POM encoder wheel – 1 sandblasted and 1 unsandblasted wheel
Sample 3: POM encoder wheel – 1 sandblasted and 1 unsandblasted wheel

Specular reflectance measurements at 90 degrees relative to the sample surface were performed on each sample on both the top and bottom sides of the samples. Specular reflectance was measured at 90 degrees with diffuse reflectance measurements for some of the samples at 45 degrees relative to the sample surface using the RPH-1 reflection probe holder. The shiny Zn plated encoder wheels (50% sandblasted sample and sample 1) were analyzed with the STAN-SSH high reflectivity specular reflectance standard as a reference. The dull black encoder wheels (samples 2 and 3) were analyzed with the WS-1 diffuse reflectance standard as a reference. One additional set of data was acquired for the 50% sandblasted Zn plated and POM (sample 3) encoder wheels with the unsandblasted samples used as a reference for the sandblasted samples.

Since the samples had different shapes, various methods were used to acquire the reflection spectra. Measurements of the Zn plated encoder wheels (50% sandblasted and sample 1) were configured as shown in Figure 1 with measurements of the sandblasted and unsandblasted ABS and POM encoder wheels configured as shown in Figure 2. Measurements of the taller POM sandblasted and unsandblasted encoder wheels were configured as shown in Figure 3.

Hardware Used:
HR4000-CG (HR4C349) high resolution spectrometer
DH2000-BAL tungsten deuterium light source
R400-7-UV-VIS reflection probe
RPH-1 reflection probe holder
WS-1 diffuse reflectance standard
STAN-SSH high reflectivity specular reflectance standard
Experimental Parameters:
Zn plated samples (50% Sandblasted and Sample 1)
Integration Time (msec): 45
Spectra Averaged: 50
Boxcar Smoothing: 1

ABS or POM samples (Sample 2)
Integration Time (msec): 375
Spectra Averaged: 20
Boxcar Smoothing: 1

POM sample (Sample 3)
Integration Time (msec): Top 420 Bottom 750
Spectra Averaged: Top 20 Bottom 10
Boxcar Smoothing: 1
Measurement Mode:
Reflectance

Results:
For specular reflectance measurements at 90 degrees, reflectance always decreased following sandblasting. To provide a more quantitative assessment of the decrease, the specular reflectance at 400 nm (wavelength chosen arbitrarily) was compared for the sandblasated versus unsandblasted samples. The results shown in Table 1 are the percent reflectance values at 400 nm for an average of 3 to 5 replicates measurements at different locations on the sample. There was an ~84% difference in specular reflection for the unsandblasted versus sandblasted side of the 50% sandblasted Zn plated encoder wheel. Note (as shown in the last rows of the table) that there was an 8.5% difference in specular reflection when the top and bottom of the unsandblasted Zn encoder wheel (Sample 1) were compared suggesting that there is some variability when various locations on the sample were measured. For the ABS or POM (Sample 2) and POM (Sample 3) encoder wheels, specular reflection differed by 63 to 69% and 41 to 69%, respectively for the sandblasted versus unsandblasted samples. Note that specular measurements from the top and bottom of the same sample varied in most cases. The other data shown in the table is the specular reflection measured for the sandblasted 50% sandblasted Zn plated and POM encoder wheels using the unsandblasted samples as the reference. In this case, specular reflection of the Zn plated encoder wheel at 400 nm was only ~17% of the unsandblasted sample with the POM sample showing a decrease of 45% with sandblasting relative to the unsandblasted POM sample. Specular reflectance data are shown in Figures 4 through 7.

An additional set of measurements was done with the probe placed in the 45 degree position in the reflection probe holder for the 50% sandblasted Zn plated and ABS or POM encoder wheel (Sample 2) samples. Note that the diffuse reflection at 45 degrees was much lower than the specular reflection measured at 90 degrees. The WS-1 diffuse reflectance standard was used as a reference for these measurements. In contrast to the specular reflection measurements at 90 degrees, the diffuse reflectance increased after sandblasting. The data acquired for the 50% sandblasted Zn plated encoder wheel is shown in Figure 8.

Figure 1: Specular reflection measurements of the Zn plated encoder wheels (50% sandblasted sample and Sample 1)

Figure 2: Specular reflection measurements of the ABS or POM encoder wheels (Sample 2)

Figure 3: Specular reflection measurements of the POM encoder wheels (Sample 3)

Table 1: Summary of Specular Reflectance Results

Figure 4: Specular reflection from the 50% Sandblasted Zn Plated Encoder Wheel

Figure 5: Specular reflection from the ABS or POM Encoder Wheel (Sample 2)

Figure 6: Specular reflection from the POM Encoder Wheel (Sample 3)

Figure 7: Specular reflection from sandblasted samples (unsandblasted samples used for reference)

Figure 8: Diffuse Reflection Measurements for the 50% Sandblasted Zn Plated Encoder Wheel

Introduction
Irradiance is the amount of energy at each wavelength emitted from a radiant sample. Absolute irradiance is the measure of light in absolute terms. Relative irradiance is a comparison of the fraction of energy the sample emits and the energy the sampling system collects from a lamp with a blackbody energy distribution.

Measurement of absolute solar irradiance is relevant in a number of applications: monitoring the sunlight itself, perhaps in the context of its relationship to greenhouse gases in the atmosphere; investigating the effect of solar radiation on ecological systems and crops; and evaluating the effect of UV sunlight on our skin and eyes.

Solar radiation includes spectral response across the broad UV-NIR region. We offer a number of spectrometer options that will measure solar irradiance over various portions of the solar spectrum between 200-1100 nm. A spectrometer now in development will combine both UV-Vis CCD-array and NIR InGaAs-array detectors in the same unit, extending the possible measurement range considerably.

Experimental Conditions
To measure a series of strong absorption lines in the solar spectrum, we configured our Jaz spectrometer with a grating optimized to 200-850 nm, a detector collection lens to increase light collection, a 50 µm slit and a battery module for portable operation. The Jaz is a self-contained unit that includes a microprocessor and low-power OLED display in place of a PC. Also, we added SpectraSuite spectrometer operating software and our Jaz-A-IRRAD irradiance-measurement application software to complete the basic irradiance setup.

For absolute irradiance measurements, we added a radiometrically calibrated light source (LS-1-CAL) and cosine corrector (several options are available), which also is our sampling optic. The cosine corrector collects sunlight from 180º and attaches directly to the SMA 905 connector on the Jaz. Accessory options such as a fixture for mounting Jaz in various positions and a shoulder holster for carrying Jaz are also available.

The Jaz spectrometer is radiometrically calibrated against the NIST-traceable LS-1-CAL Light Source, with the calibration file stored on an SD card. This SD card comes with the Jaz and fits into a slot in the battery module. We used this calibration file in software to determine the absolute irradiance values of the solar spectrum.

Results
Integration time for the measurement was 4 milliseconds, with spectral averaging set at 10 and boxcar smoothing at 2. The resulting spectrum showed a number of strong absorption lines from atmospheric elements in the range from 300-900 nm, including hydrogen, helium and sodium.

Similar results can be achieved using different spectrometer models with comparable optical bench configurations, including a USB2000+ Spectrometer that has our extended-range (200-1050 nm) grating or our USB2000+RAD, an application-ready setup with all the components needed for absolute solar irradiance measurements.

Conclusions
Because of its form-factor and battery operation, Jaz is a useful option for solar irradiance measurements of all types. The system is easily portable and extremely robust as well, having traveled to the top of Mt. Everest and successfully making solar radiation measurements for researchers studying ozone depletion.

Additional Resources
http://www.oceanoptics.com/Products/jazulm.asp
http://www.oceanoptics.com/Products/usb2000rad_compare.asp
http://www.oceanoptics.com/applications/samplesetups_upwellingdownwelling.asp
http://www.oceanoptics.com/Products/jaz_el350_200.asp
http://www.oceanoptics.com/Products/usb2000+rad.asp

It took thousands of years, following the first questioning gaze at the sky, to understand why the clear daytime sky is blue [1]. The theory of Rayleigh scattering, however, does not explain the entire palette of sky colors that delight us at dawn or dusk. It was only realized in the middle of the last century [2]that the unstable oxygen molecule ozone (O3) has a profound impact on twilight colors.

It is well known that the ozone layer protects the surface of our planet from the damaging effects of ultraviolet radiation but few people appreciate that, without ozone, the color of the zenith (overhead)sky at twilight would be a pale green/straw yellow rather than the deep, steely blue that we observe. The electronic Chappuis absorption band of ozone is intrinsically weak and has little effect on the color of the daytime sky. This band, extending from 450 to 850 nm, only becomes significant when the pathlength of sunlight through the atmosphere is dramatically increased around sunrise and sunset. At these times, the Chappuis band becomes by far the strongest feature in the visible spectrum of the sky or the setting sun.

In preparation for future ‘transit spectroscopy’ (analysis of the light transmitted by the atmosphere of a planet transiting its parent star) we have performed relative spectrophotometry of the sky during twilight. This can be compared with the observations of the eclipsed Moon [3] which examine light that has grazed the Earth’s atmosphere during a lunar eclipse. The eclipse geometry results in a much stronger influence of Rayleigh and aerosol extinction in the light reflected from the eclipsed Moon than in scattered light from the horizon sky at twilight, resulting in strong suppression of the blue end of the spectrum.

The twilight spectra appearing in this report were obtained with an Ocean Optics JAZ spectrometer covering 350–1000 nm and using a single optical fiber input pointed about 10° above the western sky in overcast conditions. The altitude of the observing site was 560 m at a latitude of +47.8°.

Figure 1 The experimental setup for the twilight observations showing the JAZ spectrometer, the fiber feed and the data-taking laptop. These photographs, taken around the time of sunset with a ‘daylight’ white balance setting on a Canon 5D MkII, illustrate the high color temperature of the ambient light at this time and caused predominantly by the ozone absorption (see Fig 2).

Figure 2 The variation of sky brightness at 700 nm, in units of counts per 8192 ms, and color temperature, measured with a Gossen 2F COLORMASTER.

Figure 3 A sequence of ‘Relative Irradiance’ spectra of a cloudy western sky at approximately 10 min intervals from an hour before, to 15min after, sunset. Also plotted is the Lunar eclipse spectrum from [3]. The rapid development of the ozone Chappuis absorption, centered at 600 nm, is apparent together with the dramatic bluening of the sky color during this period. The somewhat irregular behavior of the blue end of the spectrum is due to the variable cloud thickness during these measurements. The reference spectrum is with a solar altitude of+13° and the final spectrum of the sequence is with an altitude of -3°.

Figure 4 The observed ratio (dark blue), with a normalized continuum, of a spectrum taken with a solar altitude of-3° to one at +13°. Atmospheric models [4], using the HITRAN database [5], with a similar but pure transmission geometry, are over plotted (red: pure ozone absorption; light blue: ozone + O2 + H2O). The models have been scaled in intensity by a factor of 1.7 as a way of accounting for the simplification of the model geometry.

This simple experiment clearly illustrates the profound influence of the ozone Chappuis absorption on the color of the twilight sky (the color temperature of the western sky varied from 6,000K to14,000K during the course of the observations) and the very strong visible spectral signature that could be expected in transit spectroscopy of an oxygen-rich exoearth atmosphere.

The absence of strong signatures of O2 and H2O in the spectral ratios probably indicates that most of the light entering the spectrometer has passed above the cloud layer, in the stratosphere (~25km), where ozone dominates the absorption.

An interesting exercise with these data is to invert them to derive the ozone Chappuis absorption cross-section times the column density of the atmospheric pathlength that is characteristic of these twilight observations.

Figure 5 The inversion of the observation (ln[observed ratio] * const) compared with the data in the radiative transfer code we used for the modeling, remembering that we have neglected the water and diatomic oxygen contributions which probably accounts for some of the deviations in the band wings.

This project, designed for high-school education, will go on to explore different illumination geometries involving long absorption pathlength through the atmosphere.

Acknowledgments: We thank Enric Pallé for providing the Lunar eclipse digital data.

References

1              Pesic, P., “Sky in a bottle”, The MIT Press

2              Hulbert, E. O., 1953, “Explanation of the Brightness and Color of the Sky, Particularly the Twilight Sky”, Journal of the Optical Society of America, 43, 113-118

3              Pallé, E. et al. 2009, “Earth’s transmission spectrum from lunar eclipse observations”, NATURE, 459, 814-816

4              Clough, S.A., et al. 2005, “Atmospheric radiative transfer modeling: a summary of the AERcodes”, Journal of Quantitative Spectroscopy and Radiative Transfer, , 91, 233

5              Rothman L.S., et al. 2009, “The HITRAN 2008 molecular spectroscopic database”, Journal of Quantitative Spectroscopy and Radiative Transfer, 110, 533

Robert (Bob) Fosbury [a] & Andreas Seifahrt [b]

a. Space Telescope – European Coordinating Facility, Garching bei München, 85748, Germany(rfosbury@eso.org)

b. Physics Department, Univ. of California, Davis, CA 95616, USA (seifahrt@physics.ucdavis.edu)

A 2-meter 35?m bifurcated borosilicate bundle was used inside a T-300 sleeve, and an LS-1 tungsten halogen light source was used. All components were placed inside an environmentally controlled chamber, along with a second beaker of seawater being monitored by a Ross pH electrode. The beakers were sealed with Parafilm®, and the probes kept a tight seal with the film to prevent evaporation. The experimental setup is pictured below:

Galvanic corrosion became apparent with the use of the stainless steel sleeve, most likely due to improper sealing of the mirror’s metal from the steel. This led to accumulation of rust and sediment on the mirror, steadily causing a continued tilt in the absorbance curve. The typical pH algorithm looks at two wavelengths, an analytical wavelength and a baseline correction to account for vertical offset.

Though, a tilt in the absorbance spectra skews these values and yields incorrect measurements. To correct for this, an accompanying algorithm was used to eliminate this tilt effect, bringing the absorbance spectra back to its appropriate position, knowing that 750nm and 509nm (isosbestic point) should be pegged at zero absorbance. The algorithm is shown below, followed by the plot showing the corrected absorbance curve:

Knowing that it is required to monitor three wavelengths in order to account for the tilt, a long-term experiment was run to monitor pH patch stability. Analysis was performed that calculated the continuous pH assuming the original algorithm alone using a 750nm baseline correction, as well as a 509nm (isosbestic point) correction, and also using the tilt correction algorithm that looks at all three wavelengths. The plot of these three trends is shown below:

Clearly the original approach of using a single wavelength for baseline correction is not feasible for long-term measurement; rather, we see that the tilt algorithm is able to account for these absorbance curve distortions and produces a very coherent trend. The probe originally reads sea water as being roughly pH 8.2, and then drifts downwards to just above pH 6, where it equilibrates. This drift is most likely due to galvanic corrosion reactions occurring, which alter the pH of the system.

Ocean Optics also makes a transmissive probe of the same design using chemically inert peek material. This experiment was repeated using this alternate model, the TP-300, again with the Ross pH electrode running in parallel.

The accumulation of sediments and particles on the mirror and/or lens led to the absorbance curve tilt phenomenon previously seen, though this occurs much more slowly than the stainless steel probe. The system equilibrates after roughly 20 hours, and remains at a constant pH for the following 30 hours, showing the potential for strong long-term stability. A comparative plot shows the readings of the optical pH probe and the Ross electrode, with the electrode’s values being recorded manually at five discrete times:

The drift of the Ross pH electrode is slightly over 0.03 pH unit per day, which is roughly 15 times more drift than the specification reported in the user manual. While the Ocean Optics pH probe showed enhanced stability over the Ross electrode, the measurement was cut short due to light source failure. The LS-1 light source bulbs are offered in 900 hour and 10,000 hour lifespans; the longer-use bulb is recommended for long-term measurements. LED(s) may also be used with these sensors as another alternative. This study has been vital in determining a number of aspects and limitations for this optical pH probe system, some notable points include:

- The peek TP-300 probe should be used over the stainless steel version for pH measurement in seawater or high salinity environments; corrosion occurs extremely quickly with the stainless steel and sediment accumulates on the mirror.

- As sediments form on the mirror and/or lens, the absorbance curve may show a tilting distortion; this can be corrected via a dynamic algorithm that looks at three wavelengths to eliminate the effect.

- The light source should be relatively new and stable when performing long-term measurements. As the light source begins to die, there is a non-uniform intensity decay that causes an incorrect pH reading drift, followed by the light source finally dying completely. LED’s may be used as an alternative.

Algorithms Used:

pH Calculation:

Absorbance Spectra “Tilt” Correction:

The ability to provide UV-NIR coverage in a single miniature spectrometer has always been a challenge. Trade-offs inherent to most diffraction gratings – most noticeably, the effect of blaze angle on the efficiency of the diffraction – can pose challenges for certain applications. While gratings were available that diffracted over a wide range, this came at the expense of decreased optical resolution and increased problems associated with second- and third-order overlap.

Newer gratings such as the XR-1 provide good efficiency over a wider wavelength range (200-1050 nm) than is otherwise possible with standard gratings. What’s more, good optical resolution (<2.0 nm FWHM for most setups) can be maintained, and second- and third-order effects are eliminated by applying proprietary filtering technology to the CCD-array detector window. Transmission efficiency is affected only marginally by this filtering.

Broad spectral response in a single spectrometer offers convenience for those who regularly make measurements in both the UV-Vis and Vis-NIR, yet it also offers a solution for applications where samples are responsive across that same broad range. Examples include certain plasmas, solar irradiance, atomic emission lines and broad-range light sources.

Experimental Conditions

To test the response of the XR-1 grating, we installed the 500 lines/mm groove density grating in the optical bench of our USB2000+ Spectrometer. The spectrometer’s optical bench also included a 25 µm slit and order-sorting detector filter. The grating provides 850 nm of spectral range and is blazed at 250 nm.

The test sample for the experiment was our DH-2000-BAL Deuterium Tungsten Halogen Light Source. The DH-2000-BAL combines the continuous spectrum of deuterium and tungsten halogen light sources in a single optical path to produce a powerful, stable output from 215-2000 nm (we observed only the region from 200-1050 nm). A UV-Vis optical fiber collected the signal from the light source. We recommend our QP450-2-XSR optical fiber, which is a 455 µm core diameter fiber with excellent solarization resistance properties. Integration time of 10 milliseconds is typically sufficient for measuring a light source such as the DH-2000-BAL.

Results

The emission spectrum of the UV-NIR light source measured with the USB2000+XR matched the anticipated spectral output. The XR-1 grating showed good efficiency across the 200-1050 nm spectral range, with the best efficiency in the UV. Optical resolution was calculated at ~1.7 nm (FWHM) with a 25 µm slit (the standard slit option for the USB2000+XR) and at ~1.2 nm (FWHM) with a 5 µm slit. Other expected spectrometer performance characteristics were unaffected by the presence of the grating.

Conclusions

Results demonstrate that an Ocean Optics spectrometer configured with the XR-1 extended-range grating will provide spectral coverage across the 200-1050 nm spectral range without sacrificing optical resolution performance or being subject to second- and third-order diffraction effects. The XR-1 is available in the application-ready USB2000+XR, USB4000-XR1 and JAZ-EL200-XR1 Spectrometers (each has a 25 µm slit and order-sorting filter) or as a custom option in one of our other spectrometers. For applications requiring broad range and sub-nanometer optical resolution (FWHM), our HR2000+CG and HR4000CG-UV-NIR composite-grating spectrometers are recommended.

Introduction:
Different mixtures of similarly colored detergent must be shuttled along a conveyor line, presumably in a manufacturing application. As detergents display fluorescence wavelengths similar to those emitted by the light source near 365nm, it was necessary to determine whether differences in the fluorescence of such detergents could be identified using the reflection probe. By measuring samples of distinct detergent mixtures with the probe in combination with a lowpass UV filter, it was determined that the fluorescence of the detergent was not only distinguishable from the light source, but distinguishable from every other sample as well.

Hardware Used:
USB2000-USB2E4066, grating 1, 200 micron slit, L2 lens
PX2 Light Source
R600-7-SR/125F Probe
LVF – FHS
P1000-2-UV/Vis Fiber
UV-LVF-Lowpass-300nm

Measurement Mode:
Relative Irradiance

Experimental Conditions:
Samples of detergent were individually placed in plastic weighboats, approximately 1cm below the reflection probe. The probe was suspended from a clamp, and shielded with dark cloth to minimize outside light interference. In order to block wavelengths of light from the source suspected to coincide with the wavelength of fluorescence, a UV-LVF lowpass filter was placed in between the light source and fiber at around 300 nm. A P1000 UV/Vis fiber in conjunction with an LVF-FHS was used in order to gather more light. Measurements were then taken, replacing the sample each time.

Results:
Using relative irradiance mode, fluorescence peaks of gradually increasing intensity were observed at approximately 406nm, 422nm, and 426 nm depending on each sample. Peaks were also observed below 300nm, and above 590nm, but this was a result of the emissions from the light source outside the range of the lowpass filter. The resulting fluorescence peaks were different than the estimated value of 365 nm. These fluorescence peaks corresponded to the wavelength of light emitted by the detergent when excited by the PX-2 light source as indicated in figure 1 below. Figure 2 is a close up of fluoresced emission.

Conclusions:
Based on the resulting data, it can be concluded that the reflectance probe compounded with a lowpass filter is an appropriate apparatus for determining the fluorescence of detergent samples.

INTRODUCTION:
Various biological samples exhibit UV-Vis reflectivity and color characteristics of interest to researchers. Applications are diverse: For example, among some species of birds, insects and reptiles, UV reflectance and color play a role in mating behavior, recognizing species and assessing predator risk. Color as an indicator of fruit and vegetable ripening is significant; also, chlorophyll distribution in crops, measured using reflectance, can tell growers something about optimum fertilizer amounts. Many more similar applications, both in the field and in the lab, can be classified as bioreflectance applications.

Jaz provides a particularly compelling option for bioreflectance applications in the field, where portability, flexibility and ease of use are critical. Jaz is a modular spectrometer-based system that integrates into a single stack those components that otherwise would have to be handled separately: the spectrometer, microprocessor with low-power display (in place of a PC), light source, battery pack and even Ethernet capability for remote measurements. Reflection probes and other sampling optics connect easily to the Jaz, keeping the overall system footprint compact and manageable.

Experimental Conditions
A typical Jaz configuration for portable UV-Vis reflectance comprises the Jaz spectrometer set from 200-850 nm, with a 25 µm slit and L2 detector collection lens. Also installed in the Jaz stack is the Jaz-B battery module, which has two slots for SD card data storage, and the Jaz-PX, a high-intensity pulsed xenon light source with up to four hours battery life on a single charge. SpectraSuite spectrometer operating software is also recommended. Depending on experiment considerations, other options to consider are the Jaz-E Ethernet module and the SpectraSuite-PAR add-on software application, which is used to calculate Photosynthetically Active Radiation (PAR) values of horticultural samples.

Most bioreflectance applications involve diffuse reflection of solid surfaces. Our fiber optic reflection/backscattering probes can measure diffuse or specular reflectance from a surface; a good choice for most UV-Vis bioreflectance applications is our QR600-7-SR/125F, a premium-grade probe with 600 µm core diameter in a six-around-one fiber configuration. Also, the probe is solarization-resistant and has a 1/8” ferrule.

Results
Bioreflectance setups using miniature portable spectroscopy have become so simple to perform that even high school science students have little problem with such setups. In one example, a student measured the reflection at 90º of philodendron plant leaves, theorizing that reflectance values could be correlated to fertilizer levels. The results suggested that plant reflectance at wavelengths >700 nm was insensitive to the stress of over-fertilization (at 4x the recommended amount of fertilizer), while the peak within the 530-630 nm range was noticeably sensitive (i.e., had greater reflectivity) to stress. The increased reflectivity related to a decrease in chlorophyll and to the effects of osmosis. Water collected between the leaf cell membrane and cell wall and exposed more of the leaf surface, which increased reflectivity.

Conclusions
The inherent flexibility of the Jaz sensing system can be exploited for a number of UV-Vis bioreflectance applications simply by mixing and matching Jaz modules and selecting sampling optics most appropriate for your application. A high-intensity, low-power pulsed xenon source and solutions to system power requirements make Jaz an extremely reliable choice for field and other measurements.

Introduction
Near-infrared spectroscopy is a common analytical technique for chemistry and process control, where typical applications include species identification and water and fat content determination. In cases like those, absorbance peaks are often broad and optical resolution requirements of lesser concern than performance parameters such as low noise and high sensitivity.

Yet there also are number of NIR applications where optical resolution of <5.0 nm (FWHM) is critical. Characterization of laser lines – examples include solid state lasers at 1064 nm and at wavelengths from 1020-1050 nm, as well as semiconductor lasers with response in the 900-1800 nm range – often require even better optical resolution. Optical fiber characterization and determination of atomic emission lines are other high-resolution NIR applications.

Experimental Conditions
To test the optical resolution performance of our NIRQuest512-2.2 Spectrometer, which has a Hamamatsu G9206-512W InGaAs-array detector and is responsive from 900-2200 nm, we measured the spectrum of a xenon wavelength calibration source. The low-pressure gas-discharge source has a number of closely aligned emission lines in the region from ~820-2000 nm, making it a good choice for our experiment.

The NIRQuest512-2.2 was configured with a 100 lines/mm grating set to 900-2050 nm, with a 25 µm slit and gold-coated collimating and focusing mirrors for enhanced reflectivity. This is the standard setup for the NIRQuest product line, which includes models for 900-1700 nm, 900-2050 nm, 900-2200 nm and 900-2500 nm. Additional grating options and slit sizes are available for custom setups.

We used a 50 µm Vis-NIR optical fiber to collect signal from the xenon source. (For optimum results, we recommend the use of a 50 µm diameter or smaller optical fiber with all of our spectrometer wavelength calibration sources.) NIRQuest512-2.2 integration time was set to 350 ms and spectral averaging set to 5.

Results
The spectrum from the xenon calibration source illustrates that optical resolution of ~4.6 nm (FWHM) is possible with the NIRQuest512-2.2 in its standard configuration. What’s more, even better optical resolution is possible in a NIRQuest512-2.2 configured with a grating that has a narrower spectral bandwidth. For example, a NIRQuest512-2.2 with a 600 l/mm grating set over a 100-nanometer bandwidth and configured with a 25 µm slit would yield optical resolution of <0.5 nm (FWHM). Resolution would improve even more with a 10 µm slit, but at the expense of throughput. For most laser applications, that’s likely to be an acceptable trade-off.

Conclusions
New NIR detectors and optical bench options allow researchers to experience high optical resolution performance in the region from 900-2500 nm. This elevates the versatility of smaller footprint, more modular NIR spectrometers for applications previously thought to be out of reach.

Hardware Used:
USB2000-FLG
EG&G pulsed xenon light source
CUV-ALL-UV
600 micron optical fibers

Acquisition Parameters:
Integration Time (msec): 10
Spectra Averaged: 100
Boxcar Smoothing: 20

Measurement Mode:
Gated Fluorescence

Experimental Conditions:
The sample used was Bacillus globigii spores in terbium reagent. Data acquisition delayed from 5 to 500 usec after the lamp was triggered.

Results:
The effect of gating on the spectral shape is shown in the figures below. As the delay between lamp trigger and data acquisition is increased, the terbium/DPA photoluminescence becomes more distinct. Shorter lived background fluorescence and lamp pulse are not observed. Longer delay times (above 50 usec) lead to a decrease in sensitivity as a portion of the terbium/DPA photoluminescence is missed at long delay times.

Conclusions:
The optimal delay for these samples is 40 microseconds. A 40 microsecond delay between the lamp trigger and data acquisition minimizes the contribution of the xenon source (and fluorescence due to other fluorophores) to the photoluminescence spectrum while maximizing the photoluminescence signal.

Images:

Introduction:
Currently, the materials generated during the zinc refinement process are analyzed with wet chemistry and LA-ICP-MS for CaO, MgO, SiO2, Al2O3, TiO2, Cr205 and F to monitor the refinement process. Typical concentrations are 0.5 to 35%. Four samples were sent representing various stages in the refinement process – Calcine, Zinc Ferrite Cake, Cobalt Cake and Copper/Cadmium Cake. The elements of interest for Calcine and Zinc Ferrite were Fe, Pb, S and Zn with the presence of Cu, Co, Cd, Ni, Pb and Zn of interest in the Cobalt and Copper/Cadimium Cakes.

Hardware Used:
LIBS2000+ broadband, high-resolution spectrometer
200 mJ Nd:YAG Big Sky laser
LIBS-SC sampling chamber with imaging module

Acquisition Parameters:
Laser setting 8 (highest setting ~200 mJ)
Analysis in air
-1 Q-switch setting for all samples except Calcine (Q-switch delay was increased to -2.5 setting to elminate the bright continuum background)
Element ID Parameters: +/-1 pixel search width and 50 count peak height

Measurement Mode:
LIBS

Experimental Conditions:
The four samples varied in color and consistency. Calcine was a brown powder, Zinc Ferrite was large chunks of brown material similar in consistency to clay and the Cobalt and Copper Cadmium Cakes were dark grey material with the consistency of thick mud. All four samples were analyzed on double-sided photo mounting tape adhered to glass microscope slides. Calcine and the Cobalt and Copper Cadmium Cakes were easily dispersed on the tape.

Due to an inability to spread the Zinc Ferrite sample, it was analyzed as large chunks. Three single shot spectra were acquired for different locations on each sample with two additional locations analyzed with 5 and 10 shot spectra for all samples except Calcine. Three and five shot spectra were used for Calcine due to the thin layer of sample present on the tape.

The use of 10 and possibly even 5 shot spectra would have drilled through the thin sample and into the tape and microscope slide. The other samples provided a thick enough layer that analysis of the underlying tape and microscope slide substrates was not a concern.

Results:
The results from the Elemental Identification software are summarized for each of the samples below. The complete list of elements identified by the software are found in an Excel spreadsheet with the results for each sample found on a separate worksheet.

Calcine
All the elements of interest (Fe, Pb, S and Zn) were detected in all replicates.
Decreased intensity and inability to detect S was observed when the dusty powdered sample coated the optics following the laser events. Compressed air was used to clean the sampling optics probe between each measurement.
Signal averaging did not improve detection (no additional lines were detected when spectra were acquired as the accumulation of multiple laser shots).

Zn Ferrite
Fe, Pb and Zn were detected in all replicates.
S was detected in one single shot replicate.
The sample was moist as observed visually and by the strong H alpha line at ~656 nm. If S was expected in this sample, the moisture in sample may have quenched the plasma making it more difficult to see some elements. Note that the Cobalt and Copper/Cadium Cakes appeared to have a higher moisture content.
Signal averaging did not improve detection.

Copper/Cadmium Cake
Less elemental lines were detected for the Copper/Cadmium Cake than for the Calcine and Zn Ferrite samples.
All elements of interest were detected in all replicates (Cd, Co, Cu, Ni, Pb and Zn).
The Cu lines were very intense (even stronger than the hydrogen alpha line).
The sample was moist as observed visually and by the strong H alpha line at ~656 nm.
Signal averaging did not improve detection.

Cobalt Cake
The least number of elemental lines was observed for the Cobalt Cake sample.
Cd, Co, Cu, Ni and Zn were detected in all replicates.
Pb was not detected in any of the replicates.
The sample was moist as observed visually and by the strong H alpha line at ~656 nm.
Signal averaging did not improve detection.

Introduction:
It may be necessary in certain applications to determine the elemental composition of chip components, including the core metals and coating plastics. In this experiment, LIBS was used to assess whether gold and silver were present in the core of chips, and whether the core could be distinguished from the plastic coating in resulting spectra. It was found that silver was present in the core, while gold was present only in protruding wires from certain chips. It was also found that the coating could be distinguished based on elements present in the core but not in the coating.

Hardware Used:
LIBS 2000+ broadband, high resolution spectrometer
200 mJ Nd:YAG Big Sky laser
LIBS-SC sampling chamber with imaging module

Acquisition Parameters:
3 scans/average
Laser level 8
Q switch delay: -2.5 microseconds

Experimental Conditions:
All samples were placed individually on double sided tape within the sampling chamber to prevent movement from laser ablation. The plastic sheath was assessed on all samples, while two samples with protruding filaments were assessed for metal content by creating a hole in the plastic sheath with the laser through which the metal core could be ablated.

Measurement Mode:
LIBS

Results:
Using the elemental analysis tool in OOILIBS, All samples of coating were shown to contain silicon to varying degree by the presence of corresponding peaks at 251.61, 288.15, and 252.81nm. Elemental analysis also revealed that the coating of sample 2 contained high counts of magnesium as well as silicon. High peak values and strong correlation to elemental peaks in sample 4 suggested a strong presence of hydrogen and carbon, while sample 5 also suggested carbon in high presence. However, none of the coatings displayed any detectable amounts of silver. When the coating on sample 3 was degraded down to the core via repetitive laser strikes, a strong presence of silver was detected, both in the number of peak correlations and high signal counts. Once the core had been reached, sodium peaks no longer appeared. In addition, lasing of wires protruding from sample 4 showed noticeable presence of gold via close correlation to peaks. In all coating samples, sodium and potassium were also detected, however this is likely attributable to contamination from handling.

Conclusions:
It was shown that LIBS compounded with the elemental analysis function in OOILIBS is a capable tool for distinguishing between elemental components of the core and coating of computer chips. By correlating peak intensity counts and the number of elemental peaks present in each spectrum, it was possible to distinguish the major elements within each chip coating of samples 1-7, and the core of sample 3. Further ablation of sample 3 showed that the presence of silver distinguishes core material from coating, and ablation of sample 4 on protruding wires showed that metal components present on the chip or in the core of the chip can be differentiated using LIBS.

A reflective pH patch was affixed to the inner wall of a small 7mL tube, and was held in place using a ring stand and clamp. A 600um bifurcated reflective probe was connected to an LS-1 light source with a blue filter, as well as a USB2000 spectrometer with grating #1, 200um slit, and no lens installed. The first feasibility test used clear buffers instead of the muddied buffers, in order to observe the performance. The experimental setup is pictured below, along with the plot of the resulting titration.

Although the curves showed a slight distortion, they followed the expected trend and produced a linear calibration plot. Dirt was obtained and mixed in with the buffer solutions; the experimental setup is shown below.

This yielded a very interesting titration plot, unlike any that had been seen before:

Typically for reflective pH patches, the peak is observed at 620nm and the baseline correction is done at 509nm. In this case, however, we see the peak had shifted closer to 635nm, and the 509nm region was distorted completely. As a result, 750nm was used as the baseline correction wavelength, which produced an incredibly linear calibration plot, as seen below.

This application has been critical in determining the effect of turbidity on the absorbance curves for the reflective pH patches, and has concluded that the correct wavelengths for acquisition and baseline need to be chosen based on the nature of the analyte solution.

Background
The Chemistry Department at the University of Washington is performing a research study where the measurement of oxygen in the headspace of Acetonitrile is critical. Since acetonitirle is an organic solvent and the vapors of acetonitrile are harsh, a sensor which can resist the fumes is applicable for this environment. The Hioxy oxygen sensor design my Ocean Optics was tested for this application.

Equipment
* Phase Fluorometer Electronics (MFPF)
* Bifurcated Fiber Optic Cable
* Hioxy-R Sensor

Feasibility Test Procedure
The MFPF electronics is an instrument platform for measurement of fluorescence lifetime and phase. This frequency domain electronics uses a blue LED excitation and a photodiode for detection. A fluorescence method is used to measure the partial pressure of dissolved or gaseous oxygen. A bifurcated optical fiber carries excitation light produced by the blue LED to the thin-film coating of the Hioxy-R sensor.. Fluorescence generated at the tip of the probe is collected by the probe and carried by the optical fiber to the detector of the MFPF. The phase shift between the blue LED excitation and emission signal of fluorescence is used to calculate the lifetime.  The Lifetime is an essential parameter for the calculation of oxygen.

To test the feasibility of the Hioxy sensor in the Acetonitrile headspace, the Hioxy Sensor is evaluated for its performance before exposure to the acetonitrile vapor.

The Hioxy sensor is exposed to Nitrogen and then to Air as show in Figure 1. The ratio of the lifetime from Nitrogen to Air is noted to be approximately 4.01. Now the sensor is exposed to the vapors of acetonitrile for about 2 hours. After the exposure the sensor is pulled out from the vapors and left in ambient conditions. The ratio of Nitrogen to air is measured again in terms of lifetime. The ratio after this exposure has changed to 4.27. The sensor is again exposed to acetonitrile vapor and the ratio is measured after exposure to be the same at 4.27.

Results
As shown in Figure 1, the Hioxy Sensor looks very stable in the headspace of the Acetonitrile container. The ratio of Nitrogen to Air has changed after the first exposure to Acetonitrile vapor. After the first exposure the ratio stays the same. This means there is a one time effect on the sensor when exposed to acetonitrile vapor.

Conclusion
The Hioxy sensor can be used to measure oxygen in the headspace of Acetonitrile. When a sensor is sold for this application, the sensor needs to be cured with acetonitrile vapor before calibration.

Also note: The sensor needs to be placed in acetonitrile vapor for at least 2 hours before use.

1    INTRODUCTION

1.1    Microorganisms in blood

Microorganisms are one celled organisms such as viruses, fungi and bacteria. Presence of microorganisms is harmful and cause diseases. The presence of microorganisms in blood cultures plays an important role in the diagnosis of different diseases. Different methods have been in existence to detect the presence of microorganisms in blood cultures. Early detection of such organisms is of primary importance to the selection of appropriate therapies and doses to be adopted on patients . Blood culturing systems are bioreactor system which involves the process of selectively growing microorganisms under optimized conditions. Blood culturing systems are closed culture systems where blood along with the growth media is operated under constant temperature along with continuous mixing. The numbers of microorganism increase due to respiration process and establish reactions with blood components changing the forms of hemoglobin. In the absence of microorganisms irrespective of the growth media present, the blood components do undergo changes due to aging of the red blood cells. As the microorganism’s density increases in the blood culture, partial pressure of oxygen is reduced and partial pressure of carbon dioxide is increased as a part of respiration process.

Automated systems are being developed to continuously monitor the different metabolic changes happening in the blood contents along with the changes in the partial pressure of oxygen/carbon dioxide consumed/generated respectively. The instruments primarily constitute the detection system to capture the data points at different intervals to form mathematical models to study the behavior of microorganisms and their growth patterns. The information collected using such systems helps us to understand the time period when the microorganisms have grow and aid in the selection of system parameters optimum to detect the different microorganisms. Some of the changes such as conversion of oxy to deoxyghemoglobin within the red blood cells have been detected using spectroscopy methods which provide growth behavior of organisms . As oxygen is necessary for cell respiration and is consumed during the growth phase of a cell processes for aerobic microorganisms. The cells reproduce and their cell density increases during the growth phase leading to increased oxygen consumption by the cells. The cells consume the dissolved oxygen from the liquid media (blood culture).

This paper presents the application of smart cuvette coated with oxygen sensitive sol gel coating which acts as a detection system to measure the dissolved partial pressure of oxygen in blood culture systems and the trend in oxygen consumption in response to the increasing density of microorganisms

2    SYNTHESIS OF NANO POROUS SOL GEL MATRIX AS A MOLECULAR PROBE FOR DISSOLVED OXYGEN

A ruthenium compound was immobilized in an organically modified silicate (ORMOSIL) using sol gel process. Methyltrimethoxysilane (MTMS) was used as the sol gel precursor. Appropriate amount of water and alcohol is added to the precursor to induce hydrolysis and condensation polymerization. Sub ppb levels of DO were able to be detected using the sol gel coating. Organically modified silicate (ormosil) sol-gel thin films have many advantages over their inorganic sol-gel and polymeric counterparts for sensing applications .

3    MATERIALS AND METHODS

3.1    Cell culture system design (Bioreactor)

An optical system is integrated to monitor the oxygen levels in a bioprocess system in a continuous fashion. The system built is a small scale version of the bioreactor. The integration involves the optical oxygen sensing system with the bioprocess system built to grow cells at constant temperature. Each of the components is described in detail in the following section:

3.2    Smart Oxygen Cuvette

Smart oxygen is a revolutionary oxygen sensing product designed for monitoring the dissolved oxygen in samples for biological and medical applications. Smart oxygen cuvette consists of a sensor coating formulation trapped in a sol gel matrix, immobilized and protected from the package contents. .The cuvette (Glass flourometer cell, Rectangular, Starna Cells Inc, CA) is the cell growth container of the bioprocess system. The Smart Oxygen cuvette has oxygen sensor coating formulation integrated with the cuvette on the inner lining of one of the side as shown in Figure 1

3.3    Qpod

The qpod is a complete sample compartment for fiber optic spectroscopy, including a peltier-controlled cuvette holder with magnetic stirring, and fused silica lens systems with SMA fiber optic connectors. The collimating /imaging/mirror optics enables the collection of rays and focus on the collection fiber. The qpod is equipped with Quantum Northwest TC125 Temperature Controller for temperature control and magnetic stirring to enable the cells in the cuvette to be in continuous stirring mode. As the cells have to be in a continuous stirring mode in a bioreactor, so magnetic stirring feature enables a good control on the stirring aspect integrated into the system .

3.4    NeoFox

The NeoFox Phase Fluorometer is an instrument platform for measurement of fluorescence lifetime and phase. This frequency domain electronics uses a blue LED excitation and a photodiode for detection. A fluorescence method is used to measure the partial pressure of dissolved or gaseous oxygen. A bifurcated optical fiber carries excitation light produced by the blue LED to the thin-film coating of the Smart Cuvette. Fluorescence generated at the surface of the patch is collected by the probe and carried by the optical fiber to the detector of PF. The phase shift between the blue LED excitation and emission signal of fluorescence is used to calculate the lifetime. The Figure 2 below is a representation of the phase measurement.  A new compact phase flourometer, NeoFox developed by Ocean Optics is used in this system design.

Oxygen as a triplet molecule is able to quench efficiently the fluorescence and phosphorescence of certain luminophores. This effect (first described by Kautsky in 1939) is called “dynamic fluorescence quenching.” Collision of an oxygen molecule with a fluorophore in its excited state leads to a non-radiative transfer of energy. The degree of fluorescence quenching relates to the frequency of collisions, and therefore to the concentration, pressure and temperature of the oxygen-containing media. When oxygen in the gas or liquid sample diffuses into the thin-film coating, it quenches the fluorescence. The degree of quenching correlates to the level of oxygen pressure.

4    SENSOR CALIBRATION

In order to make accurate oxygen measurements inside the cuvette, the calibration of the Smart Oxygen Cuvette was performed using the Linear (Stern-Volmer) algorithm. Since this experiments were performed at room temperature (~25C), temperature compensation during the calibration was not required.
Temperature does not affect the fluorescence decay time, fluorescence intensity, collision frequency of the oxygen molecules with the fluorophore, and the diffusion coefficient of oxygen as long as the temperature is maintained between ± 1 °C of the calibrated temperature.

Linear (Stern-Volmer) Algorithm: The Linear (Stern-Volmer) algorithm requires at least two standards of known oxygen concentration. The first standard must have 0% oxygen concentration and the last standard must have a concentration in the high end of the concentration range. The Smart Oxygen Cuvette patch was calibrated at 0% and 20.9% oxygen. The calibration curves were generated from these standards and the linear algorithm was used to calculate oxygen concentration values for unknown samples.

The fluorescence lifetime (?) can be expressed in terms of the Stern-Volmer equation where the fluorescence is related quantitatively to the partial pressure of oxygen:

Where t0 is the lifetime of fluorescence at zero pressure of oxygen, ? is the lifetime of fluorescence at a pressure p of oxygen, and k is the Stern-Volmer constant.

For a given media, and at a constant total pressure and temperature, the partial pressure of oxygen is proportional to oxygen mole fraction. The Stern-Volmer constant (k) is primarily dependent on the chemical composition of the sensor formulation. The Stern-Volmer constant (k) is temperature dependent. All measurements should be made at the same temperature (± 1 °C) from the calibration experiments. If temperature compensation is needed, then the relationship between the Stern-Volmer values and temperature is defined as:

The lifetime of fluorescence at zero pressure of oxygen depends on details of the optical setup: the power of the LED, the optical fibers, loss of light at the probe due to fiber coupling, and backscattering from the sample. It is important to measure the lifetime of fluorescence at zero pressure of oxygen (I0) for each experimental setup .

5    OPTICAL SENSING SYSTEM INTEGRATION WITH CELL CULTURE SYSTEM

The Smart Oxygen cuvette is placed in a qpod and the side of the cuvette which has the oxygen sensor coated material is interfaced with the bifurcated reflectance probe. The bifurcated reflectance probe is connected to the NeoFox system. The LED source on the NeoFox provides the excitation light and is coupled to one of the legs of the bifurcated optical probe. The reflected florescence light is coupled back into the other leg of the bifurcated probe and terminated at the detector surface on NeoFox. The NeoFox interfaces with the NeoFox Viewer Software (Ocean Optics Inc.) which measures the oxygen levels. The complete system used to measure oxygen levels is shown in Figure 3

5.1    Experimental Setup

The oxygen sensing experiment was carried out in a Smart Oxygen Cuvette. To build a two point calibration, the nitrogen gas is diffused into the cuvette for 0% oxygen and then air is diffused into the cuvette for 20.9% oxygen. The two points are captured and a calibration curve is built to quantify the oxygen levels in the range of 0 – 21% from the life time measurements. We start our experiment by placing Whole goat blood and water (1:1.5) in the cuvette, magnetic stirrer is placed in the cuvette and the stirring speed is set to a maximum using the qpod temperature and magnetic controller interface. The temperature is set at room temperature. The NeoFox viewer software starts logging the data from the instant diluted blood is placed in the cuvette. The oxygen concentration in blood starts at a low concentration of oxygen and increases until almost air saturation. Once the oxygen level increases and is stable, yeast cells are added to the blood in the cuvette. The oxygen quenching is observed over a period of time. After each run all of the dissolved oxygen sensor data is logged. The cuvette is washed and dried and placed back into the qpod for the next run. The experiment is conducted 3 times.

To replicate the bioprocess conditions, nutrients were added to the blood to study the rate of dissolved oxygen in the cell culture media. The experiments were repeated with the yeast cells of 200mgrams. The sensor data was logged for a period of 30 minutes and after each run, the cuvette was rinsed and dried with vacuum for the next run. The experiment was repeated three times.
Another set of experiments was run to study the time it takes to quench the dissolved oxygen in a closed cuvette. Different amounts of yeast were added to the diluted blood and the time it takes to quench the dissolved oxygen is recorded.

Figure 3 shows the bioreactor setup with oxygen sensor patch interfaced with bifurcated fiber optic probe. The legs of the fiber optic probe are connected to the LED excitation source and detector on the NeoFox. The USB interface on the NeoFox transfers the data to the NeoFox Viewer Software for the data logging process

6    RESULTS AND DISCUSSION

The Smart Oxygen cuvette is a small-scale system used to study the effects on the oxygen partial pressure of the blood sample in the presence of microorganisms in the blood. During the experiment while the blood is diluted with water and added to the cuvette very low concentration of dissolved oxygen is present. Due to stirring the blood in a closed system cuvette the oxygen level in the dissolved blood eventually rises up to air saturation. Once the dissolved oxygen level is stabilized at air saturation, the yeast cells are added to observe the consumption of the oxygen

The yeast cells when dissolved in blood started consuming the oxygen through the liquid cell membrane interface by the diffusion process. The system is calibrated and the dissolved oxygen levels are monitored when the yeast cells are added and the measurements have been carried out for a time period of approximately 30 minutes. The experimental results (n = 3) in Figure 4 show the performance of Smart Oxygen Cuvette in measuring the oxygen levels continuously as the bioprocess happens in the cell culture system. As the cells are consuming the oxygen in the liquid media through diffusion, the oxygen depleted in the liquid media is what the Smart Oxygen Cuvette is really sensing. The same experiment can be extended to a single cell, in a micro fluidic well culture system. The one side of the cuvette has the oxygen sensing coating which measures the oxygen level depleted in the liquid media surrounding the cell. Using the diffusion parameters of the cell, one can calculate the oxygen consumed by each cell. It is observed that adding 200 milligrams of yeast to about 2.5mL of diluted blood can quench the oxygen to approximately 1 % within 20 minutes. The three runs show very similar results as shown in Figure 4.

Figure 4 shows the dissolved oxygen levels in a bioreactor system measured using a Smart Oxygen Cuvette

The small scale culture applications have the advantage of the studying the effect of multiple nutrients/environmental conditions on the oxygen levels consumed and also on the process throughput. With an objective to study the performance of the performance of Smart Cuvette in sensing oxygen levels in the cell culture, we have performed another set of experiment varying the amount of yeast dissolved in blood. The oxygen is consumed by the cells faster if the amount of cells is more. Figure 5 shows the performance of Smart Oxygen Cuvette in measuring the dissolved oxygen levels in cell culture environment with different yeast amount added to diluted blood.

Figure 5 shows the oxygen consumed by cells when different amounts of yeast is added to diluted blood as measured by Smart Oxygen Cuvette

7    CONCLUSION

A Smart Oxygen cuvette is reported to provide superior measurements of dissolved oxygen in important biological experiments such as in blood culture/bioreactor systems. The integration of Smart oxygen cuvette when combined with advanced phase fluorometry can be used to develop portable systems to measure presence of bacteria in different blood cultures. The fluorescent technology based on oxygen quenching has already proven it success in the mycobacterial growth indicator(TB test)  and is used to accurately identify mycobacteria .Development of a cost effective system integrated with multiplexing capabilities would open a new approach to study the presence of microorganisms in blood culture  system. As healthcare costs are rising and especially with the increasing incidence of TB cases, the proposed system can be used in the preventive healthcare to diagnose the presence of bacteria at an early stage from blood sample. Systems of this nature would accelerate the intervention procedures and facilitate the reduction of healthcare costs.

[ii] Debra E. Huffman, et al, “J.New method for the detection of micro-organisms in blood: application of quantitative interpretation model to aerobic blood cultures”, Biomed. Opt. 14, 034043 (2009), DOI:10.1117/1.3156837

Hardware Used:
•    Phase Fluorometer Electronics (MFPF)
•    Bifurcated Fiber Optic Cable
•    HiOXY-R Sensor.

Abstract:
This application note explains how partial pressure of oxygen is measured even in the presence of gases like Methane.

Background:
Methane is a chemical compound with the chemical formula CH4. It is the simplest alkane, and the principal component of natural gas. Burning methane in the presence of oxygen produces carbon dioxide and water. The relative abundance of methane makes it an attractive fuel. Several oil companies have approached Ocean Optics to provide a solution of measuring oxygen in the presence of Methane.

The HiOXY oxygen sensor design by Ocean Optics was tested for this application.

Feasibility Test Procedure:
The MFPF electronics is an instrument platform for measurement of fluorescence lifetime and phase. This frequency domain electronics uses a blue LED excitation and a photodiode for detection. A fluorescence method is used to measure the partial pressure of dissolved or gaseous oxygen. A bifurcated optical fiber carries excitation light produced by the blue LED to the thin-film coating of the Hioxy-R sensor. Fluorescence generated at the tip of the probe is collected by the probe and carried by the optical fiber to the detector of the MFPF. The phase shift between the blue LED excitation and emission signal of fluorescence is used to calculate the lifetime.  The Lifetime is an essential parameter for the calculation of oxygen.

A short term feasibility test has been performed using HiOXY sensor in Methane gas.

The chart below first shows the response of the HiOXY sensor in Nitrgoen and Air first.  The sensor is again exposed to nitrogen and a small flow of methane is introduced. There is no observed change in the lifetime due to the introduction of methane in the nitrogen flow.
The sensor is now exposed back to air to see if there are any effects or changes caused in the lifetime due exposing the sensor to Methane gas. It is observed that there is no change in the lifetime in Air. A small flow of Methane is introduced in the air stream and a slight increase in the lifetime caused from the methane.

When only methane is exposed to the sensor, it behaves in the same way as nitrogen as the sensor does not see any oxygen.

In conclusion, the HiOXY sensor can be used to measure oxygen in the presence of Methane gas without affect the intensity or the lifetime of the sensor.

To measure oxygen concentration is measured in the headspace of organic solvents such as Acetonitrile) using a phase fluorometer systemMethod:
O2 Sensing

Hardware Used:
Phase Fluorometer Electronics (MFPF)
Bifurcated Fiber Optic Cable
Hioxy-R Sensor

Feasibility Test Procedure
The MFPF electronics is an instrument platform for measurement of fluorescence lifetime and phase. This frequency domain electronics uses a blue LED excitation and a photodiode for detection. A fluorescence method is used to measure the partial pressure of dissolved or gaseous oxygen. A bifurcated optical fiber carries excitation light produced by the blue LED to the thin-film coating of the Hioxy-R sensor.. Fluorescence generated at the tip of the probe is collected by the probe and carried by the optical fiber to the detector of the MFPF. The phase shift between the blue LED excitation and emission signal of fluorescence is used to calculate the lifetime.  The Lifetime is an essential parameter for the calculation of oxygen.

To test the feasibility of the Hioxy sensor in the Acetonitrile headspace, the Hioxy Sensor is evaluated for its performance before exposure to the acetonitrile vapor.
The Hioxy sensor is exposed to Nitrogen and then to Air as show in Figure 1. The ratio of the lifetime from Nitrogen to Air is noted to be approximately 4.01. Now the sensor is exposed to the vapors of acetonitrile for about 2 hours. After the exposure the sensor is pulled out from the vapors and left in ambient conditions. The ratio of Nitrogen to air is measured again in terms of lifetime. The ratio after this exposure has changed to 4.27. The sensor is again exposed to acetonitrile vapor and the ratio is measured after exposure to be the same at 4.27.

Results
As shown in Figure 1, the Hioxy Sensor looks very stable in the headspace of the Acetonitrile container. The ratio of Nitrogen to Air has changed after the first exposure to Acetonitrile vapor. After the first exposure the ratio stays the same. This means there is a one time effect on the sensor when exposed to acetonitrile vapor.

Conclusion
The Hioxy sensor can be used to measure oxygen in the headspace of Acetonitrile. When a sensor is sold for this application, the sensor needs to be cured with acetonitrile vapor before calibration.

Note:
The sensor needs to be placed in acetonitrile vapor for at least 2 hours before use.

Method:
pH Sensing

Hardware Used:
USB2000 Spectrometer: Grating #1, 200um slit, no lens installed
LS-1 Light Source
Bifurcated Reflective Probe, 600um
SpectraSuite Software

Measurement Mode:
Absorbance

Notes:

There was an interest in non-intrusively monitoring pH in tubes containing wet soil samples. This was significant for a number of reasons; this was the first time the reflective patches were to be tested in something other than a cuvette, on a curved surface rather, and this was also the first time an analyte solution was observed that contained sediment or turbidity.

A reflective pH patch was affixed to the inner wall of a small 7mL tube, and was held in place using a ring stand and clamp. A 600um bifurcated reflective probe was connected to an LS-1 light source with a blue filter, as well as a USB2000 spectrometer with grating #1, 200um slit, and no lens installed. The first feasibility test used clear buffers instead of the muddied buffers, in order to observe the performance. The experimental setup is pictured below, along with the plot of the resulting titration.

Although the curves showed a slight distortion, they followed the expected trend and produced a linear calibration plot. Dirt was obtained and mixed in with the buffer solutions; the experimental setup is shown below.

This yielded a very interesting titration plot, unlike any that had been seen before:

Typically for reflective pH patches, the peak is observed at 620nm and the baseline correction is done at 509nm. In this case, however, we see the peak had shifted closer to 635nm, and the 509nm region was distorted completely. As a result, 750nm was used as the baseline correction wavelength, which produced an incredibly linear calibration plot, as seen below.

This application has been critical in determining the effect of turbidity on the absorbance curves for the reflective pH patches, and has concluded that the correct wavelengths for acquisition and baseline need to be chosen based on the nature of the analyte solution.

Algorithm Used:

Method:
LIBS

Hardware Used:
LIBS2000+ spectrometer
Big Sky Nd:YAG 50 mJ 1064 nm laser
LIBS Sample chamber with 125 mm focal length lens
OOILIBS software

Acquisition Parameters:
Data was acquired for different levels of surface cleaning (0 to 10 laser shots before acquiring data) and for samples where 10 laser shots were averaged. All of the spectra were collected with a 1.5 microsecond delay between when the laser fired and when data acquisition began.

Measurement Mode:
LIBS

Experimental Conditions:
The copper sample was placed in the LIBS sample chamber and the probe was focused until the maximum intensity was observed from the plasma. Three replicates at different locations on the sample were acquired using the following data acquisition parameters:

No surface cleaning (OOILIBS has a cleaning function that allows the user to fire several cleaning shots to clean contaminants from the surface of the sample before acquiring spectral data.)

10 cleaning shots

3 cleaning shots

10 laser shots averaged with no cleaning shots

Single laser shot resulted in surface pitting of the sample.

Results:
The average spectral data for the three replicates acquired using varying data acquisition parameters are shown in the figure below. The spectra were offset to facilitate comparison of spectral features.

For the bottom spectrum where no cleaning shots were fired, it appears that there is a significant amount of material on the surface of the sample as evidenced by the region between 500 and 600 nm. This region of the spectrum is filled with broad bands and elemental lines unique to the uncleaned surface. In the remaining spectra, when the samples are cleaned 3 or 10 times or 10 laser shots are averaged at the same location, the spectral data look similar. Even though the top spectrum where 10 laser shots at the same spot were averaged is similar to the condition where the surface is pre-cleaned with 10 shots before acquiring data, the top spectrum represents the average of 10 individual laser shots at the same location so it includes a contribution from the uncleaned spectrum shown at the bottom of the plot.

The elements identified in one of the replicate measurements for each set of data acquisition parameters are shown in the table. As anticipated based on the spectral data, the number of elements identified decreases with increasing cleaning shots. For example, numerous magnesium (Mg) and calcium (Ca) lines are observed when no cleaning shots are used to remove contaminants from the sample surface (first two columns in the table). With surface cleaning, the number of lines observed for these elements decreases. The data shown in the final two columns of the table for 10 shots averaged together without any surface cleaning still shows the Ca and Mg lines since the surface was not cleaned before the measurements.

Note that the library used to identify the spectral lines in the OOILIBS software is a partial library containing the persistent lines of the elements. With a more complete library, like the MIT Wavelength Tables containing thousands of spectral lines, additional elemental lines would be identified. As expected, several copper lines were observed for each measurement. In the case of the uncleaned sample, a larger number of lines was detected. It is important to note that these samples were measured in the presence of air. The sample chamber was not purged to remove the air. For this reason, the oxygen and nitrogen lines observed result from ambient air in the plasma in addition to any oxygen and nitrogen present in the sample.

Method:
Absorbance

Introduction:
Feasibility testing was done to assess the detection of bleach with OOI hardware and to determine the optimal hardware configuration for detecting 10, 20 and 30 ppm sodium hypochlorite.

Attempts to measure chlorine with another system (USB2000 (USB2E3017) with grating #2, 25 um slit, UV2 detector upgrade, OFLV order sorting filter and L2 detector collection lens, DT1000-S source, CUV-UV cuvette holder, 400 um SR fiber on one leg, 200 um UV/VIS fiber on the other leg, CVD-UV1S cuvette were not successful at the 30 ppm sodium hypochlorite level.

Hardware Used:
USB2000 (USB2E214) with grating #1, 50 um slit, L2 detector collection lens and OFLV order sorting filter
CUV-UV cuvette holder
P400-025-UV-SR optical fibers
DT1000 deuterium tungsten halogen

Acquisition Parameters:
Integration Time (msec): 7
Spectra Averaged: 100
Boxcar Smoothing: 3

Measurement Mode:
Absorbance

Experimental Conditions:
Generic ultra bleach for household use was diluted with water to provide sodium hypochlorite samples. According to information on the internet, ultra bleach is 6% sodium hypochlorite or 60,000 ppm. Water was used to dilute the 6% solution down to 10, 20 and 30 ppm.

Results:
The plot below contains spectral data for 10, 20 and 30 ppm sodium hypochlorite. A peak at 295 nm is observed. This peak intensity decreases with dilution of the sodium hypochlorite.

Conclusions:
The use of a larger slit and UV optimized grating in the spectrometer (50 um slit versus 25 um and grating 1 versus grating 2) resulted in excellent detection of 10 to 30 ppm sodium hypochlorite. These concentrations are easily detectable with OOI hardware.

Method:
Fluorescence

Hardware Used:
USB2000-FL (USB2E3295)
USB-LS-450 (U45EA229)
CUV-ALL-UV
P400, P600, P1000 UV/VIS optical fibers

Acquisition Parameters:
Integration Time: 100 msec
Spectra Averaged: 1
Boxcar Smoothing: 10

Measurement Mode:
Fluorescence

Experimental Conditions:
10 uM fluorescein contained in a disposable cuvette (CVD-UV1S) (cuvette oriented with 1 cm pathlength towards spectrometer)

Results:
Fluorescein fluorescence measured with various combinations of 400 to 1000 micron optical fibers is shown in the figure below.

Conclusions:
For fluorescence measurements with the CUV-ALL-UV (90 degree configuration), the optimal optical fiber sizes for detecting fluorescein fluorescence are a 1000 micron fiber on the illumination/excitation side (from light source to cuvette holder) and 1000 micron fiber on the read/detection side (from cuvette holder to spectrometer). As shown in the figure, the use of a 600 micron fiber on the detection side does not have a significant impact on the fluorescence intensity measured.

These results can most likely be extrapolated for other fluorophores meaning ideal fiber sizes for fluorescence measurements are 1000 um fibers.

Method:
LIBS

Introduction:
Elemental analysis of alloy beads composed primarily of aluminum (80% Al) and iron (20% Fe) was carried out with LIBS to provide an elemental profile for the sample. Detection of contaminants present in the samples in addition to the major components was desired.

Hardware Used:
LIBS2000+ broadband, high-resolution spectrometer
50 mJ Big Sky Nd:YAG laser
LIBS sampling chamber with 125 mm focal length lens

Measurement Mode:
LIBS

Experimental Conditions:
The alloy beads were pressed onto a piece of double-sided, photo-mounting tape attached to a microscope slide. A total of 10 single shot spectra were acquired at different locations on the slide. An average spectrum was generated from the single shot spectra. The elements present in the average spectrum were identified with the OOILIBS Elemental Identification module.

Results:
The average spectrum (n=10) for the aluminum alloy beads is shown in Figure 1. The elements identified for the average spectrum are shown in Table 1 with the number of elemental lines identified per lines in the Elemental Identification library shown in Table 2. As shown in Table 2, 3 out of 9 Al lines were detected with all 15 of the Fe lines included in the LIBS Elemental Identification library detected with LIBS.

Other elements detected included potassium (P), gold (Au), iodine (I), lead (Pb), tin (Sn), calcium (Ca), magnesium (Mg), cadmium (Cd), phosphorus (P), chromium (Cr) and barium (Ba). In addition to the lines detected (see Table 1 for a complete list), numerous other emission lines were not identified by the library. The identity of these lines could be determined with a resource like the MIT Wavelength Tables. The identity and concentration of the elements present in the alloy beads (as determined by an alternative analytical method) was not provided with the samples for comparison.

Method:
LIBS

Introduction:
The target analyte for the samples is the element Lanthanum (La). There are two spectral regions of interest. In the region centered at 324 nm, there is a La doublet at 317 nm and an emission line at 332 nm. The second spectral region contains La emission lines at 427 nm and 433 nm.

Hardware Used:
LIBS2000+ broadband high-resolution spectrometer
50 mJ Big Sky Nd:YAG laser
LIBS sampling chamber with 125 mm focal length lens

Acquisition Parameters:
2 microsecond Q-switch delay between laser event and data acquisition

Measurement Mode:
LIBS

Experiment Conditions:
Samples were prepared by evaporation of a series of 3 microliter aliquots of La at three different concentrations. Concentrations were 10,000, 1,000 and 50 ppm, labeled 10k, 1k, and 0.05, respectively. Two different substrates were used, 6061 aluminum (0.125″ thick), and 304 stainless steel (0.025″ thick). The discrete spots where La was deposited on the metal surfaces were easily visible (with the naked eye) on the aluminum substrate at all concentrations, and on the stainless steel substrate at the 10k concentration. The La spots were not visible (with the naked eye) on the stainless steel substrate for the 1k and 0.05 La concentrations.

The La deposition pattern for the stainless steel samples were all the same, a 5 x 5 matrix, with one spot missing at the label. The 10k aluminum sample had a 4 x 5 spot pattern with one spot missing at the label, while both the 1k and 0.05 concentrations had a full 5 x 5 spot pattern. Due to differences in spot diameter these samples were not suitable for development of a traditional calibration curve.

Results:
Single and multi-shot LIBS spectra were acquired for several La spots on each sample. For the 10k La samples, extra data was acquired to profile the effect of subsequent shots at the same spot. The 10k samples were analyzed the most extensively.

Results are shown in the plots below. The LIBS analysis of La deposited on 6061 aluminum is shown in Figure 1. Each spectrum shown is the average of multiple single shot spectra (see legend for number of single shot spectra averaged). Two spectral regions that contain the La lines of interest are shown.

LIBS analysis of La deposited on 304 stainless steel is shown in Figure 2. Each spectrum shown is the average of multiple 3-shot spectra. As shown for the aluminum substrate, the spectral regions containing the La lines of interest are shown. In Figure 3, the effect of taking multiple shots at the same location is shown. Note that the richest spectrum is achieved with the second shot.

In Figures 4 and 5, the LIBS spectra for the 6061 aluminum and 304 stainless steel substrates are shown, respectively. Each spectrum represents the average of three 3-shot spectra. Note that the spectrum for stainless steel has more elemental lines indicative of differences in the plasmas obtained for the different materials.

Conclusions:
For La deposited on 6061 aluminum, 3 of 6 La peaks included in the Elemental Identification library were detected at the 50 ppm La concentration. At the 1000 ppm La concentration, 4 out of 6 peaks were detected with 5 of 6 La peaks detected in the 10,000 ppm concentration. With a 304 stainless steel substrate, 5 of the 6 La lines were detected at all La concentrations. The data show that La deposited on aluminum and stainless steel substrates is detectable down to the 50 ppm level with more peaks detected at higher La concentrations. Improved detection on the stainless steel substrate illustrates the impact of the matrix on detection. Differences in the plasma characteristics for the two substrates may have resulted in the detection of additional elemental lines (example of matrix effects on detection).

For the elemental lines of interest, the La doublet around 317 nm was detected on both the aluminum and stainless steal substrates at the 10,000 and 1000 ppm La concentrations. The elemental line around 332 nm was not detected at any La concentration on either substrate. The peaks around 427 and 433 nm were detected on the stainless steel substrate at all three La levels. For the aluminum substrate, the peak around 433 nm was detected at all three La levels with the peak around 427 nm detected at the 1000 and 10,000 ppm levels.

Method:
Reflectance

Introduction:
Skin is an optically dense scattering material. Non-invasive measurements of hemoglobin using a reflection probe and tungsten halogen source indicate that sampling depths are sufficient to monitor changes in the oxygenation state of hemoglobin contained in blood vessels beneath the skin. To address the question of how deep light penetrates during non-invasive reflection measurements of the skin, colored ceramic tiles were used to simulate material below the skin with beef and chicken breast cut to different thicknesses to mimic varying depths of skin.

Experimental Conditions:
Colored ceramic tiles were used to simulate material below the skin with beef and chicken breast used to provide varying depths of skin. Reference measurements included the white tile alone with other trials using the meat and white tile as a reference. Reflection probe was positioned 90 degrees relative to the sample.

Hardware Used:
R400-7 UV/VIS reflection probe with RPH-1 reflection probe holder
LS-1 tungsten halogen light source
USB2000 spectrometer (USB2E214- grating # 1, OFLV-2, L2, 50 um slit)
Experimental Parameters:
Integration time: 3 to 18 msec
Averages: 10 to 25
Boxcar: 3
Measurement Mode:
Reflectance

Results:
Baseline reflection spectra were acquired from the colored tiles to determine the impact of tile color on the shape of the reflection spectra. The data for the colored tiles are shown in Figure 1 below. Note that the spectra were different depending on the tile color. The white tile was used as the reference for these measurements. To avoid specular reflection from the shiny tiles, measurements were made at 45 degrees relative to the sample using the RPH-1.

For the measurements made with meat shown in Figure 2 below, the probe was positioned 90 degrees relative to the sample. Strong absorption in the region between 500 and 600 nm was observed. These peaks were most likely due to the presence of high concentrations of myoglobin in the beef. When the plot shown in Figure 2 for reflectance through beef is compared to the plot shown in Figure 1 for reflectance of the ceramic tiles only, similar shapes and trends due to tile color were observed in the region from 600 to 900 nm. The beef was approximately 0.5 to 1 mm thick.

When a thicker piece of beef (approximately 3 mm) was used, the spectra did not show any evidence of the colored tiles beneath the meat. As shown in Figure 3, the spectra are all very similar in shape and magnitude. As shown in Figure 4, when a different less fatty piece of meat was used, the spectra are more different than those shown in Figure 3 but the differences did not appear to be related to the color of the tiles.

The results for colored ceramic tile reflectance through chicken were not consistent with either the ceramic tile or beef and ceramic tile spectra. As shown in Figure 5, the spectral trends suggested that we were not seeing tile color through the chicken. Note that the strong absorption due to myoglobin is much lower in chicken due to the lower levels of myoglobin found in poultry. The chicken was approximately 1 to 2 mm thick.

In Figure 6, the spectra for various types of beef and reflection through fat (absorbance should not be as significant) are shown. Note the presence of a strong absorption band around 650 nm for the older beef. This peak is most likely related to the presence of oxidized myoglobin and hemoglobin in the older sample (met form of the heme group). This spectrum also had an interesting shoulder that occurred near 500 nm. Measurements for the thick slice of beef yielded spectra that did not contain the doublet expected between 500 and 600 nm (consistent with the presence of deoxygenated hemoglobin and myoglobin).

Conclusions:
For the first set of trials with 0.5 to 1 mm beef and 1 to 2 mm chicken, reflectance measurements through a very thin section of beef (approximately 1 mm) suggested that colored ceramic tiles could be detected through the beef. Reflectance through chicken did not yield the same detection of the colored ceramic tiles beneath the chicken. One reason for this may be that the chicken was a thicker section of meat than the beef. Additional measurements with 3 mm thick beef showed that the colored tiles were not detected with a thicker piece of beef. These data suggest that the light from the LS-1 is not reflected from the colored tiles when the meat (steak and chicken breast) is more than 1 mm thick.

Method:
Absorbance

Introduction:
Several compounds were purchased to provide UV/VIS absorption data for a spectral library of data for biological samples. Small molecules included Adenosine-5′-triphosphate (Sigma #A2283), Bilirubin mixed isomers (Sigma #B4126), Dipicolinic acid (Sigma #D0759). Several heme proteins were also analyzed including Cytochrome c from bovine heart (Sigma #C3131), Human methemoglobin (Sigma #H7379), Human hemoglobin-Ao, ferrous(oxyhemoglobin – Sigma #H0267) and Myoglobin from horse skeletal muscle (Sigma #M0630).

Experimental Conditions:
A 5 mg/mL stock solution was prepared for each heme protein in blood bank saline (VWR # 48212-270). A 1 mg/mL stock solution of ATP was prepared in blood bank saline. A 0.5 mg/mL stock solution was prepared for bilirubin in 0.1% NaOH (insoluble material removed with a 0.2 um syringe filter (Nalgene # 195-2520)). A 10 mM CaDPA solution was prepared in 10 mM NaOH/5 mM CaCl2.

Hardware Used:
ISS-UV-VIS integrated sampling system
USB2000 (USB2E214 – with Grating 1, OFLV-2, L2 lens, 50 um slit)
1 cm pathlength quartz cuvette (CVD-Q-10)
P300-1-SR 300 um solarization resistant fiber

Experimental Parameters:
ATP and Bilirubin
Integration time: 30 msec
Averages: 10
Boxcar: 3

DPA
Integration time: 3 msec
Averages: 100
Boxcar: 3

Heme proteins
Integration time: 45 msec
Averages: 10
Boxcar: 3

Measurement Mode:
Absorbance

Results:
Spectral data for the small molecules is shown in Figure 1 below. The ATP concentration shown is 0.0417 mg/mL in saline with Bilirubin at a concentration of 0.015 mg/mL in 0.1 N NaOH and 0.5 uM solution CaDPA in 10 mM NaOH/5 mM CaCl2.

Spectral data for the heme proteins are shown in Figure 2. A concentration of 0.238 mg/mL MetHemoglobin and OxyHemoglobin in saline are shown. Myoglobin and Cytochrome c are shown at concentrations of 0.111 and 0.122 mg/mL in saline, respectively.

Figure 1: Absorbance spectra for biological molecules

Figure 2: Absorbance spectra for heme proteins

Method:
LIBS

Introduction:
Following the promising results obtained for the ulexite/clay pellets, additional pellets prepared from XPI-263 grab samples were prepared with a narrow boron concentration range. The pellets were prepared to assess the sensitivity of LIBS to boron concentration.

Experimental Conditions:
Ulexite pellets were prepared with closely spaced boron concentrations. The pellets were prepared with a new grinding technique. Unlike the previous set of pellets, each of the new pellets was received intact. Each pellet was placed in the LIBS sampling chamber. Ten single shot spectra were acquired at different locations on each pellet.

Data analysis involved calculating the ratio of the boron (249.5422 nm) and silicon (251.40066 nm) lines for each single shot spectrum and then calculating average ratio and standard deviation values for each sample.

Hardware Used:
Single channel LIBS spectrometer (HR2000CG-UV-NIR with LIBS upgrade serial # HR2B338)
BIF600-UV/VIS with LIBS focusing lens at the tip
Big Sky 50 mJ Nd:YAG Laser
LIBS SC Sampling Chamber with 125 mm focal length lens
Radiometrically calibrated spectrometer

Experimental Parameters:
Q-Switch Delay (delay between laser firing and data acquisition): -1.5 microseconds
Single laser shot at a fresh spot on the pellet (10 single shot spectra averaged together for standard curve calculations)

Measurement Mode:
LIBS

Results:
The complete data set acquired previously was reassessed to determine the optimal measurement parameters. Based on the standard curve generated and the standard deviation of the values, the best results were obtained with a radiometrically calibrated spectrometer for results averaged from 10 single shot spectra. Due to changes in the LIBS system configuration (probe was removed and replaced) since the last set of pellet measurements, a new standard curve was generated using the previous set of pellet samples. The new standard curve is shown in Figure 1 below. It is similar to the standard curve reported previously. As shown in Figure 1, when the ratio of these lines was taken at each ulexite concentration, the intensity trend increased with increasing boron concentration.

In Figure 2, the ratio of boron and silicon lines calculated for the new set of XPI-263 pellets is shown. Note that these pellets cover a much narrower range of concentrations (10.15 to 11.70% boron). As anticipated, based on the error associated with the wet analysis technique, the trend is noisy especially in regions where the boron concentrations were very closely spaced (e.g. 10.29 to 10.36% boron). Overall, an increasing trend in intensity is observed with increasing boron concentration. The standard deviations of the values are higher than observed for the previous set of pellets.

Conclusions:
It is difficult to determine the sensitivity of the LIBS technique from this data based on the error associated with the wet assay technique. Sensitivity at the 0.5% boron concentration may be possible based the results shown above.

Figure 1: Ratio of the average intensities (n=10) for boron and silicon lines as a function of boron concentration: Original set of ulexite pellets measured previously

Figure 2: Ratio of the average intensities (n=10) for boron and silicon lines as a function of boron concentration: New set of XPI-263 pellets

Method:
LIBS

Introduction:
Three features of interest can be distinguished visually in the glued wood composite. The very thin dark lines are a phenol formaldehyde resin bondline (sodium catalyst). The light tan (earlywood) and darker brown (latewood) lines represent different growth rates for the wood.

Experimental Conditions:
For the initial analysis to assess the discriminating capabilities of LIBS, a series of LIBS spectra were acquired at random locations on the sample. The goal was to obtain data that could be subjected to chemometrics analysis to determine if the different layers could be distinguished using LIBS and PCA and PLS analysis. Five different spots were measured at random locations on the sample with 7 spectra acquired at each spot to provide a depth profile. Each spectrum was single laser shot. To provide more specific data for chemometrics analysis, additional analysis was carried out on the specific features of the composite.

Hardware Used:
LIBS2000+ broadband, high-resolution spectrometer
50 and 200 mJ Nd:YAG Big Sky lasers
LIBS-SC sampling chamber with a 38 mm focal length lens (to provide a small spot size since spatial resolution is important)

Experimental Parameters:
-2 microsecond Q-switch setting

Measurement Mode:
LIBS

Results:
Based on the chemometrics analysis of the original set of data acquired with the 200 mJ laser at random locations on the glued wood composite (PLS and PCA analysis carried out by the customer – proprietary data), PCA and PLS analysis of the LIBS spectra were not sufficient for discrimination of the three layers of interest. PCA analysis showed greater differences between spectra acquired at the same spot than for the different areas sampled. PLS analysis showed a limited correlation between the depth and spectra suggesting that the actual laser pulse may have created a limited “memory” effect as it changed the wood (i.e. crater formation).

Since the first set of measurements were made at random locations on the composite, it was difficult to determine whether or not each of composite layers was sampled. To provide a more specific set of data to assess the discrimination capabilities of LIBS, an additional set of data was acquired for each of the specific layers in the glued wood composite. Analysis was carried out with the 50 mJ laser to minimize the spot size and allow sampling of the individuals layers. The average of 3 single shot spectra are shown in Figure 1 for each of the glued wood composite layers. Additional data for 6 consecutive LIBS spectra acquired at the same spot on the light tan layer are shown in Figure 2 to provide a pseudo-depth profile of this layer.

Conclusions:
A quick assessment of the spectral data showed a unique spectral line around 586 nm for the thin black bondline. This line is most likely a sodium line indicative of the sodium used to cure the phenol formaldehyde resin. Compared to the early and latewood layers, the black bondline had more sodium lines (as detected by the Elemental Identification software) and more intense sodium lines around 589 nm.

Unique spectral lines were observed for the early and latewood layers around 821, 822 and 857 nm. The identity of these lines is not known but all three of them occur near reported lines for nitrogen. The early and latewood layers also had a larger hydrogen alpha line around 656 nm related to the moisture content of the layer.

All of these unique spectral features, along with others that were not observed in the quick data assessment done, provide potential discriminating features for chemometrics analysis.

Figure 1: LIBS Spectra for Different Layers of Glued Wood Composite: Average of 3 Single Shot Spectra

Figure 2: LIBS Depth Profile for Light Tan Layer of Glued Wood Composite

Method:
LIBS

Introduction:
Two steel samples were tested for the presence of magnesium (Mg), manganese (Mn), sulfur (S) and silicon (Si) to assess the ability of LIBS to detect dirt in steel. Two samples were analyzed: approximately 38 mm diameter metal cylinder (approximately 50 mm tall) and approximately 25 mm diameter metal disk (less than 12 mm tall).

Experimental Conditions:
The smaller steel sample was measured on the flat surfaces on either side of the sample. The larger sample was measured on two of the curved surfaces. Sand was observed on the outside of the larger sample. Several single shot spectra and multi-shot spectra were acquired for each of the surfaces chosen for analysis.

Hardware Used:
LIBS2000+ broadband, high-resolution spectrometer
50 mJ Nd:YAG Big Sky laser
LIBS-SC sampling chamber with a 125 mm focal length lens

Experimental Parameters:
-2 microsecond Q-switch delay setting
1 to 10 spectra averaged

Measurement Mode:
LIBS

Results:
Average spectral data for the different surfaces measured for each of the samples are shown in Figures 1 and 2. As expected for alloy samples, the spectral data are very rich in elemental emission lines.

Results for LIBS detection of the elements of interest are presented in separate tables for each sample. The number of elemental lines identified by the Elemental Identification mode of the OOILIBS software is shown in Table 1. Note that the elemental identification library contains multiple lines for each element taken from the persistent lines in the MIT wavelength tables. The values shown represent the number of lines identified out of the total number of lines contained in the elemental identification library.

Conclusions:
Based on the results shown in Table 1 for LIBS analysis carried out with a 50 mJ laser, all of the elements were detected for the smaller steel sample. Note that sulfur was only observed for the single shot spectra acquired at fresh spots on the sample. When 10 shots were averaged at the same location on the sample, sulfur was not detected. This suggests that sulfur is only found on the surface of the samples. For the larger sample, all of the elements except sulfur were detected. The use of a more powerful laser may improve detection by allowing for the detection of additional emission lines for the elements of interest.

Figure 1: Average spectral data (n=3) for larger steel piece

Figure 2: Average spectral data (n=3) for smaller steel piece

Method:
Absorbance

Introduction:
UV/VIS spectra were acquired for diluted sheep, goat and chicken whole blood (Colorado Serum Company). Whole blood from these animals was chosen for differences in the properties of the red blood cells (RBCs) from each animal which could be used to assess the impact of these properties on the spectral data. Red blood cells in sheep blood are 4.5 um in diameter with limited central pallor (related to distribution of hemoglobin in the cell). Goat blood contains 3.2 um diameter RBCs with limited central pallor. Red blood cells in chicken blood were the largest of the samples at 7.7 um wide by 8.1 um long. In addition, chicken RBCs contain a nucleus and are oval in shape.

Experimental Conditions:
The optically dense whole blood samples were diluted to provide samples appropriate for UV/VIS analysis with a 1 cm pathlength cuvette. A 0.9% NaCl solution at approximately pH 7.5 (0.9 g NaCl in 100 mL, sodium hydroxide used to adjust pH) was prepared for diluting blood samples. The blood samples were diluted as follows:

Sheep blood – 5 uL blood + 3. 5 mL diluent (0.143%)
Goat blood – 3 uL blood + 3 mL diluent (0.102%)
Chicken blood – 8 uL blood + 3 mL diluent (0.267%)

Hardware Used:
ISS-UV-VIS light source
1 cm pathlength quartz cuvette (CVD-Q-10)
300 um UV/VIS solarization resistant fiber (P300-1-SR)
USB2000 spectrometer(USB2E214- grating # 1, OFLV-2, L2, 50 um slit)

Experimental Parameters:
Integration Time (msec): 36
Spectra Averaged: 10
Boxcar Smoothing: 3

Measurement Mode:
Absorbance

Results:
Visual observations were carried out on the settled whole blood samples to assess cell lysis (rupture) and hematocrit (concentration of red blood cells (RBCs)). The presence of a red tinge in the plasma portion of the settled blood samples indicated that RBC lysis (rupture) had occured resulting in the release of hemoglobin into the plasma. Goat blood had the highest level of red tinge in the plasma and the highest hematocrit based on the amount of settled RBCs (note that the containers were different sizes so these observations were highly subjective).

Spectra for the diluted whole blood samples are shown in Figure 1. The absorbance spectra shown are a combination of absorption due to the chromophores present in the sample (i.e. plasma proteins, hemoglobin) and light scattering due to the presence of large particles (red blood cells, white blood cells and platelets). The slope of the spectrum (observed most easily above 400 nm) is related to the size of the particles present in the sample. For the smallest RBCs found in the goat sample, the slope was steeper than for the other blood samples.

Peaks observed around 414 nm and between 500 and 600 nm, indicating free hemoglobin in solution, confirm that all three blood samples had some degree of cell lysis. Chicken blood appeared to have the most cell lysis. Note for the chicken blood that the peak expected at 260 nm due to the presence of nucleic acids was not observed for the only RBCs containing a nucleus. The expected peak may have been masked by light scattering due to the presence of particles in the sample or by the presence of the strong hemoglobin peak around 280 nm caused by RBC lysis.

For Figure 2, the whole blood samples were diluted directly into deionized water resulting in cell lysis and release of hemoglobin into the plasma. Under these conditions, goat and sheep blood lysed completely as observed by a shift of the entire spectrum to the baseline. The decrease in spectral intensity was caused by a decrease in scattering as the largest and most numerous particles in the sample were ruptured. Chicken red blood cell lysis also occured but not to the same extent as evidenced by a spectrum that remained shifted above the baseline due to scattering from the unlysed red blood cells remaining in the sample. When the lysed blood spectra were normalized to remove intensity differences, the peak around 280 nm is broader in chicken blood than in the other lysed blood samples. The broader peak observed may be due in part to the presence of nucleic acids (absorb light at 260 nm) in the chicken RBC.

Figure 1: Animal blood diluted in 0.1% NaCl (pH 7.5)

Figure 2: Animal blood diluted in deionized water

Method:
Fluorescence

Experimental Conditions:
Five fluorescein samples were prepared at Megabase ranging in concentration from 1 mM to 1 nM. Fresh capillary tubes were also sent for the analysis. The capillaries were dipped into the fluorescein solution to draw sample into the capillary. Each capillary was sampled with the bifurcated fiber pointing at the open end of the tube. Note that the fluorescein solution was very close to the opening of the tube (approximately 1 to 3 mm from the opening). A dark cloth was used to exclude room light from the measurements.

Hardware Used:
USB2000-FLG (USB2E4066)
USB-LS-450
LS-450
BIF600-VIS/NIR
Experimental Parameters:
Integration Time: 3 to 1000 msec
Spectra Averaged: 1
Boxcar Smoothing: 5
Measurement Mode:
Fluorescence

Results:
Results are reported below for two different versions of our LED light source (direct attach USB-LS-450 in Figure 1 and stand alone LS-450 in Figure 2), the USB2000-FLG and a 600-micron bifurcated fiber.

Due to slight variations between the light sources (most likely related to filter placement within the light source), detection down to 1 nM was achieved with the stand-alone light source (Figure 2 for the LS-450) but the emission band was not as resolved with the direct attach source (Figure 1 for the USB-LS-450). At the longer integration times necessary for detection of the lowest fluorescein concentrations, the LED band broadens. Without the appropriate filter in place (shortpass or narrow bandpass filter), the broad LED band will overlap the emission band. While both light sources contained the same filter, the stand-alone light source (Figure 2) had a sharper cutoff than the direct attach module (Figure 1 – compare the LED peaks on the two plots) which resulted in better filtering of the bleed through from the LED for the stand alone source (Figure 2 for the LS-450). The slightly different cutoffs observed for the same filter in the different light source housings is most likely related to differences in the placement of the filter relative to the LED.

For the purpose of providing the least complicated hardware setup for integration into the Megabase instrument, the direct attach module is preferred (smaller housing that requires no external power – power comes from the USB port). After the initial Megabase R & D effort to configure the system, a custom filter with a sharp cutoff (i.e. narrow bandpass interference filter 480 nm +/- 10 nm) can be customized so that it will fit inside the direct attach module to provide the necessary filtering.

It is important to note that for the data reported above, the position of the bifurcated fiber relative to the open tip of the capillary tube had a significant effect on the signal measured with the spectrometer. Parameters such as the angle of the fiber relative to the opening, distance of the sample from the opening and position of the opening relative to the fibers in the SMA connector were all important. Greater detection limits with less backscatter of the LED signal into the fiber are most likely possible with careful sample interface design.

Conclusions:
The UBS2000-FLG, LS-450 and BIF600-VIS/NIR have sufficient sensitivity to detect nanomolar fluorescein solutions contained in capillary tubes. Additional accessory filters should be tested with the LS-450 light source to determine which filter provides sufficient filtering while maximizing the detection limits.

Figure 1:

Figure 2:

Method:
Fluorescence

Introduction:
FITC is excited at 494 nm and emits at 525 nm. Cy5 is excited at 652 nm and emits at 667 nm. For the final sensor developed for Cy5 detection, it is critical that the sample is not exposed to wavelengths below 610 nm so that the fluorescein present in the sample does not fluoresce. Ultimately, detection will be done in a volume of 10 to 50 uL (typical PCR reaction volume) The sample holder/cuvette design will be carried out by the customer and marketed with their instrument and primers. The applications for the technology range from biological warfare agent detection to agricultural and nosicomial (hospital acquired infections) agent detection. The primers they are using for DNA amplification are selected to give a 0% false positive rate and to be very specific (can differentiate between pathogenic and non-pathogenic forms of anthrax). They already have primers necessary for the identification of 18 significant organisms.

Experimental Conditions:
Customer sent serially diluted samples of FITC and Cy5 in PCR buffer for analysis with our hardware. The samples were poured into UV transmissive disposable cuvettes (CVD-UV1U) and analyzed with a fluorescence based spectrometer (USB2000-FL) and the appropriate LEDs and optical filters. The cuvettes used were chosen to allow for analysis of small samples (less than 100 uL) even though they were not optimal for fluorescence analysis (2 clear windows and 2 frosted windows).

Hardware Used:
USB2000 (Grating #3, 200 um slit, L2 detector collection lens and OFLV order sorting filter – USB2E3295)
CUV-ALL-UV cuvette holder
P600-2-VIS/NIR optical fibers
LS-450 and USB-LS-450 blue led light sources
LS-450 with 640 nm led bulb light source
Disposable UV transparent cuvettes
Experimental Parameters:
Integration Time: 1000 to 30,000 msec
Spectra Averaged: 1 to 10
Boxcar Smoothing: 10
Measurement Mode:
Fluorescence

Results:
Results for FITC are shown in Figure 1. For the FITC data shown in Figure 2, a 515 nm longpass filter was included on the emission side of the sample. Based on the peak at 525 nm, the filter reduced the FITC emission by approximately 45%. In Figure 3, the data shown in Figure 2 is compared to data collected with an R600 reflection probe dipped into the cuvette containing the sample. By dipping the reflection probe into the sample, the FITC signal more than doubled in intensity. In Figure 4, preliminary data collected for Cy5 using an LS-450 containing a 640 nm LED, 300 msec integration time, 10 averages and 10 boxcar is shown. The data was collected for samples contained in the polypropylene tubes in which they arrived. No optical filters were used for this preliminary data but appropriate filters will be necessary to ensure that there is no signal below 610 nm for FRET analaysis.

Conclusions:
The hardware described above was used to detect 3000 to 30 picomoles of FITC (S1 – S3). Cy5 was detected in the 600 to 60 picomole range (S6 and S7).

Figure 1

Figure 2

Figure 3

Figure 4

Method:
Fluorescence

Introduction:
Customer is hoping to use a probe to measure chlorophyll in water (algae) and on rocks (lichens). He currently has a USB2000-VIS-NIR and a 400 micron optical fiber. He would like to purchase a reflection probe and light source for measuring chlorophyll in situ.

Experimental Conditions:
Measurements were made with the reflection probe held in our probe holder at 90 degrees relative to the sample surface. The probe holder was placed on the leaf or lichens sample for the fluoresence measurements.

Hardware Used:
USB2000-FLG (USB2E4876)
LS-450
R400-7-VIS/NIR
RPH-1

Experimental Parameters:
Integration Time (msec): 25 – 200
Spectra Averaged: 1 – 10
Boxcar Smoothing: 3
Measurement Mode:
Fluorescence

Results:
For the leaf measurements shown in Figure 1, we were able to make measurements through glass and in water. The large peaks you see at 470 nm and 920 nm are related to the LED with the peak at 920 nm due to second order effects from the grating. Order sorting filters are not included on our fluorescence spectrometers because the filter will decrease your sensitivity to low level fluorescence. When the measurements are made through glass, there is a lot of LED light scattered back into the reflection probe.

For the lichen samples shown in Figure 2, the peaks were not as high intensity or resolved. This may be related to either the type or concentration of chlorophyll in the samples. When you compare the lichen spectra to the spectra measured for the other side of the lichen samples (bark and bark2), there are spectral differences which seem to be related to the presence of chlorophyll. When I tried to measure the lichen samples through glass, I didn’t see a lot of difference between the bark and lichen side of the sample. With the lower level of fluorescence observed for the lichen samples, we may not be sensitive enough to detect the chlorophyll in the lichen samples when we are making measurements through glass.

Figure 1:

Figure 2:

Experimental Conditions:
The coupons were sent with tape protecting the analysis surface. The tape was peeled back and 3 spectra were acquired at different locations on each sample. Additional spectra for silicone RTV were acquired to provide a reference for the silicone peaks.

Hardware Used:
LIBS2000+ broadband, high-resolution spectrometer
200 mJ Nd:YAG Big Sky laser
LIBS-SC sampling chamber with imaging module
Experimental Parameters:
Laser at full power (setting 8)
-1.5 Q-switch setting (1.5 microsecond delay between firing laser and data acquisition)
Measurement Mode:
LIBS

Results:
LIBS spectra for the coupons and silicone RTV are shown in Figure 1. In Figures 2 and 3, the regions where several silicone peaks occur are shown. The LIBS spectrum for silicone RTV is included with the coupon spectra for comparison.

All of the silicone spectral lines contained in the Element Identification software (5 lines total) were detected in samples labeled 0.2, 0.5, 1, 1.5 and 2. Four out of five spectral lines were detected in the sample labeled 0.1. For the 0 sample, 1 – 2 silicone lines were detected although they are difficult to see in the spectral data (low intensity). Note that it may be possible to obtain a standard curve for silicone by taking the ratio of each silicone peak to the carbon peak at ~247 nm. This ratio may provide a method for addressing shot-to-shot reproducibility issues.

Figure 1: LIBS spectra for coupons

Figure 2: LIBS spectra for coupons: Region where silicone is detected

Figure 3: LIBS spectra for coupons: Region where silicone is detected (cont)

Method:
LIBS

Introduction:
The ability of LIBS to detect the following elements was addressed for several glass and glass-related samples:

• Filter dust: chlorine (Cl), fluorine (F), sulfur (S)
• Colemanite: calcium (Ca), boron (B )
• Green glass cullet: silicon (Si), sodium (Na), potassium (K), Ca, magnesium (Mg), aluminum (Al), iron (Fe), titanium (Ti), chromium (Cr)
• White glass cullet: Si, Na, potassium (K), Ca, Mg, Al, Fe, Ti, Cr
• Glass wool: Si, Na, K, Ca, Mg, Al, Fe, B
• Glass R: Si, Na, K, Ca, Mg, Al, Fe, B

Note that the green and white glasses had a coating on the outer side (convex side). The coating consisted of a tin oxide layer of about 45 angstroms with a polyethylene coating on top with unknown thickness. The concave side of the glass was of more interest since it was not coated. The samples are described in more detail in the Experimental Conditions section below.

Experimental Conditions:
The first samples analyzed were the White and Green cullets. The White cullet sample contained three large fragments. LIBS spectra were acquired for all three fragments. For both cullets, LIBS spectra were acquired on the concave and convex side. When possible (in some cases the curvature of the surface prohibited the averaging of multiple shots), signal averaging was also carried out to improve the signal to noise for the spectral data. Note that the Green cullet pitted less (showed less damage following LIBS analysis) than the White cullet samples.

The Glass R sample was deposited on double-sided, photo-mounting tape adhered to glass microscope slides for LIBS analysis. The particle size of the Glass R sample on the tape varied from large chunks to much smaller fragments. LIBS analysis was carried out for 3 large chunks and several different spots on the tape containing the smaller fragments.

For the remaining samples, the Glass wool was pressed onto the double-sided, photomounting tape using tweezers and a thin film of Filter dust and Colemanite were deposited on separate pieces of the double-sided tape.

Hardware Used:
LIBS2000+ broadband, high-resolution spectrometer
50 mJ Nd:YAG Big Sky laser
LIBS-SC sampling chamber with a 38 mm focal length lens

Experimental Parameters:
-2 microsecond Q-switch delay setting
1 to 10 spectra averaged
Measurement Mode:
LIBS

Results:
Average spectral data for each of the samples is shown in Figure 1. The results from the Elemental Identification mode in OOILIBS are arranged into tables for each sample. The number of elemental lines identified by the software is shown in the table for each sample replicate. Note that the element library contains multiple lines for each element taken from the persistent lines in the MIT wavelength tables. The values shown represent the number of lines identified out of the total number of lines contained in the library.

Conclusions:
Based on the results shown in the tables below for LIBS analysis carried out with a 50 mJ laser, B, Ca, K, Mg, Na, Si were easily detected in the varying matrices of the samples. A small number of Al and Cr lines were also detected for some samples. Elemental emission lines for Fe, Ti, Cl, F and S were not detected and will probably require a more powerful laser. Note that in some cases, signal averaging of 3 to 10 laser shots at the same location enhanced the detection of the elemental lines of interest.

Figure 1: Average Spectral Data for Glass Samples

Elements Identified in Glass R

Method:
Fluorescence

Experimental Conditions:
Three samples were prepared by the customer for FRET analysis. Measurements were carried out in the dark room on the first floor. Sample 1 was tested with the LS-450 and blue laser. The other samples were tested with the blue laser. A 630 longpass optical filter was used on the emission side of the measurements to filter out as much of the fluorescein signal as possible.

Hardware Used:
USB2000 (Grating #3, 200 um slit, L2 detector collection lens and OFLV order sorting filter – USB2E3295)
CUV-ALL cuvette holder
P600-2-VIS/NIR optical fibers
LS450 blue led light sources
DPSS-473 blue Laser
RG-630 Longpass optical filter
Disposable UV transparent cuvettes
Experimental Parameters:
Integration Time: 50 msec
Spectra Averaged: 1
Boxcar Smoothing: 10
Measurement Mode:
Fluorescence

Results:
For the plot below, the samples were kept frozen and in the dark until just prior to analysis with the blue laser. The samples were pipetted into UltraVette, ultra-micro, 15 mm disposable UV-transmissive cuvettes for analysis (CVD-UV1U). Even though these cuvettes were not optimal for fluorescence measurements (2 optically windows and 2 frosted windows), they were chosen to allow for analysis of the small samples provided (less than 100 uL). No Cy5 signal (667 nm) was observed when Sample #1 was analyzed with the blue LED (LS-450) so the remaining samples were only measured with the blue laser.

The position of these peaks suggests that the emission is caused by the excitation of Cy5 by fluorescence energy transfer from fluorescein. In order to confirm whether or not we are actually seeing Cy5 fluorescence, samples containing only Cy5 should be measured to determine the location of the Cy5 peak. Based on the shape of the peaks and the shifting of the peak maxima, the huge background signal due to the excitation of fluorescein with the blue laser is most likely a component of the Cy5 emission peak. The 630 nm longpass filter used for these measurements has a stopband of 580 nm (0.001% internal transmittance at 580 nm) and cut-off of 630 +/-6 nm (50% internal transmittance).

Conclusions:
As anticipated, the huge background fluorescence signal caused by fluorescein present in the sample does appear to interfere with the measurement of Cy5. Two possibilities to deal with the background fluorescence are a lower intensity excitation source or a different optical filter. Based on the analysis of Sample #1 with the blue LED, it is important to note that the sensitivity of the measurement will decrease with a lower energy excitation source. Alternative filter options from Edmund Optics are a narrow bandpass interference filter centered at 671 nm (671 +/-10.8 nm – L30-930 or L43-139) or a longpass filter at 665 nm (RG-665 – note that the transmission at 665 nm will only be 50% with this filter).

Figure 1