Analysis of FRET Samples
Goal:
To assess feasibility and determine the optimal system configuration for detecting Cy5 fluorescence in samples labeled for fluorescence energy transfer from fluorescein to Cy5
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
