Our plan was to use these slides to characterize he throughput of our microscope objectives at different wavelengths. However, because we know that the Hg long arc lamp is not stable, we thought we should take a few exposures of each field. We noticed a trend of decreasing intensity in these exposures. Therefore, we hypothesized that the plexiglass fluorescence might not be as stable as expected.
Slide labeled "UV-BLUE ex:DAPI" Olympus 40X N.A.0.75 PH2 UplanFL excitation & emission with standard LP dapi filter set Cooke Sensicam QE camera Method: Results show that there is initial rapid bleaching which plateaus.
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Slide labeled "RED" Olympus 40X N.A.0.75 PH2 UplanFL excitation & emission with standard narrow pass Cy3 filter set Cooke Sensicam QE camera What this graph shows most of all is that a Hg long arc lamp that has been on for less than an hour jumps all over the place (magenta curve) whereas after being left on for more than 6 hours, it is less jumpy (dark blue line). After doing the time lapse, we noticed that there was a ND filter in the excitation light path, so the bleaching can't be compared directly with the results above.
And this example has the same ND filters in the light path, but the excitaion and imaging is with a long pass Dapi block.
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Page last revised 27 Feb 2002