Fluorescent Sample with Cuvette but not with Plate -- Why?

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Discussion Overview

The discussion revolves around the differences in fluorescence detection of a compound when using a cuvette versus a 96 well plate in a fluorometer. Participants explore potential reasons for the observed discrepancy in fluorescence, considering various factors related to the equipment and materials used.

Discussion Character

  • Exploratory
  • Technical explanation
  • Debate/contested

Main Points Raised

  • One participant questions whether the cuvette itself might be contributing to the fluorescence, suggesting the need to consider the material of the cuvette.
  • Another participant asks for clarification on the type of fluorometer being used, specifically whether the excitation is from above or below.
  • Concerns are raised about the materials of both the cuvette and the well plate, with inquiries into whether the cuvette is made of fused silica, borosilicate glass, or quartz.
  • There is uncertainty expressed regarding the excitation method of the 96 well plate reader, with one participant doubting that it could excite from the side.
  • One participant suggests that differences in sensitivity and background noise between the instruments could explain the variation in fluorescence detection.

Areas of Agreement / Disagreement

Participants express multiple competing views regarding the factors affecting fluorescence detection, and the discussion remains unresolved with no consensus reached.

Contextual Notes

Limitations include missing information about the specific materials of the cuvette and well plate, as well as the exact configuration of the fluorometer used.

RooneyChemistry
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TL;DR
Fluorescent Sample with Cuvette but not with Plate
I am doing an assay where the formed compound is fluorescent when I use a cuvette in a fluorometer, but not when I put the same sample into the well of a 96 well plate. Why is this?

The fluorometer reads from the top and the plates I am using are 96 well plates, black, flat-bottomed well, opaque bottom, fluotrac from Greiner.

Exc. 468 nm, Em. 572 nm.
 
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What kind of fluorometer are you using? Exc from above or below?
 
Yeah we lack information here. What is the cuvette made out of? Fused silica? Borosilicate glass? Quartz? How about the well plate? I am also unsure what fluorometer excites from above. Most of them are sideways, where detection are perpendicular to the excitation path.
 
A 96 well fluoro reader couldn’t excite from the side, could it?
 
chemisttree said:
A 96 well fluoro reader couldn’t excite from the side, could it?
Maybe with tomography, LOL. A most expensive method.
 
Probably the sensitivity and different background noise of each instruments. Plate readers used in bio-assays are generally much less sensitive than traditional cuvette fluorometers. You could also get some background noise if your detector is not at a right angle from the light source.
 

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