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

In summary, there seems to be a discrepancy in the fluorescence readings between using a cuvette and a 96 well plate in a fluorometer assay. This could be due to the type of material used for the cuvette and well plate, as well as the positioning of the excitation and detection paths in the fluorometer. Additionally, the sensitivity and background noise of each instrument may also play a role in the differing results. Further clarification and experimentation may be necessary to determine the exact cause of this inconsistency.
  • #1
RooneyChemistry
1
0
TL;DR Summary
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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  • #3
What kind of fluorometer are you using? Exc from above or below?
 
  • #4
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.
 
  • #5
A 96 well fluoro reader couldn’t excite from the side, could it?
 
  • #6
chemisttree said:
A 96 well fluoro reader couldn’t excite from the side, could it?
Maybe with tomography, LOL. A most expensive method.
 
  • #7
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.
 

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

Why is my fluorescent sample showing a signal with a cuvette but not with a plate?

Fluorescent samples can behave differently depending on the material they are in contact with. Cuvettes are made of materials that are less likely to interfere with the fluorescence signal, while plates may contain substances that can absorb or scatter the fluorescent light, leading to a weaker signal.

Could the plate material be causing the issue?

Yes, the material of the plate can have a significant impact on the fluorescence signal. Some materials may absorb or scatter the fluorescent light, reducing the intensity of the signal. It is recommended to use plates made of materials that are compatible with fluorescent measurements.

Is the thickness of the cuvette affecting the signal?

The thickness of the cuvette can affect the fluorescence signal, but typically cuvettes are designed to minimize interference with the signal. However, if the cuvette is too thick, it may affect the path length of the excitation and emission light, leading to a weaker signal.

Could the shape of the cuvette be influencing the signal?

The shape of the cuvette is unlikely to have a significant impact on the fluorescence signal. As long as the cuvette is designed for fluorescence measurements and has optically clear surfaces, the shape should not affect the signal. However, irregularities in the shape or surface of the cuvette may lead to signal distortion.

What are some troubleshooting steps to improve the fluorescence signal with a plate?

To improve the fluorescence signal with a plate, you can try using plates made of different materials, ensuring that the plate is clean and free of any contaminants that may interfere with the signal, optimizing the excitation and emission wavelengths, and adjusting the plate positioning in the fluorescence reader to minimize signal loss.

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