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.
 

1. Why does fluorescence work well in a cuvette but not in a plate?

Fluorescence detection can be less effective in a plate compared to a cuvette due to several factors. Plates, especially those with multiple wells, can suffer from higher background fluorescence and cross-talk between wells. This happens because the material of the plate and the proximity of the wells can allow light to scatter and fluorescence signals to bleed from one well to another. Cuvettes, typically having larger volumes and being made from materials like quartz or optical-grade glass, minimize these issues, resulting in clearer and more accurate fluorescence measurements.

2. What material differences affect fluorescence measurements between cuvettes and plates?

The material of the container can significantly impact fluorescence measurements. Cuvettes are often made from quartz or high-quality glass that has minimal autofluorescence and high optical clarity, enhancing sensitivity and accuracy. On the other hand, plates are commonly made from polystyrene or polypropylene, which can have higher autofluorescence and may distort or absorb light, thereby affecting the fluorescence signal detected.

3. How does the geometry of cuvettes and plates influence fluorescence detection?

The geometry of the container plays a crucial role in the effectiveness of fluorescence detection. Cuvettes usually have a rectangular shape that allows a clear path for light beams to pass through with minimal distortion. Plates, however, with their shallow and wide well design, can cause more light scattering and reflection, which can interfere with the detection of the fluorescence signal.

4. Can the volume of sample affect fluorescence detection in cuvettes versus plates?

Yes, the volume of the sample can greatly affect fluorescence detection. Cuvettes typically accommodate larger volumes, allowing for more uniform sample illumination and more stable fluorescence measurements. In contrast, the wells in plates hold much smaller volumes, which can lead to rapid photobleaching and less reliable measurements due to the increased surface-to-volume ratio, which affects the interaction of fluorescent molecules with light.

5. Are there specific applications where plates might be preferred over cuvettes for fluorescence measurements?

Despite the disadvantages in some aspects of fluorescence detection, plates are often preferred in high-throughput screening scenarios where many samples need to be analyzed simultaneously. Their design allows for multiple assays to be run concurrently, significantly reducing the time and cost per test. For applications where sensitivity and accuracy are not the paramount concerns, or where rapid screening is required, plates provide an efficient solution.

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