It sounds like your platereader is a spectrophotometer and not a fluorimeter--or that you are using the wrong measurement mode.

So you are likely measuring absorbance at 480 and absorbance at 520, while what you want to do is measure fluorescence, so you need input light at 485-490 (excitation) and measure the emitted light at 520 (emission).

Dear Peter, You got me right. In some of the research papers, it is mentioned that we can also detect the signal by using microplate reader. How? I don't know! so I think I just captured the absorbance. Did I ruin my experiment or still there is a way to convert absorbance taken at 480 & 520 into excitation and emission, respectively?

There are microplate readers that can measure fluorescence, perhaps even yours---the software is often not very clear in the case of multifunctional instruments. You may want to triple check what features/functions your instrument has.

Your assay works on the principle that ROS oxidize your non-fluorescent reagent into a fluorescent one, so you detect an increase in emitted light at the emission wavelength, following input of the excitation light.

Measurements of absorbance (negative logarithm of transmittance) and fluorescence (intensity of emitted light by the relaxation of an excited orbital electron) are based on different phenomena; there is no way to interconvert the values.

Now it is possible that the oxidation also results in changes somewhere in the absorbance spectrum, but you would have to develop and validate an alternative assay to ensure your measuring something real. Be aware that the sensitivity of such an absorbance assay will likely be 10-1000 times worse, since many many different species interfere and overlap in absorbance, while far fewer exhibit high quantum yield fluorescence.

Hello Peter Sobolewski,

A very nice & descriptive explanation, I just want to understand the point that " if I have florescence based (Excitation & Emission) assay, And I do not have microplate reader which can detect florescence, Can I take absorbance of the sample only at a EMISSION wavelength?

really sorry, question may sound like baseless or pointless.

Hi Saurabh,

Your question is fine! No such thing as a bad question!

If you don't have the option to read the fluorescence, you may be able to get some meaningful data out of an absorbance measurement. Typically, your best bet is to measure the absorbance in the *excitation region*, since you can be sure the dye/assay molecule absorbs light in this region. Briefly, fluorescence relies on the principle of an electron absorbing energy from the incident photon and entering an excited state. The emission we measure is a result of the relaxation of this excited state. If you can't detect the emitted photons (fluorescence), your best bet is to measure the absorbance, which should have a peak around the excitation wavelength. Then, assuming minimal interference, you should be able to prepare a standard curve of concentration vs. absorbance and be able to quantify some data thanks to the Beer-Lambert law.

Now I recommend doing some googling or other lit research on your specific assay--it's possible, even likely, someone has optimized an absorbance based protocol, perhaps using an alternate wavelength or adding a 2nd reading to correct for some background or other effect.

Thank you so much Peter Sobolewski for explaining the situation.