By hemolysing blood from the same mouse multiple times, I find that my fluorescence gets quenched proportionally to how much absorbance I can measure at 410nm compared to just having PBS. I have been trying to figure out how I can use Absorbance at 410nm to normalise for this. Any help would be greatly appreciated.
This paper describes a method to correct for quenching of fluorescence in an assay:
http://jbx.sagepub.com/content/14/8/1008.long
To use this method, you will need to make a measurement of the amount of quenching under the same conditions as the assay. In your case, you can do this by replacing the substrate with the product. Measure the ratio of the fluorescence of the product in PBS versus in the hemolyzed sample. This becomes the correction factor by which to multiply the fluorescence in the reaction.
Thank you Adam - reading your publication is helping quite a bit. Thank you for your suggestion as well. Unfortunately, the substrate also has background fluorescence, and the hemolyzed sample absorbs at both the excitation and emission wavelengths.
I have samples that should have zero activity. Would it be reasonable to use loss of fluorescence that comes from the substrate to derive my correction factor for losing excitation? And then derive the correction factor for emission by using the same samples with product, as you suggested? How would I combine these correction factors?
Quenching of the excitation and emission are probably both due to the inner filter effect, which is simply the absorbance caused by the hemolyzed sample. It affects excitation and emission to different extents, but that doesn't matter for the correction factor that you are going to apply to the product fluorescence. You should still use the change in fluorescence intensity of the product in the hemolyzed sample.
A separate concern is whether the hemolyzed sample is itself fluorescent. I don't think the correction method can handle interference consisting of a combination of fluorescence background and quenching.
I tested your latter question - there is no fluorescence from the samples themselves in my range of measurement.
How would I accommodate for variable hemolysis between samples? I don't get enough of each sample to run it in duplicate with product, which is what I think you are suggesting. That's why I was trying to find a way to use absorbance (which I can measure after fluorescence has been measured) to normalize.
Various methods have been proposed to correct for the inner filter effect. Here is a paper that describes one such method, that also has references to other methods:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4114876/
Thank you Adam - that last reference is exactly what I needed.
(1+(ABS-ABSblank))*fluorescence
for absorbance at both emission and excitation wavelengths reduces variability between my wells - I use the same spectrometer for both measurements
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