Can FRAP Be Performed in Water with Small Molecules?

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SUMMARY

The discussion centers on the feasibility of performing Fluorescence Recovery After Photobleaching (FRAP) in aqueous solutions with small molecules to determine diffusion coefficients. The participant, Lindsay, expresses challenges in evaluating FRAP data due to the absence of discrete regions of interest (ROIs) in liquid environments, which complicates the analysis compared to cellular contexts. Lindsay suggests that techniques like fluorescence correlation spectroscopy may be more appropriate for studying diffusion in low-viscosity solutions. A reference to a relevant paper on FRAP in cell-free conditions is provided for further exploration.

PREREQUISITES
  • Understanding of Fluorescence Recovery After Photobleaching (FRAP) methodology
  • Familiarity with diffusion coefficient measurement techniques
  • Knowledge of fluorescence correlation spectroscopy principles
  • Basic concepts of regions of interest (ROIs) in imaging
NEXT STEPS
  • Research the application of fluorescence correlation spectroscopy for diffusion studies
  • Explore advanced FRAP techniques in non-cellular environments
  • Study the mathematical derivation of FRAP equations from first principles
  • Review literature on FRAP applications in low-viscosity solutions
USEFUL FOR

Researchers in biophysics, chemists studying molecular diffusion, and anyone interested in advanced fluorescence microscopy techniques.

Lindsayyyy
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Hello everyone,

I have the following question: I'm doing FRAP in water with a quite small molecule in order to get the diffusion coefficient. I'm quite new to FRAP but I have a feeling that this doesn't work very well. I couldn't find any papers or publications in general about someone doing FRAP in a solution. FRAP seems more like a technique for biological cells. Can anyone confirm this or tell me about papers where they perfom FRAP in solution with low viscosity?

thanks for your helpLindsay
edit: also I did some measurements, but it seems like quite the impossible task to do the evaluation of the data, because I need a whole cell ROI and background ROI which isn't possible in a liquid because I have no discrete borders like I would have in a cell.
 
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thanks for your answer.

Basically I can detect a typical FRAP "spectrum" but I don't know if I can evaulate correctly because of the problems mentioned above (no full 'cell' ROI) and also no background is available to measure.
 
thank you very much, I will take a look
 

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