Question

1. In one paragraph or less, propose the best solution to the following experimental problems:
   A) You are trying to do a two-colour FRAP experiment using epifluorescence microscopy. You make FP constructs of your two proteins and confirm that each retains its function within your cells. One of the two proteins is coupled to a green emitter and one is coupled to a red emitter. You test each FP construct separately within the cell line (cell-FPgreen and cell-FPred), and find that each protein (based on the FP signal) localizes to the nucleus, showing a bright fluorescence emission in either the green (strain-FPgreen) or red (strain-FPred), and you find that you are able to track the movement of each protein separately during a FRAP experiment. However, when you express both FP constructs together in the same strain (strain-FPgreen-FPred), you see predominantly red fluorescence. What might be happening, and how could you remedy it?

Answer 1

In the above two colour FRAP experiemnt, cell coexpressing the (strain-FPgreen and FPred) ,if you happen to see the predominant red fluorescence, then Spectral bleed through phenomenon is happening.

This occurs due to broad bandwidth and assymtrical spectral range exhibited by common flurophores.In the above condition,emission of red fluorophore is getting dectected by photomultiplier channel or may be through filter combination for green fluorophore.

In order to remove the chances of cross bleed through, instrumental approach can be adapted to separate the fluorophore emission separation.Here we need to make changes manually and allocate the emission spectra with minimum degree of overlap for the signal from each fluorophore to be separated with chosen bandpass filters.Thereby ,emission spectra of flurophore should have shortest excitation wavelength of other fluorophore to avoid bleed through of red and green signal.Confocal microscopy with two different laser can be used to overcome bleed through,one allocated for green fluorphore and other for red fluorophore.