Hi all. I'm doing some TIRF analysis of lipid membranes and layers, with and without dyes. I've long been pondering about some "strange" phenomena I've encountered. The internet isn't much help. It seems TIRF in practice is a closely guarded secret...
When I excite my sample (could be anything, even something that shouldn't fluoresce at all!) at either 488 nm or 640 nm, I get two very different results. At 488 I get a hazy outline of the sample morphology. However, if I excite with 640 I get a myriad of vibrating, glowing spots, as if I'm inducing fluorescence in a small % of the molecules (although there aren't any obviously fluorescent compounds involved). The density of the spots also roughly correlates with the surface morphology. Why does this happen?
And secondly, why are small impurities on the surface so extremely bright? Does it have something to do with the way it interacts with the light?
And finally, how come I can excite the dye Atto655 perfectly well with low intensity at 488 nm, although spectra suggest that it's impossible? Is there some configuration with the microscope I'm overlooking (aside from using the wrong laser entirely. But I'm pretty sure I'm not. It looks pretty blue to my eyes)?
All solvents and samples are of highest purity grade, and equipment strictly cleaned and used for specific purposes only to avoid any type of contamination.
1) Along with absorption and fluorescence, there is also another very important optical effect - scattering. This is what you see with fluorescence microscope in (presumably) absence of any fluorophors. You observe it at 488 as a haze image and at 640 as a bright image because you seem to be using the dichroic red filter (makes sense for your dye) - the scattered light at 640nm passes very well through it, but not the light scattered at 488 nm.
2) Scattering on inhomogeneities is very strong, that's why the intensity of scattered light closely follows the surface morphology (roughness). Scattering is quite a complex phenomena, please, check the literature.
3) One possible reason is the the absorption of dye at 488 nm - it might be very low compared to the main absorption bands, but it is not zero. Note that bands may shift depending on the local environment of dye molecules. Another reason could be the fluorescence of the organic matter - most biological matter will fluorescence green when illuminated by blue light. This fluorescence can be reabsorbed by dye and cause its excitation.
Hi Johannes,
I am not sure what kind of fluorescence signal are you trying to see? Are you trying to image single dyes? In that case impurity and the material of the slide plays very important role. Are you using a slide or a cover glass to image the sample? if it is a slide, is it quartz or glass?
If it is a glass slide and you are imaging the slide surface, you are expected to get a very strong background in the 640 range due to auto fluorescence from glass.
You didn't mention any hardware you are using, check the if the emission filters are strong enough OD8+ (TIRF-compatible), it appears you observe some laser Excitation interference from the laser.
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