Single molecule fluorescence microscopy and limits

[HAYAO]

I want to ask the limits on the molecule side for single molecule fluorescence microscopy. I am writing a proposal but I lack experimental knowledge since no one in my lab have ever used one.

At least how much of an absorption coefficient, with at least how much of a radiative rate is necessary for detecting on fluorescence microscope if combined with spectrometer and you want decent wavelength resolution?

*I know it also depends on other factors such as bleaching and the length of the experiment one is expecting, but I want some guidelines.

 

[Andy Resnick]

I want to ask the limits on the molecule side for single molecule fluorescence microscopy. I am writing a proposal but I lack experimental knowledge since no one in my lab have ever used one.

A proposal to whom?

Single-molecule fluorescence is all about the signal to noise ratio, and there are many different approaches, depending if you want to (for example) spatially localize the molecule (imaging) or not (detection). Combining imaging with spectroscopy will decrease the signal even further.

 

[HAYAO]

Thank you Andy.

Research proposal for funding.

I do need to spatially localize the molecule and combine spectroscopy.

I am thinking about confocal technique with Nd:YAG laser as excitation source but I know not everyone has this setup. It is fine if you have other setup; I still want to know the order of magnitude of the limits on the molecule side (and I would appreciate it if one could briefly provide the setup).

 

[Andy Resnick]

Research proposal for funding.

That's obvious- why else spend the time writing a proposal? To whom are you submitting? I ask because reviewers will quickly see that you have no expertise and so the probability of you successfully carrying out the work is low.

Hyperspectral imaging (which is what you want to do) minimizes the available signal. Using an Nd:YAG in a confocal requires modification of most commercial units, and restricts the fluorophores you can use. If you don't even know what hardware is required, I don't understand why you would bother to submit a proposal.

 

[HAYAO]

That's obvious- why else spend the time writing a proposal? To whom are you submitting? I ask because reviewers will quickly see that you have no expertise and so the probability of you successfully carrying out the work is low.

Hyperspectral imaging (which is what you want to do) minimizes the available signal. Using an Nd:YAG in a confocal requires modification of most commercial units, and restricts the fluorophores you can use. If you don't even know what hardware is required, I don't understand why you would bother to submit a proposal.

Yes, I have no expertise, which is why I am asking. I'm sure I've said that before. I don't know who is going to read (I am not supposed to) but will likely be some professor in photochemistry.

Like I said, I know the thing is technical. People make a career out of the machine itself. So I am not expecting it to be simple.

It doesn't have to be Nd:YAG. It was a example only because I have the diode pumped Nd:YAG right now so I qualitatively know the output power in CW mode for THG wavelength, and have been using that for the molecules I am working on. But argon lasers and nitrogen lasers are still fine (or maybe some diode-laser), argon works better for my molecules in terms of excitation wavelength. It's just that I don't have it right now so I don't have any idea how strong they are going to be.

I know hyperspectral imaging is going to minimize the available signal, qualitatively.