What is a Diffraction Limited Spot in Fluorescent Molecule Imaging?

[Alice Jin]

Dear everyone,

I'm wondering what the diffraction limited spot is.
When a laser(CW) is used to excite a fluorescent single molecule, the fluorescence from a single molecule is shown in the CCD camera. The size of fluorescence molecule is determined by Airy Disc which comes from diffraction limited spot. Is it right?
Does anyone explain this? Please~~ Thank you.

 

[mgb_phys]

A diffraction limited spot is the best/smallest spot you can achieve with the system with perfect focus and perfect optics. Whether you can get this in real use depends on the system design/alignmnet.
It is often used for calcualtions of the limiting signal.

 

[Alice Jin]

Thank you.
Then what's the factor to improve the limited spot I can see in terms of NA or focal lenth?

 

[mgb_phys]

Not sure what you mean?
You want to collect as much of the fluorescence signal as possible, so assuming that the emission is uniform (it emits into a sphere) you want as large a numerical aperture as posible.
But then in signal-noise you want to put the spot onto as few CCD pixels as possible (ideally only one) because there is a readout noise associated with each pixel.
Which is where the diffraction limited bit comes in - for this you want a short focal length.

Unfortuantely Numerical Aperture and focal length are linked (for a given design of objective). Dependign on how weak the signal is and how much money you have - you might want to look at a schwarschild objective

 

[Andy Resnick]

Dear everyone,

I'm wondering what the diffraction limited spot is.
When a laser(CW) is used to excite a fluorescent single molecule, the fluorescence from a single molecule is shown in the CCD camera. The size of fluorescence molecule is determined by Airy Disc which comes from diffraction limited spot. Is it right?
Does anyone explain this? Please~~ Thank you.

I think you are mixing concepts- the size of the fluorophore is not related to any optical properties of the system, it's a physical size of a molecule.

"diffraction limited" is taken to mean an aberration-free optical system. In this case, luminous points are imaged as Airy disks (YMMV- the assumption is that the aperture stop is rotationally symmetric and the optical system is linearly shift-invariant). The 'size' of the Airy function, which can also have several meanings, is usually taken to be similar to a full width half maximum and is given as 0.61*wavelength/(numerical aperture)

http://en.wikipedia.org/wiki/Airy_disc