What is a Diffraction Limited Spot in Fluorescent Molecule Imaging?

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Discussion Overview

The discussion centers around the concept of a diffraction limited spot in the context of fluorescent molecule imaging, particularly how it relates to the size of the fluorescence observed from a single molecule when excited by a continuous wave laser. Participants explore the implications of diffraction limits on imaging systems and the factors that influence the size of the observed fluorescence spot.

Discussion Character

  • Technical explanation
  • Conceptual clarification
  • Debate/contested

Main Points Raised

  • One participant inquires about the relationship between the diffraction limited spot and the size of fluorescence from a single molecule, suggesting it is determined by the Airy Disc.
  • Another participant defines a diffraction limited spot as the smallest achievable spot with perfect optics and focus, noting that practical results depend on system design and alignment.
  • A question is raised about factors that can improve the visibility of the diffraction limited spot, specifically regarding numerical aperture (NA) and focal length.
  • One participant explains that a larger numerical aperture is desirable for collecting fluorescence, but also emphasizes the need to minimize the number of CCD pixels used to capture the signal due to readout noise.
  • A later reply challenges the initial assumption that the size of the fluorophore is related to optical properties, clarifying that the physical size of the molecule is distinct from optical considerations, and provides a formula for the size of the Airy function.

Areas of Agreement / Disagreement

Participants express differing views on the relationship between the size of the fluorophore and the optical properties of the imaging system. There is no consensus on the implications of diffraction limits for practical imaging scenarios, and the discussion remains unresolved regarding the optimal balance between numerical aperture and focal length.

Contextual Notes

There are assumptions regarding the uniformity of emission and the design of optical systems that are not fully explored. The discussion also touches on the complexities of signal-to-noise ratios and the impact of system design on imaging outcomes.

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

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