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Hi Claire,

In microscopes equipped with lasers, it is known that the laser 
polarization influences the resolution.
In confocal microscopes, the PSF is the product of the illumination PSF 
and the detection PSF.
The illumination PSF is broader in the direction of the beam’s original 
polarization.
The detection PSF is not influenced by the polarization, because the 
fluorescence is usually not polarized.

Some confocal laser scanning microscopes on the market illuminate the 
sample with linearly polarized light, some with circularly polarized light.
1) In the first case, the linear polarization gives rise to an 
elongation of the PSF along the direction of the incident linear 
polarization. In this article from Jonkman and co-workers [J. E. N. 
Jonkman et al., Methods in Enzymology 360, 416–446 (2003)], this 
elongation has been theoretically quantified to about 17 %. In practice, 
it can be more. There is another interesting paper from Li and 
co-workers [Q. Li et al., Advanced Device Materials 1, 4–10 (2015)], in 
which you can find both the theory about the influence of the incident 
polarization on the illumination PSF and a comparison of measurements 
from gold beads with the theory.
2) In the second case, the circular polarization gives rise to an 
isotropic PSF.

Does this mean that microscopes in which the illumination is circularly 
polarized provide a better resolution than those in which the 
illumination is linearly polarized? Not necessarily, because there is no 
such thing as a free lunch. Indeed, dichroic mirrors in the microscopes 
are optimal for linear polarization, which results in better 
signal-to-noise ratio (SNR) in the images. Since the resolution is 
limited by the SNR, the "bad thing" (the elongation of the PSF in one 
direction for linear polarization) can be compensated by a "good thing" 
(a better SNR in the images).

This is why our leitmotiv at Argolight is: when one wants to measure 
resolution, it is important to associate the minimum measurable 
separation distance to a contrast criterion, as well as to  
signal-to-noise and signal-to-background ratios, because all these 
parameters influence what we usually call "resolution".

Best regards,
Arnaud

*Arnaud ROYON, Ph.D.*
/CEO & CSO, Co-founder/
Argolight
Cité de la Photonique, Bat. Elnath
11 avenue de Canteranne
33600 Pessac, FRANCE
Email: [log in to unmask]
Tel: (+33) 5 64 31 08 50
Web site: www.argolight.com <http://www.argolight.com>
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Le 06/02/2024 à 02:38, Claire Brown, Dr. a écrit :
> *****
> To join or leave the confocal microscopy listserv or to change your email address, go to:
> https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1
> Post images onhttp://www.imgur.com  and include the link in your posting.
> *****
>
> Hello All,
>
> I'm posting this for a colleague who is not on the list and is trying to do FCS on a Zeiss LSM980.
>
> "When constructing the PSF to measure its dimensions in the Zeiss LSM 980 using 100 or 20 nm diameter fluorescent beads, there is large astigmatism in the y dimension compared to the x dimension where y is up to 30% larger than x. The same problem is witnessed for the Airyscan PSF on the same microscope. Has anyone else witnessed this? Is that normal? Can that be fixed without impacting the sensitivity or the resolution limits? And how does that impact FCS measurements where the 3D fitting diffusion model assumes x, y symmetry."
>
>
> All the best,
>
> Claire