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Stefan: I am almost sure that Sebastian is talking about a 5mm thick crystal to built the dichroic on to, so all the dichroics will have this thickness. Best regards
 > Date: Thu, 28 Jun 2012 15:04:44 +0200
> From: [log in to unmask]
> Subject: Re: calibration of confocal spinning disk setup (yokogawa)
> To: [log in to unmask]
> 
> *****
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> 
> Hi Sebastian,
> 
> I am simply calculating the x/y fwhm of each particle. I already used different objectives, still
> giving the same results, thus it's probably not a matter of the objective. Also we use no
> aftermagnification.
> 
> Unfortunately, I don't understand what you mean with using 5mm substrates on the dicroics. Maybe you
> can explain this in more detail?
> 
> Thanks!
> Stefan
> 
> Sebastian Rhode wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> > 
> > Hi Stefan,
> > 
> > from my experience so far, there can be a couple of things go wrong. When I
> > found asymmetric PSF, the most common problem were caused by:
> > 
> > - the objective itself - Did you check the PSF of those beads using the same
> > objective on different setups?
> > 
> > - Do you use a CMount with built-in optics (Aftermagnification) since use a
> > CCD with rather large pixels?
> > 
> > - Dichroic - especially the CSU-X1 use very thin (0.5mm) dichroics, which
> > will not be very flat (for this reason we use 5mm substrates for our
> > dicroics) - so this might induce a curvature in one direction and finally
> > lead to astigmatism
> > 
> > By the way, what are the values you calculated?
> > 
> > By the way, no commercial interests here.
> > 
> > Cheers,  Sebi
> > 
> > Dr. Sebastian Rhode
> > Project Manager Research & Development      
> > 
> > TILL Photonics GmbH
> > an FEI Company
> > 
> > 
> > 
> > On Thu, 28 Jun 2012 12:08:02 +0200, Stefan Sokoll <[log in to unmask]>
> > wrote:
> > 
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Hello,
> >>
> >> I am doing single particle tracking using an upright confocal spinning disk
> > setup. The setup
> >> basically consists of an Olympus TIRF100x oil objective, an Olympus
> > microscope and on top a Yokogawa
> >> spinning disk CSU-X1 with an Andor EMCCD camera.
> >>
> >> Analyzing the 3D PSF of latex beats I discover that the PSF is not
> > symmetrical in xy direction,
> >> instead the x expansion is roughly 50nm larger than in y direction.
> >>
> >> I wonder what might be the main cause for this effect? Tilted cover slip,
> > adjustment of the devices,
> >> the CSU itself or ...? So far I just put the devices on top of each other
> > but due to the upright
> >> setup I cannot check the symmetry of the laser beam e.g. over long distance
> > at a wall.
> >> Any ideas on how to best start calibrating the setup are very welcome.
> > Thanks in advance!
> >> Best,
> >> Stefan
> >>
> >> --
> >> ---------
> >> Dipl. Ing.-Inf. Stefan Sokoll
> >> Lab. Molecular Physiology
> >> Dept. Neurochemistry & Molecular Biology
> >> Leibniz Institute for Neurobiology (LIN)
> >> Brenneckestrasse 6
> >> 39118 Magdeburg
> >> Germany
> >>
> >> Mail: [log in to unmask]
> >> Tel.: +49 - 391 - 626393171
> 
> -- 
> ---------
> Dipl. Ing.-Inf. Stefan Sokoll
> Lab. Molecular Physiology
> Dept. Neurochemistry & Molecular Biology
> Leibniz Institute for Neurobiology (LIN)
> Brenneckestrasse 6
> 39118 Magdeburg
> Germany
> 
> Mail: [log in to unmask]
> Tel.: +49 - 391 - 626393171