Posted by
Arne Seitz on
Aug 23, 2012; 2:55pm
URL: http://confocal-microscopy-list.275.s1.nabble.com/focused-laser-beam-size-on-stage-tp7578908p7578910.html
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy*****
Dear Yan,
the size of the excitation beam in confocal microscopy is usually diffraction limited. In the easiest case the minimum size depends on the numerical aperture of the objectives and the used wavelength. Under the following link you can find a PSF generator with which you can calculate the size of your excitation spot (even with rather complicated models).
http://bigwww.epfl.ch/algorithms/psfgenerator/In practice changing the size is only possible if you under fill the back focal aperture of your objective. In Leica SP2 models this was possible via the so called beam expander. But I'm not sure whether this is also true for the SP5 microscopes (I do not think so). The effective size of your excitation spot in MP microscopy is even more difficult to calculate. I suggest reading the following articles in order to get an idea about it.
Nat Biotechnol. 2003 Nov;21(11):1369-77.
Nonlinear magic: multiphoton microscopy in the biosciences.
Zipfel WR, Williams RM, Webb WW.
Deep tissue two-photon microscopy.
Helmchen F, Denk W.
Nat Methods. 2005 Dec;2(12):932-40. Review.
But you can easily find numerous other articles dealing with this topic.
By no means the size of the excitation beam will change if you change your sampling frequency (pixel size).
I hope that helps.
Best regards
Arne
---------------------------------------------------------------
Arne Seitz
Head of Bioimaging and Optics Platform (PT-BIOP)
Ecole Polytechnique Fédérale de Lausanne (EPFL)
Faculty of Life Sciences
Station 15, AI 0241
CH-1015 Lausanne
Phone: +41 21 693 9618
Fax: +41 21 693 9585
http://biop.epfl.ch/---------------------------------------------------------------
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:
[hidden email]] On Behalf Of Tong Yan
> Sent: jeudi 23 août 2012 15:58
> To:
[hidden email]
> Subject: focused laser beam size on stage
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Dear list,
>
> We have one Leica TCS SP5 MP confocal system and I would like to find out
> how the size of the laser beam on the stage is controlled? When we change
> the pixel size to meet Nyquist criteria, does the laser focal point on the
> specimen change its size accordingly? Will the focused beam has same size to
> the pixels? What is the smallest focus size of the laser beam on the stage we
> can achieve? Does two photon excitation has the same scenarios?
> Thank you very much in advance for your kind advice.
> Best regards,
>
> TONG Yan (Ms) :: National University of Singapore :: 14 Science Drive 4,
> Singapore 117543 :: (65) 6516 7202 (DID) :: (65) 6779 2486 (Fax) ::) ::
>
[hidden email] (E) :: www.nus.edu.sg (W)Company Registration No:
> 200604346E