Posted by
Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/microbead-calibration-to-set-psf-tp2560403p2562178.html
Autoquant doesn't usually need an experimental psf, though maybe it can use one. To get a reasonably true psf (that is, free from the influence of the bead) the bead needs to be well below the resolution limit. I'd suggest 100nm if you are using immersion objectives. Some deconvolution software can use larger beads and mathematically extract the psf from the resulting image. Since I've always done 'blind' deconvolution with Autoquant I don't know if it can do that. Check your manual. It it can it's best to go that way - you'll get better signal/noise.
Bang's Labs have just about every form of bead. Pick one that is close to the excitation you'll be using. To prepare a slide dilute the suspension with distilled water and let a drop dry on a #1.5 coverslip. Mount it (bead side down ...) with your favourite mountant.
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
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Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
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http://www.guycox.net-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Matiar Jafari
Sent: Tuesday, 31 March 2009 9:59 AM
To:
[hidden email]
Subject: microbead calibration to set psf?
hey again,
I'm trying to find out how and what the best way to setup my own psf is in AutoQuant. I know i need microbeads and im wondering what brand and what product i need and then how i need to shoot it and load it.
Also what diameter does it need to be. A cat# would be great too also i don't know if it matters on what im shooting but just to put it out there im shooting gfp slices. Sorry if this is vague. Any help would be greatly appreciated. Thanks in advance
Thank You
--
Matiar Jafari
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