Re: PSF bead shot but X Y and Z dont match
Posted by
Chris Baumann on
URL: http://confocal-microscopy-list.275.s1.nabble.com/PSF-bead-shot-but-X-Y-and-Z-dont-match-tp4589883p4590006.html
Matiar,
What is happening is that you are up against the limit of resolution of
the optics in your system. To correct for this, you would use a
technique called deconvolution. There are a number of resources
available on deconvolution but a good place to start will be
http://micro.magnet.fsu.edu/primer/digitalimaging/deconvolution/deconvolutionhome.html.
Good luck,
Chris
On 2/17/2010 10:32 PM, Matiar Jafari wrote:
how would i then go about correcting the Z blur for all my other
images. if the XY resolution is just distorted because of the
limitations of optics? sorry im really new to this kind of stuff.
Thank You
-------
Matiar Jafari
On Wed, Feb 17, 2010 at 7:29 PM, Fred Mast [hidden email] wrote:
Is this the diametre of your deconvolved bead? If it is not than you have
diffraction to thank for the increased width. The .17 micron beads should be
sub-resolution on your microscope and are representative of a point source
of light. The resulting "blurred" image that you have captured is the result
of the physical properties of light and the optical constraints of your
microscope. A good source for further information can be found at the
following links:
http://support.svi.nl/wiki/SubResolution
http://en.wikipedia.org/wiki/Diffraction
But also try the Handbook for Biological Confocal Microscopy, James Pawley
is the editor, for further information.
Best,
Fred
On 17-Feb-10, at 8:14 PM, Matiar Jafari wrote:
Hey Fellow ListServers,
I purchased a PSF kit from Invitrogen and imaged the beads and when i
loaded the beads to correct the Z blur i measured the XY of the bead
to make sure the diameter matched but it turns out it doesnt. The
beads are supposedly .17um with +-.005 but whatever angle i measure
the bead at in xy i keep getting .4 um. Any ideas as to why this is
happening?
Thank You
-------
Matiar Jafari
Fred D. Mast
Department of Cell Biology
Medical Sciences Building Room 5-14
University of Alberta
Edmonton, Alberta, T6G 2H7
Canada
Tel: 1-780-492-7407
[hidden email]