Re: Perfect Focus

Posted by Chris Tully on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Perfect-Focus-tp1098084p1098297.html

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Scott,

The perfect focus systems usually rely on a laser that is reflected off of a cover slip and automatically adjusts the focus of the microscope such that the cover glass is always the same distance from the objective.  They ARE NOT autofocus devices - you have to set the initial focus and then trigger the system.  For long term experiments and especially for experiments with multi well plates perfect focus systems can be VERY useful.  While taking a z stack can help, I have seen more than one multi well plate with enough variation from well to well (especially from one end of the plate to the other) to require 11 or more slices to insure you get the infocus plane.  (NOTE: I always recommend using an odd number of slices, based on the idea that if you focus on a specific plane and then do say a four slice stack centered on that plane, you will actually miss the plane the you focused on!)   The beauty of the perfect focus system is that it gaurantees that you always start at the same relative plane.  If the samples are stable, you may be able to get away with a single plane, but if not you can still use a much smaller stack say three or five planes that actaully bracket the sample ratherthan 11 or more in the hope that the few planes you need will fall somewhere in that range.

Chris Tully


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Chris Tully
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On Thu, Sep 18, 2008 at 10:10 AM, Mayandi Sivaguru <[hidden email]> wrote:

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Scott, it looks like you have a fully automated system. Personally, I do not rely on autofocus, I would suggest, if you take multi position, multi channels over multiple time point, then instead of one frame position/well also include a small z stack, say 4 images (2 above and 2 below) from the best focus of each position, so latter even if there is a drift, you will have an option to select the best plane out of those four.
Shiv

At 07:56 AM 9/18/2008, you wrote:

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All,

Have new faculty coming aboard and a major application will be 48 hour
timelapse studies in 96 well plates using multichannel collections.
Therefore my question is how critical is it to have a system that has
a perfect focus mechanism to meet this application? Are there decent
work arounds if the perfect focus is not used? Have never actually
worked with this type of system so would like input on this matter
from the list. Thanks. 


Scott J. Howell, Ph.D.
Manager, Imaging Module
Visual Sciences Research Center
Case Western Reserve University
2085 Adelbert Rd.
Institute of Pathology Room 106
Cleveland, Ohio 44106
216-368-2300
http://www.case.edu/med/vsrc/

Mayandi Sivaguru, PhD, PhD
Microscopy Facility Manager
8, Institute for Genomic Biology
University of Illinois at Urbana-Champaign
1206 West Gregory Dr.
Urbana, IL 61801 USA

Office: 217.333.1214
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