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1  Principles of Confocal Scanning

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1.4   Pinhole Size - optical thickness - resolution  -  light transmission

Smaller pin hole size gives better rejection of out of focus information and hence better x-y & z axis resolution.  In terms of the z-axis smaller pin hole size gives a thinner optical section.  Loss of out of focus information also causes less light to reach the detector, hence confocal views are dimmer.  Loss of intensity can be compensated for by increasing the excitation power and increasing the gain of the detector.  

Actual optical section thickness is a function of N.A., pin hole size and light wavelength.  The exact relationship is complex and is best estimated with test samples.  The figure shows an X-Z scan (side view) of a 10 um solid fluorescent bead impregnated with rhodamine taken with 10X, 20X, 40X and 100X objectives (from left to right) on a LeicaTCS-NT confocal microscope.  Pin Hole size for each objective was 1.0 Airy discs units.  These views demonstrate the different optical sectioning abilities of the objectives.

 

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Last updated 10/24/06


Icon_Inverted_Laser.GIF (1500 bytes) Icon_book_white.gif (1143 bytes) 1998-2003 Michael Chua, Cell & Molecular Physiology, UNC.  All rights reserved. 
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