CONFOCALMICROSCOPY Archives

January 2014

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Subject:
From:
Julio Vazquez <[log in to unmask]>
Reply To:
Confocal Microscopy List <[log in to unmask]>
Date:
Wed, 29 Jan 2014 11:48:15 -0800
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Yu may want to have a look at these three: 


Robert H. Webb: Confocal optical microscopy. Rep Prog Phys 59: 427-471 (1996). (can be Googled; can send you a copy if you like)



Appl Opt. 1994 Feb 1;33(4):603-15. doi: 10.1364/AO.33.000603.
Background rejection and signal-to-noise optimization in confocal and alternative fluorescence microscopes.
Sandison DR, Webb WW.
Abstract
In the confocal microscope, tightly focused illumination and spatially filtered detection are combined to reduce out-of-focus background and to produce high-quality images that display thin optical sections within thick fluorescent specimens. We define background as the detected light that originates outside a resolution volume and signal as the detected light that originates within the same volume. Background rejection is measured by the signal-to-background ratio (S/B) and is calculated for confocal, spinning-disk, line-illumination, slit-detection, and conventional fluorescence microscopes as a function of both the spatial filter size and the specimen thickness. Spatial filter sizes that reject background and optimize the signal-to-noise ratio (S/N) are calculated for each microscope. These calculations are normalized so that the time-averaged illumination at each point in the specimen is the same for each microscope. For thick specimens, we show that the S/B obtained with a confocal microscope can be more than 100 times greater than the S/B available with a conventional microscope, and we find that the optimal confocal S/N can be a factor of 10 greater than the S/N in the conventional microscope.


Appl Opt. 1995 Jul 1;34(19):3576-88. doi: 10.1364/AO.34.003576.
Quantitative comparison of background rejection, signal-to-noise ratio, and resolution in confocal and full-field laser scanning microscopes.
Sandison DR, Piston DW, Williams RM, Webb WW.
Abstract
Development of a laser scanning microscope for simultaneous three-dimensional imaging in both a full-field laser scanning mode (FLSM) and a confocal laser scanning mode (CLSM) permits the direct comparison of axial resolution and out-of-focus background rejection as a function of sample thickness for both FLSM and CLSM with varying detector aperture (pinhole) radii. The sample-dependent detector aperture radii that optimize the signal-to-noise ratio (S/N) in the CLSM are experimentally determined. The results verify earlier calculations [Appl. Opt. 33, 603 (1994)]. Using these results, we discuss the practical and theoretical limits on the S/N in the CLSM and compare them with those of a full-field epifluorescence microscope (FEM) that is enhanced by image deconvolution. The specimen volume over which the FLSM exhibits imaging properties that are equivalent to a FEM is calculated in the appendices.

Julio 
--


On Jan 29, 2014, at 9:45 AM, John Oreopoulos wrote:

> Thanks Julio, I think the equation I'm looking for is in this document. Does anyone know if equation (4) in this document is derived anywhere in the literature? Is it in any of the books that people have mentioned previously? I'd like to get a copy of that for my records.
> 
> John

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