Carol,

the actual thickness by which an object can or cannot be optically
sectioned, especially in widefield microscopy has been a well
discussed/disputed subject. One has to make useful assumtions when defining
the term resolution. With respect to widefield microscopy which I assume you
are referring to, the situation is not straight forward. The optical
resolution of a microscope is laterally and axially limited by the size of
the diffraction spot. Viewing that spot in the Fourier domain reveals that
some spatial object frequencies are not transmitted at all (also referred to
as 'missing cone'). Due to that, it is impossible to focus on an object
which has no lateral spatial detail and is much thicker than the diffraction
spot. Without any quantitative suggestions - increasing lateral detail
increases the ability to focus on objects inside thicker specimens. Because
of that a resolution term for optical sectioning in widefield microscopy can
not be given. If (unrealistically) the specimen plane is thinner than the
diffraction spot, you can define the axial size of it as criterion. The
diffaction spot is inversely proportional to the square of the NA and
proportional to the wavelength. A commonly used criterion is the one that is
based on Rayleigh's and is given as (peak to null):

d = 2 * n * Lambda / NA^2

n denotes the RI of the immersion media. In the literature you will find a
number of approximate 'definitions'. E.g. a factor of 1.67 instead of 2,
estimates the FWHM. By dropping the factor of 2 completely one approximates
about ~80% of the maximal intensity as 'wave-optical depth resolution'
(Zeiss booklet "Confocal laser scanning microscopy"). The latter is also
referred to as depth of field - provided that one can recognize a 20%
decrease in intensity by visual observation. A good literature reference is
always Jim Pawley's (ed.)"Handbook of biological confocal microscopy".

As for the term 'depth of focus' in Barabara's book, I can only guess, that
she used that term to express the impact of varying magnifications on the
image target plane (camera or ocular). However, the magnification does not
have anything to do with resolution, only indirectly - when the NA varies
with magnification. For instance, in the object plane a 40x/1.4 Oil lens has
the same diffraction spot size as a 63x/1.4 Oil objective.

I hope this was of some help

Lutz

______________________________________
Lutz Schaefer
Advanced Imaging Methodology Consultation
16-715 Doon Village Rd.
Kitchener, Ontario
N2P 2A2, Canada
Email: [log in to unmask]
Phone, FAX: (519)-894-8870
______________________________________



----- Original Message -----
From: "Carol Bayles" <[log in to unmask]>
To: <[log in to unmask]>
Sent: Monday, December 17, 2001 1:05 PM
Subject: depth of focus


> Dear Readers,
> in the Glossary of Barbara Foster's excellent book "Optimizing Light
> Microscopy for  Biological and Clinical Labs," she distinguishes
> between depth of field and depth of focus.  What is depth of focus
> dependent on, besides the NA of the objective?  How can one measure
> it?   I was asked to provide the depth of focus for an
> epi-fluorescence image taken on one of our microscopes.  Can I do
> this?
>
> Thank you for your help.
>
> Carol
> --
> Carol Bayles
> Microscopy,  Imaging & Fluorimetry (MIF)         Confocal and
> Multiphoton Microscopy
> Bioresource Center                       Nanobiotechnology Center
> 160 Biotech Bldg                                141 Clark Hall
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>
> Cornell University     Ithaca NY 14853
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