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>Brightness = 10 to the 4th x (Numerical
>Aperture squared/Magnification squared)
>
>So far three lens:
>
>63x/1.4NA = 9.65
>100x/1.4NA = 3.8
>40x/1.3 NA = 18.9 thus 4x brighter than 100x and twice that of 60x.
>
>From Inoue and Spring, Video Microscopy, The Fundamentals, 2nd
>Edition page 137, 138
>
>Mike Ignatius
>
>Molecular Probes/Invitrogen
Hi all,
I think that Mike quotes Inoue and Spring correctly. (My copies of
this book are at the UBC Course.)
However, some sources quote a the fourth power NA dependence and this
is really only warranted if the image of the illumination source that
should be present in the BFP of the objective is of sufficient size
to fill the BFP. This is not always true. In addition, it really only
refers to the "apparent brightness to the eye", rather than some
overarching "measure of optical goodness." Whatever you choose, I
sometimes feel that these equations are a bit misleading as they seem
to have led some to conclude that lower mag must be better for
fluorescence. Period..
At a fixed NA, the diameter of the BFP is inversely proportional to
the objective magnification. Once you know the size and shape of the
image of the source formed by the illumination system in the BFP, you
can figure out what fraction of it will pass through the BFP of each
lens. (On an inverted scope with a dry lens, you can see the relation
between the source image and the lens NA by holding a piece of lens
tissue a few cm above the lens. If the illumination is
Kohler-aligned, you you should see an image of the arc. The dark
circular border represents the rated NA of the objective.).
More than 85% of any light passing the BFP will pass the lens and
illuminate the object. However, in the same way that the area of the
BFP (and hence the amount of light passing through it) is inversely
proportional to the square of the magnification, then the area of the
specimen that is illuminated is also inversely proportional to the
square of the magnification. So assuming that the same image of the
arc is present in the BFP of both 40x and 100x lenses, the photons
striking each square micrometer of the specimen will be the same.
Assuming a uniformly-fluorescent specimen, the amount of signal that
makes it back to a given pixel of the CCD camera, will be inversely
proportional to the objective magnification. So superficially, the
40x will be about 6x brighter than the 100x.
However, when we are recording a digital image, this is not the end
of the story. Nyquist sampling theory says that we want the
dimensions of each pixel of the CCD (referred to the specimen plane)
to be about 2.5x smaller than the resolution limit of the optical
system. This means that, for a given NA (and wavelength), there
exists a "best" total magnification from the specimen to the plane of
the CCD, and if your 40x lens doesn't provide enough then you should
increase the magnification of the camera-coupling lens until Nyquist
is happy. Alternatively, if your CCD pixels are two small, you can
"bin" them 2x or 3x to make effective pixels that are 2 or 3 times
larger in linear pixel dimensions.
In any case, as long as the CCD pixels are the same size, when
referred to the specimen, the NA is the same, and the image of the
arc fills the BFP of the lower mag objective fairly uniformly, then
signal recorded in one pixel of the CCD will be the same. (i.e., the
objective mag is irrelevant because the total mag is fixed by
Nyquist.)
So why would you choose one over another? The 4Ox will look brighter
"by eye" although not necessarily 38x brighter or even 6x brighter,
because the image of the arc in the BFP usually not the optimal size.
The 40x will also have a larger field of view making it easier to
find the "good" part of the specimen. On the other hand, the image
will be smaller and harder to "see" and you will bleach a larger area
of the specimen while you are scanning around. Finally, if you fail
to use the field diaphragm to limit the illumination to include only
that part that is imaged by the CCD, you will have a lot more light
reflecting around off optical surfaces, contributing to background
haze and reducing image contrast.
In addition, as it is much more difficult to correct all of the many
aberrations over the larger field of view of the 40x, it will
probably only have "diffraction-limited" performance over the central
area. By the same token, there may be fewer optical compromises in
the 100x objective, although the 2.5x shorter focal length may impose
limits on the working distance.
Many people will base their choice on whichever lens comes closest to
Nyuquist sampling with their favorite CCD. (Cameras based on the
popular SONY chips have 6.7 micrometer pixels, while those using the
E2V 512x512 EM-CCD chip, preferred by many for disk scanning and
TIRF, has 15 micrometer pixels.)
All of this says nothing about spherical aberration that occurs when
one focuses a dry or an oil lens into fluorescent plastic etc.
Cheers,
Jim Pawley
**********************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building,
FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706
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3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/ Applications still being
accepted for Waitlist
"If it ain't diffraction, it must be statistics." Anon.
>From: Confocal Microscopy List
>[mailto:[log in to unmask]] On Behalf Of Sarah Kefayati
>Sent: Friday, May 09, 2008 2:55 PM
>To: [log in to unmask]
>Subject: 40x vs. 60x
>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>Hello all,
>
>I have checked the intensity loss by imaging a fluorescent plastic
>block with both LUMPlanFI/IR 40x/.8 W and UplanApo/IR 60x/1.2 W
>through Z axis.The drop of 60x is the way faster that 40x.As I know
>with the higher NA I should have better light collection.
>
>My question is:
>
>Could it be because of the fact of higher NA higher sensitivity to
>the mismatch and higher SA?
>or these two lenses are not comparable as 40x is a dipping lens?
>
>Any information is highly appreciated.Thanks
>
>Sarah
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