Posted by
Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Objective-for-confocal-tp590639p590650.html
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I quite agree with what Julio said. There is one
more
point, though. The NA 1.46 lens is presumably
a
TIRF lens, and so may be handy if one later
wants to
add TIRF capabilities. As such, it will probably
also
have a correction collar, and if this is used
correctly it
will significantly improve performance. At
high NA,
minor differences in coverslip thickness and
room
temperature will noticeably impact on
resolution
unless they are corrected
for.
Guy
Optical Imaging Techniques in Cell Biology
by Guy
Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello Ann,
there may be different opinions on this, but here is my take:
With a confocal microscope, you can add magnification by zooming in and
matching the image size (pixel dimensions) for optimal resolution. This means
you can get 100 x magnification with a 40x at zoom 2.5, or a 63x at zoom
1.6.
What the 100x/1.46 gives you in theory is greater resolution (20-30% more),
since resolution increases with the square of the numerical aperture. In
practice, you may not always achieve this extra resolution, unless you have
quite bright samples that are relatively thin and properly mounted, in order to
minimize spherical aberrations.
You also need to consider the type of objective and the application.
PlanApos are corrected for more colors, and therefore are best for precise
colocalization of up to four dyes. Fluars or similar objectives are corrected
for fewer colors, but tend to be brighter than PlanApos, because they are
simpler lenses with less glass... these are often better for live-cell imaging,
or for samples with fewer colors... for a more precise description,
you may want to check the tutorial on lenses and chromatic aberrations
here:
We typically use objectives in the 40x-60x range for most of our confocal
applications (in addition to a 10x or 20x for low-mag work, and possibly a
long-working distance 40x for thick/unusual samples). We rarely use a 100x,
except maybe to look at yeast cells, or some very fine cellular detail. A
100x/1.46 lens will often be less bright than a 40x/1.3, which means that your
gain in resolution may be offset by increased bleaching. In addition, a 40x is
more versatile since you can image a wider field of view at zoom 1, and then get
a close-up at zoom 2-3 if you need more mag, without having to switch
lenses.
A 100x/1.46 lens, on the other hand, would be quite nice on a widefield
microscope... also, this type of lens with high NA is what you need for
applications such as TIRF (total internal Reflection microscopy)...
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024
Tel: Office: 206-667-1215/ Lab: 206-667-4205
FAX: 206-667-6845
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On Oct 26, 2007, at 1:09 PM, Yang, Ann-Fook wrote:
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Hi All,
I am new in confocal microscopy.
We have a 40x/1.3 oil and a 63X/1.2 water objectives.
Is there any advantage to add a 100X/1.46 Oil objective?
Ann Fook Yang,
Agriculture and Agri-Food Canada/Agriculture et
Agroalimentaire Canada
EM Unit/ Unite EM
Edifice K.W. Neatby Building,
960 Carling Av /960 Boulevard Carling,
Ottawa,Ontario
K1A 0C6
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