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Dear Jeff,

Guy has already sent an answer and provided good information for you.

I would like to send some additional information:

In case of transmitted light wide field microscopy, as a rule of thumb 
the (lateral!) resolution is

d = (1.28 * lambda) / (NAobj + NAcond)

This formula is quite useful, and shows pretty well, to which degree, 
indeed, resolution is dependent on the numerical aperture of the 
condenser.  "Of course", it is never a 100% correct description of 
reality, since you will never be able to attain illumination by "just 
one" wavelength; in the very best case you might get a Lorentzian 
profile of a good single mode laserbeam.

Also, in DIC microscopy, when, as Guy already has written, close to 
perfect Koehler illumnation is a sine qua non for a "good image" - 
whatever THIS would be, :-), another issue is that you can trade 
contrast for resolution. I.e.: By closing the condenser aperture 
diaphragm - often referred to as the "A diaphragm" as opposed to the "F 
diaphragm", which is the Field Diaphragm - you will increase contrast 
while at the same time reduce resolution. In "manuals for microscopists" 
of the sixties and seventies as published in Germany, one will sometimes 
be instructed to do the following: "First, attain perfect Koehler 
illumation, then lower the condenser somewhat to attain better 
contrast." Yes, it works, and it is a kind of rather un-controlled 
oblique illumination, which one fakes in that way. But: As a real 
physicist, you would, for ideological reasons, never do this. If you are 
a pragmatic life scientist: Well, you have a better and easier life, 
anyway.

Also note: Make sure that your light source and your polarizer and 
analyzer are useful in the same wavelength band! At least on older DIC 
systems, polarizers and analyzers will do a good job in the visible 
wavelength range, while they will act as a mere 80% Transmission ND 
filter in IR and do hardly polarize! For IR, you might need separate and 
special polarizers (and analyzers, of course, which are nothing else 
than polarizers in another functional position).

Best wishes and have a good 1st of advent,

Johannes

On 2014-11-28 21:21, Jeff Spector wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your 
> posting.
> *****
> 
> Greetings,
>    This isn't exactly a "confocal" questions but I know a lot of 
> "micoscopy
> gurus" live on this list so I thought it a good place to ask this. I 
> have a
> colleague who is trying to image individual (i.e. small and 
> diffraction
> limited) microtubules in a flow chamber by using DIC. They are 
> currently
> using a 100x 1.45 oil Objective, but only a .52 LWD condenser. They 
> were
> using a solid state light source but couldn't get good image so we 
> switched
> to a lamp for illumination and the images are much better, and we can 
> now
> see the microtbules but there still isn't a lot of contrast.  My 
> question
> is, is it worth it to go to a high NA (perhaps oil immersion) 
> condenser,
> and can anyone think of why the lamp would give a better DIC image 
> than a
> solid state light source?
> thanks in advance for the help...
>  -Jeff

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P. Johannes Helm

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