Re: DIC condensers

Posted by Johannes Helm on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DIC-condensers-tp7583008p7583011.html

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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:

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>
> 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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