http://confocal-microscopy-list.275.s1.nabble.com/DIC-condensers-tp7583008p7583032.html
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>
> HI all,
> Thanks for the input. We've got a high NA oil condenser on the way so
> hopefully this solves our problem. When using both the LED and the Lamp we
> did make sure that we were in Kohler illumination in both cases. We are in
> fact able to get images using the low NA condenser but we have to stop down
> the F-stop all the way and open the A-stop all the way. We can get images
> but the contrast just isn't good enough to perform our measurements so
> hopefully going to a higher NA condenser will solve this problem..
> thanks for the input..
> -Jeff
>
> On Sat, Nov 29, 2014 at 4:26 PM, George McNamara <
>
[hidden email]>
> 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.
> > *****
> >
> > Hi Johannes,
> >
> > You do not need to add Nomarski (or Smith or anyone else's) DIC
> > components. You do need a high NA objective lens and digital image
> > processing. See
> >
> >
http://web.stanford.edu/group/blocklab/GutierrezAJP2010.pdf> >
> > Also helps to be in one of the top ten labs in the world for this kind of
> > work.
> >
> > Enjoy,
> >
> > George
> > p.s. see
http://www.ncbi.nlm.nih.gov/pubmed/17381703 for an example of
> > LED-DIC.
> > Recent IR-DIC "Dodt contrast",
>
http://www.ncbi.nlm.nih.gov/pubmed/24298032> > Earlier IR-DIC
http://www.ncbi.nlm.nih.gov/pubmed/2085783> > 1984 paper using AVEC-DIC to image single microtubules in live cells
> >
http://www.ncbi.nlm.nih.gov/pubmed/6333427> >
> >
> > On 11/29/2014 10:33 AM, Johannes Helm 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.
> >> *****
> >>
> >> 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
> >>>
> >>
> >>
> >
> > --
> >
> >
> >
> > George McNamara, Ph.D.
> > Single Cells Analyst
> > L.J.N. Cooper Lab
> > University of Texas M.D. Anderson Cancer Center
> > Houston, TX 77054
> > Tattletales
http://works.bepress.com/gmcnamara/42> >
>