http://confocal-microscopy-list.275.s1.nabble.com/DIC-condensers-tp7583008p7583048.html
Thanks for all the input. Our high NA oil condenser came today so I will
get to testing it out soon. A quick question I have is what pixel size
I still aim for ~ 90 nm pixels (as I would in fluorescence). I only ask
> *****
> 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.
> *****
>
> Make sure you are filling the aperture of your high-NA condenser when you
> get it! The LED may have been underfilling or overfilling the lens which
> could be the reason for your discrepancy between lamp and LED.
>
> Craig Brideau
>
> On Sun, Nov 30, 2014 at 9:07 AM, Jeff Spector <
[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 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> > >
> >
>