http://confocal-microscopy-list.275.s1.nabble.com/DIC-condensers-tp7583008p7583023.html
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
the F-stop all the way and open the A-stop all the way. We can get images
> *****
> 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>