Hi Eric,
Hard to believe we are still at it after 15 years. Hope we get it right soon~
Actually all the manufacturers make NA 1.4 oiled
condensers (at least for their uprights). The
optical quality isn't great but then it doesn't
have to be. The object they are trying to focus
in the blobby looking arc or a wire filament.
Neither of these are points or even flat.
So as long is the condenser shoots some light at
the right angles, all is (pretty much) well.
(Shinya Inoue used a high-NA objective with an
iris as the condenser on his homemade "Rail
Scope" but that is another matter. Ditto the 4pi
techniques. And these don't work with slides:
only coverslips).
High-NA condensers are made to work with standard
1mm thick microscope slides. (This is why their
top lenses (i.e., towards the slide) are so much
larger than that of, say a 60x 1.4 objective).
Koehler is seldom "perfect" because the light
source is usually a 3D object and so some of this
"object" must be out of focus in the "Kohler
planes" where it should be in focus. You can do a
bit better if you insert a a piece of ground
glass into the light path to act as a flatish
virtual light source. But then you lose brigtness.
Don't agonize: condenser optics are not corrected
as well as objectives anyway and so they follow
the simple optics focusing rules on the Kohler
diagrams only approximately.
Looking at some of the later posts, I am more and
more thinking that the problem is the mountant. A
mountant that is perfect for fluorescence would
have the same RI as oil and this RI is quite
similar to many of the protein components of
cells. So they disappear in terms of phase
contrast.
If you don't see it in darkfield, there is no phase contrast to see.
Jim Pawley
*********************************************************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research
Building, FAX
608-265-5315
1117 Johnson Ave., Madison, WI, 53706
[log in to unmask]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/
Applications still being accepted
"If it ain't diffraction, it must be statistics." Anon.
>
>
>Jim,
>High NA condenser/objectives come with pretty
>short working distance that imposes mounting
>samples between 2 coverslip. At least that's the
>requirement for highly efficient DIC that
>imposes perfect kölher alignment I believe. That
>+ the oil on both sides have made this practice
>less and less popular among students and
>facility managers (although not for the same
>reasons).
>Is darkfield more tolerant than DIC?
>Cheers
>Eric (13 years survivor, had to count twice to make sure, GOSH!!!)
>
>
>
>Eric Scarfone, PhD, CNRS,
>Center for Hearing and communication Research
>Department of Clinical Neuroscience
>Karolinska Institutet
>
>Postal Address:
>CFH, M1:02
>Karolinska Hospital,
>SE-171 76 Stockholm, Sweden
>
>Work: +46 (0)8-517 79343,
>Cell: +46 (0)70 888 2352
>Fax: +46 (0)8-301876
>
>email: [log in to unmask]
>http://www.ki.se/cfh/
>
>
>----- Original Message -----
>From: James Pawley <[log in to unmask]>
>Date: Thursday, May 6, 2010 3:42 pm
>Subject: Re: DIC Image
>To: [log in to unmask]
>
>> Hi Charu,
>>
>> Well, you seem to have thought of most of the suggested solutions
>> already.
>> If, as Guy suggests, the problem is that there is really not
>> enough
>> left of your sample to produce a detectable RI difference, (which
>> seems strange as video-enhanced DIC used to give good images of
>> single microtubules, or even single actin filaments.) another
>> possibility is Darkfield imaging.
>>
>> As any "survivor" of the UBC Course will tell you, darkfield is my
>> favorite transmitted light imaging method, and it will really
>> produce
>> the most contrast for a given quantity of structural material
>> present. The only problem is that you must use a darkfield
> > condenser
>> with a higher NA than that of your objective and you need to align
>> the condenser with great care. Although this isn't always easy,
>> the
>> results can be really superb.
>>
>> If you don't have a super high-NA darkfield condenser (>0.9 NA.
>> i.e.,
>> one that you have to "oil" to the slide), you might find an
>> objective
>> with an iris diaphragm that will still allow you to make the
>> objective NA lower than that of the illumination. While this will
>> reduce the resolution a bit, it will definitely allow you to see
>> the
>> edges of the cells.
>>
>> In confocal one can get a similar image using the backscattered
>> light
>> signal, however, the signal from the cell will be overwhelmed by
>> the
>> much larger reflection from the glass-water interface and this
>> will
>> make it hard to see cellular details within 3-4 micrometers of the
>> interface.
>>
>> Good luck,
>>
>> Jim Pawley
>>
>>
>>*********************************************************************************
>> Prof. James B. Pawley,
>> Ph. 608-263-3147
>> Room 223, Zoology Research Building,
>> FAX 608-265-5315
>> 1117 Johnson Ave., Madison, WI, 53706
>> [log in to unmask]
>> 3D Microscopy of Living Cells Course, June 12-24, 2010, UBC,
>> Vancouver Canada
>> Info: http://www.3dcourse.ubc.ca/ Applications
>> still
>> being accepted
>> "If it ain't diffraction, it must be statistics." Anon.
>>
>> >Hi
>> >I also suspected the same when i face this problem for the first
>> >time. I did little bit of modification in my fixation. What i am
>> >doing now is fixing my cells with 100% chilled methanol and keep
>> the
>> >cells at 4 degrees for 10min. Then i do permeabilisation with
>> 0.3%
>> >triton X-100. i need to do this step in order to stain my protein
>> >which is inside nucleus.
>> >May be this is becoming too much for the cells.
>> >Thanks
>> >Charu
>> >
>> >CHARU TANWAR
>> >Imaging Specialist
>> >Advanced Instrumentation Research Facility
>> >Jawaharlal Nehru University
>> >New Delhi 110067
>> >India.
>> >
>> >--- On Thu, 6/5/10, Guy Cox <[log in to unmask]> wrote:
>> >
>> >
>> >From: Guy Cox <[log in to unmask]>
>> >Subject: Re: DIC Image
>> >To: [log in to unmask]
>> >Date: Thursday, 6 May, 2010, 12:15 PM
>> >
>> >Charu,
>> >
>> >My guess is that your fixation is permeabilising your cells so
>> >effectively - and maybe extracting some lipids - that there isn't too
>> >much left to generate DIC contrast. As Peter suggests, meticulously
>> >checking the setup of your microscope - Koehler, polarizer,
>> analyser etc
>> >may help. But phase contrast might be a better option than DIC -
>> >generally it does better with very thin samples. Phase lenses
>> come in
>> >two forms low (L) and high (H) absorbance - if you have a choice
>> an H
>> >lens will do better with a weakly diffracting object.
>> >
>> > Guy
>> >
>> >Please help fight breast cancer by sponsoring my
>> >run in the Sydney Half Marathon on May 16th.
>> ><http://www.everydayhero.com.au/guy_cox>http://www.everydayhero.com.au/guy_cox
>> >______________________________________________
>> >Associate Professor Guy Cox, MA, DPhil(Oxon)
>> >Australian Centre for Microscopy & Microanalysis,
>> >Madsen Building F09, University of Sydney, NSW 2006
>> >
>> >Phone +61 2 9351 3176 Fax +61 2 9351 7682
>> > Mobile 0413 281 861
>> >______________________________________________
>> > <http://www.guycox.net/>http://www.guycox.net
>> >
>> >
>> >-----Original Message-----
>> >From: Confocal Microscopy List
>> >[mailto:<[log in to unmask]" target="_blank">http:[log in to unmask]>[log in to unmask]]
>> >On Behalf Of Charu Tanwar
>> >Sent: Thursday, 6 May 2010 4:27 PM
>> >To:
>> ><[log in to unmask]" target="_blank">http:[log in to unmask]>[log in to unmask]
>> >Subject: DIC Image
>> >
>> >Dear List
>> >I am doing an experiment with HeLa cells staining them with
>> antibodies>for
>> >two different proteins and trying to see localization.However,
>> >Fluorescence
>> >is not my problem but i am not able to visualize my cells in bright
>> >field.......Well, i have to really put my eyes on so much of
>> strain to
>> >visualize them in bright field and get their morphology (Cells
> > appear to
>> >become very thin and barely gives boundary demarcation) . I got my
>> >scope
>> >checked and that is in good health. I tried this with other scope and
>> >the
>> >problem remains same. I have to focus my cells through
>> epifluorescence>illumination (i.e. by looking at the expression).
>> I have never faced
>> >such a
>> >weird problem before. Even confocal microscope refuses to give a good
>> >DIC
>> >image (same scope is giving very good DIC images for RBC'S and
>> Candida).>I
>> >do see two different expressions in my cells but i am not getting
>> a good
>> >DIC
>> >image, sometimes i do not see anything in DIC channel but
>> simultaneously>i
>> >see good expression.
>> >I foresee that this is not a scope problem but either with the
>> cells or
>> >staining protocol. However, i have changed my staining protocol 4
>> times>(Fixation, time incubation for Ab's, no. of washes and
>> blocking) but the
>> >story is same.When i visualize my cells before starting the
>> experiment,>cells look healthy and appear fine.
>> >Please help
>> >Charu Tanwar
>> >
>> >
>> >CHARU TANWAR
>> >Imaging Specialist
>> >Advanced Instrumentation Research Facility
>> >Jawaharlal Nehru University
>> >New Delhi
>> >India
>> >
>> >
>> >
>> >
>> >No virus found in this incoming message.
>> >Checked by AVG - www.avg.com
>> >Version: 9.0.819 / Virus Database: 271.1.1/2854 - Release Date:
>> 05/06/10>04:26:00
>>
>>
>> --
>>
--
*********************************************************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research
Building, FAX
608-265-5315
1117 Johnson Ave., Madison, WI, 53706
[log in to unmask]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/
Applications still being accepted
"If it ain't diffraction, it must be statistics." Anon.
|
