Confocal Microscopy List

DIC Image

Classic

List

Threaded

26 messages Options

Axel Kurt Preuss

Re: DIC Image

We can explain it by physics. Guy gave us the reasons. And biology may
deliver the nice images. A few years ago I did replatings on dead cell
layers for other reasons but overall DIC or phase was improved
Let s just say
If we can explain it with physics, let biology deliver

Cheers

Axel

Axel K Preuss PhD, A*Star IMCB-Central Imaging Facility 6-19B Sent
from. +65 9271 5622

On May 6, 2010, at 9:05 PM, "Nuno Moreno" <[hidden email]>
wrote:

> What you might want to say is:
>
> When we cannot explain it by physics, let biology speak but not to
> explain.
>
> On 5/6/10 1:56 PM, Axel Kurt Preuss wrote:
>> When physics fails let biology speak! Best to let your cells grow
>> on fixed (dead) cell layers of DIC
> "friendly" cells (e.g simply dried in your sterile hood and then
> washed
> once with 80 percent of physiological buffer ).
>> The dead cells will give your probe cells a nice DIC friendly
>> environment. Once your cells have grown to the right condition
>> proceed as usual
>>
>>
>> Hope that helps
>>
>> Axel
>>
>> Axel K Preuss PhD, A*Star IMCB-Central Imaging Facility
>>
>> On May 6, 2010, at 8:12 PM, "Keith Morris"<[hidden email]<mailto:[hidden email]
>> >> wrote:
>>
>> Hi,
>>
>> Following on from Joels comments:
>>
>> I’m using live HeLa cells at the moment and via the confocal the D
>> IC image is adequate at best [naturally depends on confluency]. O
>> nce fixed and mounted the cells look a lot worse under DIC– I’ve
>> always assumed it was the anti-fade mounting media losing the cell
>> outline, as it probably isn’t optimized for transmission light e.
>> g. wrong refractive index or something [we use Vectashield liquid
>> and Prolong Gold mostly, probably out of habit]. This is also the
>> same for phase contrast [fixed cells looking poorer], although con
>> trast/image quality is superior both live cell and after fixation/
>> mounting. To be honest I’ve just thought ‘who cares’ and used
>> a plasma membrane marker if necessary to delineate cells [Invitrog
>> en wheatgerm/CellMask/Organelle Lights plasma membrane stains] or
>> DAPI/fixed and Hoescht/Live for cell number.
>>
>> So I’ve never bothered improving the DIC image for fluorescence la
>> beled fixed cells [our fixed flattened BPAE cells in particular ar
>> e largely transparent under DIC, mounted in pro-long gold – they l
>> ook great under fluorescence though]. For decent confocal transmis
>> sion images we mostly use 20x phase contrast, and I only use tend
>> to use 63x oil DIC on living cells. The confocal also seems to mak
>> e a far better job of imaging phase contrast compared to DIC as we
>> ll [as the confocal trans detector isn’t optimally placed]. Most o
>> f our confocal users don’t bother with DIC trans using the 63x oil
>> objective unless there’s a structure in the fixed cell/tissue tha
>> t responds well to DIC. You can [and probably should] adjust the c
>> onfocal gain and offset to increase DIC contrast. Our wide-field N
>> ikon microscope [that’s ‘better’ for DIC, imaged via a CCD
>> camera] can apparently be fitted with a selection of DIC options
>> [three different sliders] and one is for ‘low contras
> t thin specimens’ [i.e. cells], so possibly a change of DIC optics a
> s well as mountant might help as well.
>>
>> e.g.<http://www.microscopy-news.com/download-center/Cat_Appl_of_DIC.pdf
>> > http://www.microscopy-news.com/download-center/Cat_Appl_of_DIC.pdf
>>
>> So no help on improving the situation I’m afraid, I just don’t
>> find it a problem I guess.
>>
>> Regards
>>
>> Keith
>>
>> ---
>> ---
>> ---------------------------------------------------------------------
>> Dr Keith J. Morris,
>> Molecular Cytogenetics and Microscopy Core,
>> Laboratory 00/069 and 00/070,
>> The Wellcome Trust Centre for Human Genetics,
>> Roosevelt Drive,
>> Oxford OX3 7BN,
>> United Kingdom.
>>
>> Telephone: +44 (0)1865 287568
>> Email:<mailto:[hidden email]> [hidden email]<mailto:[hidden email]
>> >
>> Web-pages:<http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
>> > http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
>>
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of charu tanwar
>> Sent: 06 May 2010 09:26
>> To:<mailto:[hidden email]> [hidden email]
>> <mailto:[hidden email]>
>> Subject: Re: DIC Image
>>
>> 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<<mailto:[hidden email]>[hidden email]<mailto:[hidden email]
>> >> wrote:
>>
>> From: Guy Cox<<mailto:[hidden email]>[hidden email]<mailto:[hidden email]
>> >>
>> Subject: Re: DIC Image
>> To:<mailto:[hidden email]> [hidden email]
>> <mailto:[hidden email]>
>> 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:<http://in.mc83.mail.yahoo.com/mc/compose?to=CONFOCALMICROSCOPY@...
>> >[hidden email]<mailto:[hidden email]
>> >]
>> On Behalf Of Charu Tanwar
>> Sent: Thursday, 6 May 2010 4:27 PM
>> To:<http://in.mc83.mail.yahoo.com/mc/compose?to=CONFOCALMICROSCOPY@...
>> > [hidden email]<mailto:[hidden email]
>> >
>> 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 -<http://www.avg.com> www.avg.com<http://www.avg.com>
>> Version: 9.0.819 / Virus Database: 271.1.1/2854 - Release Date:
>> 05/06/10
>> 04:26:00
>>
>>
>>
>> Note: This message may contain confidential information. If this
>> Email/Fax has been sent to you by mistake, please notify the sender
>> and delete it immediately. Thank you.

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

Axel Kurt Preuss

Re: DIC Image

In reply to this post by Nuno Moreno

Oil condensor, btw, is a nice idea, worth a try

Thanks

Axel

Axel K Preuss PhD, A*Star IMCB-Central Imaging Facility 6-19B Sent
from. +65 9271 5622

On May 6, 2010, at 9:05 PM, "Nuno Moreno" <[hidden email]>
wrote:

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

James Pawley

Re: DIC Image

In reply to this post by mmodel

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
[hidden email]
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: [hidden email]
>http://www.ki.se/cfh/
>
>
>----- Original Message -----
>From: James Pawley <[hidden email]>
>Date: Thursday, May 6, 2010 3:42 pm
>Subject: Re: DIC Image
>To: [hidden email]
>
>> 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
>> [hidden email]
>> 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 <[hidden email]> wrote:
>> >
>> >
>> >From: Guy Cox <[hidden email]>
>> >Subject: Re: DIC Image
>> >To: [hidden email]
>> >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:<http://in.mc83.mail.yahoo.com/mc/compose?to=CONFOCALMICROSCOPY@...>[hidden email]]
>> >On Behalf Of Charu Tanwar
>> >Sent: Thursday, 6 May 2010 4:27 PM
>> >To:
>> ><http://in.mc83.mail.yahoo.com/mc/compose?to=CONFOCALMICROSCOPY@...>[hidden email]
>> >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
[hidden email]
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.

Johannes Helm

Re: DIC Image

In reply to this post by Phillips, Thomas E.

Good afternoon,

the old style Zeiss 1.4 NA 63X oil immersion lenses, e.g., did not read
"0.17" but used a "-" instead.

This does not mean that these lenses, despite their high NA, would
tolerate large variations in the thickness of cover slips.
However, one has to keep in mind that the refractive index of cedar wood
oil as well as modern immersion oil quite closely and over a large
wavelength range in the visible and NIR matches the refractive index of
BK7 glass, from which cover slips usually are made. Therefore, using a
cover slip not any thicker than 0.17mm and using the proper immersion oil
will, in close approximation, create a cover slip of "flexible thickness".
If you then in addition have a mounting medium with a properly matching
index, you are quite close to what is called "homogeneous immersion", the
most perfect condition for imaging.

In the deeper UV, BK7 glass absorbs to much light. Therefore, fused silica
is the material of choice for the cover slip in this case. Since fused
silica has a refractive index NOT matching that of cedar woold oil or
modern immersion oils, glycerine is the usual immersion medium for high NA
deep UV lenses as, e.g., the Zeiss Ultrafluar lenses, of which I do not
know whether they still are available but which I had used in the early
ninetees when building a CLSM for Fura-2 measurements. These lenses had
excellent chromatic properties, even in the confocal mode!

The third common case is that of water immersion. Years ago, cover slips
made out of a material named CYTOP were available on the market, matching
the refractive index of water and thus useful for - old style - water
immersion made as "dipping lenses" to be used without any cover slip in
case they nevertheless had to be used with cover slip. Ref. e.g.

http://www.wikipatents.com/US-Patent-5406421/cover-slip-for-use-in-microscope

Quit some information on all this is available in, e.g., James Pawley's
"Handbook".

Best wishes,
Johannes

> I am embarrassed to say I don't know whether high NA condensers requiring
> oil also require mounting your specimens on 0.17 mm thick coverslips. Is
> this true?
>
>
> Thomas E. Phillips, Ph.D
> Professor of Biological Sciences
> Director, Molecular Cytology Core
> 2 Tucker Hall
> University of Missouri
> Columbia, MO 65211-7400
> 573-882-4712 (office)
> 573-882-0123 (fax)
> [hidden email]<mailto:[hidden email]>
>
> http://www.biology.missouri.edu/faculty/phillips.html
> http://www.biotech.missouri.edu/mcc/
>
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Eric Scarfone
> Sent: Thursday, May 06, 2010 10:01 AM
> To: [hidden email]
> Subject: Re: DIC Image
>
>
> 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: [hidden email]
> http://www.ki.se/cfh/
>
>
> ----- Original Message -----
> From: James Pawley <[hidden email]>
> Date: Thursday, May 6, 2010 3:42 pm
> Subject: Re: DIC Image
> To: [hidden email]
>
>> 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
>> [hidden email]
>> 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 <[hidden email]> wrote:
>> >
>> >
>> >From: Guy Cox <[hidden email]>
>> >Subject: Re: DIC Image
>> >To: [hidden email]
>> >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:<http://in.mc83.mail.yahoo.com/mc/compose?to=CONFOCALMICROSCOPY@...>[hidden email]]
>> >On Behalf Of Charu Tanwar
>> >Sent: Thursday, 6 May 2010 4:27 PM
>> >To:
>> ><http://in.mc83.mail.yahoo.com/mc/compose?to=CONFOCALMICROSCOPY@...>[hidden email]
>> >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
>>
>>
>> --
>>
>

--
P. Johannes Helm

Voice: (+47) 228 51159 (office)
Fax: (+47) 228 51499 (office)

Richard Harris-6

Re: DIC Image

In reply to this post by Tobias Baskin

Two points I'd like to make
1) I'd never oil a non-oil condenser!
2) From my own experience I have to agree with other posts - the use of a
lower RI (closer to water) mounting media is critical for decent DIC images
of flat tissue culture cells with high NA lenses.
Rick,

Richard Harris, Manager - Integrated Microscopy @ Biotron
The Biotron - Center for Experimental Climate Change Research
University of Western Ontario,
London Ontario, CANADA.
N6A 5B7
Ph. 519-661-2111 ext. 86780
Fax 519-661-3935
e-mail [hidden email]
web: www.thebiotron.ca

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Tobias Baskin
Sent: Thursday, May 06, 2010 11:58 AM
To: [hidden email]
Subject: Re: DIC Image

THis is true if you make a condenser from an
objective lens. But I believe that high NA
condensers (like the dark field type that Jim
Pawley mentioned) have a working distance long
enough to accept a slide. I have been able to
fill the rear focal plane of a 1.25 NA Nomarksi
objective by oiling the high NA condenser to the
slide (I suppose might have been even better by
using a coverslip sandwich, but I don't think
that is essential).

Tobias

>Philip, the problem arises when you combine two
>short working distance lenses above and below
>the sample! If you want both of them to be
>focused on the same plane (ie Köhler
>illumination) you have no other way than to use
>coveslips on both sides!
>
>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: [hidden email]
>http://www.ki.se/cfh/
>
>
>----- Original Message -----
>From: "Phillips, Thomas E." <[hidden email]>
>Date: Thursday, May 6, 2010 5:11 pm
>Subject: Re: DIC Image
>To: [hidden email]
>
>> I am embarrassed to say I don't know whether high NA condensers
>> requiring oil also require mounting your specimens on 0.17 mm
>> thick coverslips. Is this true?
>>
>>
>> Thomas E. Phillips, Ph.D
>> Professor of Biological Sciences
>> Director, Molecular Cytology Core
>> 2 Tucker Hall
>> University of Missouri
>> Columbia, MO 65211-7400
>> 573-882-4712 (office)
>> 573-882-0123 (fax)
>> [hidden email]<mailto:[hidden email]>
>>
>> http://www.biology.missouri.edu/faculty/phillips.html
>> http://www.biotech.missouri.edu/mcc/
>>
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Eric Scarfone
>> Sent: Thursday, May 06, 2010 10:01 AM
>> To: [hidden email]
>> Subject: Re: DIC Image
>>
>>
>> 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: [hidden email]
>> http://www.ki.se/cfh/
>>
>>
>> ----- Original Message -----
>> From: James Pawley <[hidden email]>
>> Date: Thursday, May 6, 2010 3:42 pm
>> Subject: Re: DIC Image
>> To: [hidden email]
>>
>> > 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
>> > [hidden email]
>> > 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 <[hidden email]> wrote:
>> > >
>> > >
>> > >From: Guy Cox <[hidden email]>
>> > >Subject: Re: DIC Image
>> > >To: [hidden email]
>> > >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:<http://in.mc83.mail.yahoo.com/mc/compose?to=CONFOCALMICROSCOPY@LIS

TS.UMN.EDU>[hidden email]]> >On
>>Behalf Of Charu Tanwar
>> > >Sent: Thursday, 6 May 2010 4:27 PM
>> > >To:
>> >
>>
><http://in.mc83.mail.yahoo.com/mc/compose?to=CONFOCALMICROSCOPY@...
DU>[hidden email]> >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
>> >
>> >
>> > --
>> >
>>

--
_ ____ __ ____
/ \ / / \ / \ \ Tobias I. Baskin
/ / / / \ \ \ Biology Department
/_ / __ /__ \ \ \__ 611 N. Pleasant St.
/ / / \ \ \ University of
Massachusetts
/ / / \ \ \ Amherst, MA, 01003
/ / ___ / \ \__/ \ ____
www.bio.umass.edu/biology/baskin
Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243

George McNamara

Re: DIC Image

In reply to this post by Charu Tanwar

Hi Charu,

As Jim Pawley pointed out, backscattered light - aka reflected light - will enable you to see practically transparent cells. As others pointed out, your extraction method has probably reduced the refractive index difference between your cells and mounting medium to a low value. See the phase contrast images in Staudt ... Hell 2007 (PubMed 17131355 ) for example.

For reflected light imaging, a great starting point is:

Interference reflection microscopy. Barr VA, Bunnell SC. Curr Protoc Cell Biol. 2009 Dec;Chapter 4:Unit 4.23.PMID: 20013754 (if you don't have a subscription, the author's email address is [hidden email] - ask for the PDF).

You get an exquisite image of the cell-substratum interfaces by this technique.

JP: "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."

Hi Jim - I get very nice IRM on my LSM510 with coverglass-mounting medium interface brightness adjusted so it does not saturate the PMT (12-bit mode, also offset adjusted so darkest pixels >0 [I usually aim for offset of about 200]), and I have plenty of dynamic range to see structures reflected from various cell interfaces many micrometers away from the interface. Yes, i might have to play with the contrast control, but that hardly counts as "hard".

best wishes,

George
p.s. plan B - since you get great DIC with RBC's, how about sprinking some into the preparation. Or use small microspheres (fluorescent beads).

p.p.s. Speaking of IRM, could someone who is good at developing ImageJ applets please develop one for quantitative IRM. See references in Barr and Bunnell, as well as;

Absolute interfacial distance measurements by dual-wavelength reflection interference contrast microscopy.
Schilling J, Sengupta K, Goennenwein S, Bausch AR, Sackmann E.
Phys Rev E Stat Nonlin Soft Matter Phys. 2004 Feb;69(2 Pt 1):021901. Epub 2004 Feb 12.PMID: 14995485

Quantitative reflection interference contrast microscopy (RICM) in soft matter and cell adhesion.
Limozin L, Sengupta K.
Chemphyschem. 2009 Nov 9;10(16):2752-68. Review.PMID: 19816893

At 02:27 AM 5/6/2010, you wrote:

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

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara