DIC Image
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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 |
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Hello Charu,
Have you set up Koehler illumination? Pete On May 6, 2010, at 08:27 AM, Charu Tanwar 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 > > > |
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In reply to this post by Charu Tanwar
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 ______________________________________________ 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 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Charu Tanwar Sent: Thursday, 6 May 2010 4:27 PM To: [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 |
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In reply to this post by Peter Gabriel Pitrone
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In reply to this post by Charu Tanwar
On May 6, 2010, at 9:37 AM, charu tanwar wrote:
Hi Charu, Could it be because you are using plastic dishes or plastic covers? They disrupt light polarization, which you need for proper DIC. If this is the case, just switch to glass. Best jy -- Jean-Yves Tinevez PFID - Imagopole Institut Pasteur 25-28, rue du Docteur Roux 75724 Paris cedex 15 France tel: +33 1 40 61 31 77 |
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In reply to this post by Guy Cox-2
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alcohol will extract a lot of things and flatten your cells, Adehyde is a better fix fo preserving morphology and 3D structure. However if you have good images with other cells and same protocol it is puzzling. Stick to fluorescence then and use counterstaining (membrane, cytoplasm, nucleus depending on what you look for) to visualize cell structure!. > 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 > 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 > ______________________________________________ > Associate Professor Guy Cox, MA, DPhil(Oxon) > 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 > > > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]]On Behalf Of Charu Tanwar > Sent: Thursday, 6 May 2010 4:27 PM > To: [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 > 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 > simultaneouslyi > see good expression. > 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: > 05/06/1004:26:00 > > > |
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In reply to this post by Charu Tanwar
Hi, Charu,
from what you write it, indeed, seems that you have a specimen related problem. What I would try (though I cannot guarantee that this will help): a) Make sure the field diaphragm ("F diaphragm") is opened not any further then necessary to cover the entire field of view (assuming the condenser is properly centered). b) At the price of a decrease in resolution, close the aperture diaphragm ("A diaphragm") of the condenser. This will make everything a "little bit darker" and since it will reduce the NA of the condenser, it will also, as written, reduce resolution, but it will, on the other hand enhance contrast. c) In biological preparations, like your cells, the main source of scattering usually is not Rayleigh but Mie scattering. Nevertheless, there still is a square relationsship between wavelength and scattering cross section. Normally, you would prefer to use light of the near IR for thick preparations in order to maximize penetration possibilities. It might, however, be an idea to try the opposite in your case. If you can get hold of a panchromatic green filter or even blue filter - they are / were (?) usually standard items delivered together with any new microscope - to be placed in the ray path, it's a five minutes test to check whether this will help to increase your contrast. If you do not have a green filter or blue filter available, try anyway the DLF ("Day Light Filter", sometimes known as "conversion filter"), which has a reduced red transmission in order to change the spectral distribution of the light source (halogen bulb 12V, 100W) to match the spectral distribution of solar light. d) If you can make a digital image (8bits or 12bits), you can offline - on powerful systems online - change the display properties. A non-linear, possibly logarithmic, display might help to increase contrast. Best wishes, Johannes > 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 > > > > -- P. Johannes Helm Voice: (+47) 228 51159 (office) Fax: (+47) 228 51499 (office) |
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In reply to this post by Charu Tanwar
Hi Charu,
This is probably due to the mounting medium that you are using. In general, the best mounting media for high resolution fluorescence have a refractive index that is similar to that of cells. Since DIC and phase contrast both depend on refractive index differences between, say, membranes and cytoplasm, or membranes and extracellular media, you are unable to visuallize the cells. You might try diluting your mounting media, or mounting with a PBS/Glycerol (50/50) mix containing your own anti-fade, such as n-propyl-gallate. Best of luck. Joel On Thu, May 6, 2010 at 2:27 AM, Charu Tanwar <[hidden email]> wrote: Dear List -- Joel B. Sheffield, Ph.D Department of Biology Temple University Philadelphia, PA 19122 Voice: 215 204 8839 e-mail: [hidden email] URL: http://astro.temple.edu/~jbs |
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In reply to this post by Charu Tanwar
Hi, Following on from Joels comments: I’m using live HeLa cells at the moment and via the
confocal the DIC image is adequate at best [naturally depends on
confluency]. Once 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 contrast/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 [Invitrogen 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
labeled fixed cells [our fixed flattened BPAE cells in particular are largely
transparent under DIC, mounted in pro-long gold – they look great under
fluorescence though]. For decent confocal transmission 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 make a far better job of imaging phase contrast compared
to DIC as well [as the confocal trans detector isn’t optimally placed].
Most of our confocal users don’t bother with DIC trans using the 63x oil
objective unless there’s a structure in the fixed cell/tissue that
responds well to DIC. You can [and probably should] adjust the confocal gain
and offset to increase DIC contrast. Our wide-field Nikon 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 contrast thin specimens’ [i.e. cells], so possibly a change of
DIC optics as well as mountant might help as well. e.g. 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 --------------------------------------------------------------------------- Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of charu tanwar
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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 DIC image is adequate at best [naturally depends on confluency]. Once 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 contrast/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 [Invitrogen 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 labeled fixed cells [our fixed flattened BPAE cells in particular are largely transparent under DIC, mounted in pro-long gold – they look great under fluorescence though]. For decent confocal transmission 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 make a far better job of imaging phase contrast compared to DIC as well [as the confocal trans detector isn’t optimally placed]. Most of our confocal users don’t bother with DIC trans using the 63x oil objective unless there’s a structure in the fixed cell/tissue that responds well to DIC. You can [and probably should] adjust the confocal gain and offset to increase DIC contrast. Our wide-field Nikon 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 contrast thin specimens’ [i.e. cells], so possibly a change of DIC optics as 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. |
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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 DIC image is adequate at best [naturally depends on confluency]. Once 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 contrast/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 [Invitrogen 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 labeled fixed cells [our fixed flattened BPAE cells in particular are largely transparent under DIC, mounted in pro-long gold – they look great under fluorescence though]. For decent confocal transmission 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 make a far better job of imaging phase contrast compared to DIC as well [as the confocal trans detector isn’t optimally placed]. Most of our confocal users don’t bother with DIC trans using the 63x oil objective unless there’s a structure in the fixed cell/tissue that responds well to DIC. You can [and probably should] adjust the confocal gain and offset to increase DIC contrast. Our wide-field Nikon 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 > > 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. |
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In reply to this post by Charu Tanwar
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, Room 223, Zoology Research Building, 1117 Johnson Ave., Madison, WI, 53706 3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ 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 -- |
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Jim, Is darkfield more tolerant than DIC? Cheers Eric (13 years survivor, had to count twice to make sure, GOSH!!!)
> 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 > 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 > 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. > > > > > > > >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 > > > > 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 > >On Behalf Of Charu Tanwar > >Sent: Thursday, 6 May 2010 4:27 PM > >To: > ><http://in.mc83.mail.yahoo.com/mc/compose?to=[hidden email]>[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 > >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 > 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 > > > -- > |
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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) http://www.biology.missouri.edu/faculty/phillips.html From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Eric
Scarfone Jim, Is darkfield more tolerant than DIC? Cheers Eric (13 years survivor, had to count twice to make sure, GOSH!!!) |
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In reply to this post by Eric Scarfone
Charu - Have you tried closing the condenser aperture? From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Eric
Scarfone Jim, Is darkfield more tolerant than DIC? Cheers Eric (13 years survivor, had to count twice to make sure, GOSH!!!) |
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In reply to this post by Phillips, Thomas E.
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 > 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 > > 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 > > 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 > > > > 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 > > 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 > > >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 - > > 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=[hidden email]>[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=[hidden email]>[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 > > >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 > > 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 > > >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 > > > > > > -- > > > |
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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@...>[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 >> > >> > >> > -- >> > >> -- _ ____ __ ____ / \ / / \ / \ \ 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 |
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I agree for most cases this extreme setting is not required, luckily enough.
But since Charu seems to have a particularly difficult sample, he might need to make use of those high angled light rays. But again, Dark field might be more tolerant than DIC 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: Tobias Baskin <[hidden email]> Date: Thursday, May 6, 2010 5:58 pm Subject: Re: DIC Image To: [hidden email] > 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 > >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? > >> > >> > >> 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, > >> 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 > >> > >> 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. > >> > 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 > 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 > 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] > >> > 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. > >> > >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, > >> > > > >> > >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 > >> > > > >> > >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----- > >> > > >> > >[mailto:<http://in.mc83.mail.yahoo.com/mc/compose?to=[hidden email]>[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=[hidden email]>[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 > 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 > >> > >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 > >> > >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 > |
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