transmited ligth and LSM
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Hello everybody!
I`d like to ask you what I should expect from my LSM when trying
to get transmited images...
Is somewhere in the web any image gallery you could
recommend?
How much will DIC, Ph, etc improve?
Thanks a lot
Maria
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Hello Maria, I think transmitted light images (including DIC and Phase) on a confocal will look pretty much the same as they would on a normal microscope. The light path is reversed (illumination coming from the objective side, going though the sample, collected by the condenser, and detected on a PMT behind the condenser). These images will not be confocal. On our LSM, DIC is actually very nice, better than on some of our other scopes, but I think it has to do with different implementations by different vendors. We do not do phase on our confocals, so I can't comment much on that. I believe one can implement phase on a confocal, but I am not sure this is very widespread. Phase objectives have lower Numerical Apertures than their standard counterparts, and therefore are not ideal for fluorescence. DIC is more common, does not interfere with fluorescence imaging, and can substitute for Phase in most situations, except some special applications. One important issue is that you need to have very stable lasers, otherwise you may see streaks or shading in your image. We improve our transmitted light images by averaging several frames. Another issue is that confocals are generally used for the imaging of thick samples, while DIC and Phase work best with thin samples (such as cells). Transmitted light images of thick samples will generally not look very good, with Phase being the most sensitive probably. When imaging live cells and such on a confocal, however, you can have very nice DIC images in addition to your fluorescent channels. Phase and DIC all use differences in refractive index to enhance contrast. This means that it works better with some samples (such as live cells in medium, where there are nice RI gradients between water and cell membranes, for instance), and not too well for others (such as fixed cells mounted in glycerol or other medium, where RI is very homogeneous). What you can also do on a confocal, is reflected light, which uses the same confocal light path used for fluorescence (except that you detect the same wavelength band used for illumination). Works especially well for surfaces. You can find more on this and related topics here: and they have image galleries on various themes, such as DIC: -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 == On Oct 30, 2007, at 5:26 AM, Maria Jimena Ortega wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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Search the CONFOCAL archive at
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Hi Maria,
We have two confocals in our facility that have DIC on them (an Olympus FV1000
and a Leica SP5). Both produce beuatiful DIC images (providing the prisims,
filters etc are aligned properly). To be able to do transmitted light on a
confocal it will need to have a transmitted light detector on it, these are
usually seperate to the PMTs used for fluorecent imaging.
Also DIC will only be in focus in one plane of your image, you will not
be able to get any confocal effect with DIC imaging.
I
do not think phase would be able to work on a confocal due to the raster
scanning pattern of the laser. Phase relies on difraction of a solid light
beam.
Hope this helps. I can send you exapmle images of DIC captured on our
systems if you want.
Cheers
Cam
Cameron
Nowell Phone:
+61396563759 From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez Sent: Wednesday, 31 October 2007 5:03 AM To: [hidden email] Subject: Re: transmited ligth and LSM Hello Maria,
I think transmitted light images (including DIC and Phase) on a confocal
will look pretty much the same as they would on a normal microscope. The
light path is reversed (illumination coming from the objective side, going
though the sample, collected by the condenser, and detected on a PMT behind the
condenser). These images will not be confocal. On our LSM, DIC is actually
very nice, better than on some of our other scopes, but I think it has to do
with different implementations by different vendors. We do not do phase on
our confocals, so I can't comment much on that. I believe one can implement
phase on a confocal, but I am not sure this is very widespread. Phase objectives
have lower Numerical Apertures than their standard counterparts, and therefore
are not ideal for fluorescence. DIC is more common, does not interfere with
fluorescence imaging, and can substitute for Phase in most situations, except
some special applications.
One important issue is that you need to have very stable lasers, otherwise
you may see streaks or shading in your image. We improve our transmitted light
images by averaging several frames.
Another issue is that confocals are generally used for the imaging of thick
samples, while DIC and Phase work best with thin samples (such as cells).
Transmitted light images of thick samples will generally not look very good,
with Phase being the most sensitive probably. When imaging live cells and such
on a confocal, however, you can have very nice DIC images in addition to your
fluorescent channels.
Phase and DIC all use differences in refractive index to enhance contrast.
This means that it works better with some samples (such as live cells in medium,
where there are nice RI gradients between water and cell membranes, for
instance), and not too well for others (such as fixed cells mounted in glycerol
or other medium, where RI is very homogeneous).
What you can also do on a confocal, is reflected light, which uses the same
confocal light path used for fluorescence (except that you detect the same
wavelength band used for illumination). Works especially well for
surfaces.
You can find more on this and related topics here:
and they have image galleries on various themes, such as DIC:
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024
== On Oct 30, 2007, at 5:26 AM, Maria Jimena Ortega wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
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George McNamara |
Re: transmitted light and LSM
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In reply to this post by Maria Jimena Ortega
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Hi Maria,
The transmitted light images will look similar to what you see in the eyepiece. You should adjust the microscope for Koehler illumination. If your microscope has great DIC, like the current Leica SP5's, you will have great DIC on the computer, if your microscope has mediocre DIC, like my Axiovert 200M, you will have mediocre DIC. Phase contrast lenses are generally not as good in fluorescence as an objective lens that does not have a phase ring - also the latter often have higher numerical aperture (brightness being proportional to NA to the 4th power). You will probably need very low gain (I usually end up in the 200-260 V range on our LSM 510, and move the offset slider to make the scene darker. The transmitted light channel is also a handy way to see how stable - or not - your laser lines, AOTF, etc, are. Intensity tracks nicely with fluorescence specimens such as a Chroma plastic fluorescence slide, but without the possible issues of focus drift. Keep the PMT gain low! Another valuable method is to use a reflected light (another term is "backscatter") channel. Set up a laser line to a PMT with appropriate dichroics and no emission filter (or a filter that transmits that line). You can: * measure the coverglass thickness (need to account for its refractive index) * coverglass-mounting medium interface location * interference reflection contrast microscopy (IRM, aka RICM) image of cell-substratum interface - black spots are focal adhesion sites (see PubMed's Verschueren H. 1985 for details and related articles). * (sometimes) reflections of plasma, organelle and nuclear membranes * collagen and other fibers * colloidal gold (I am looking forward to seeing how well it works with Nanoprobes's EnzMet products) * medium-slide interface Different laser lines will have different reflection patterns off the coverglass etc - if not wanted, they can sometimes be subtracted or otherwise digitally removed. George p.s. not transmitted light image, but I encourage everyone to check out my Halloween image http://www.nature.com/nprot/index.html Trick or treat! For those of you not familiar with Halloween, see http://en.wikipedia.org/wiki/Halloween though frankly the holiday seems to be an excuse to sell more candy. Mexican culture has much more interesting holiday - see http://en.wikipedia.org/wiki/Day_of_the_Dead I also encourage everyone to submit images to the website - they used three of mine as images of the week in the past couple of months, so they may be pretty desperate. Tip: Submit the same size image as on the site. Payoff; besides 7 days of fame, you can add a line item to your C.V. At 08:26 AM 10/30/2007, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal George McNamara, Ph.D. University of Miami, Miller School of Medicine Image Core Miami, FL 33010 [hidden email] [hidden email] 305-243-8436 office http://home.earthlink.net/~pubspectra/ http://home.earthlink.net/~geomcnamara/ http://www.sylvester.org/health_pro/shared_resources/index.asp (see Analytical Imaging Core Facility) |
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George McNamara |
Re: transmitted light and LSM
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In reply to this post by Julio Vazquez
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Julio's comments reminded me to mention that in transmitted light, a thin object will essentially disappear when in best focus. See Zernike 1955 Science 121: 345-349 ( http://www.sciencemag.org/cgi/reprint/121/3141/345) for why and what it led to. At 02:02 PM 10/30/2007, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal - George McNamara, Ph.D. University of Miami, Miller School of Medicine Image Core Miami, FL 33010 [hidden email] [hidden email] 305-243-8436 office http://home.earthlink.net/~pubspectra/ http://home.earthlink.net/~geomcnamara/ http://www.sylvester.org/health_pro/shared_resources/index.asp (see Analytical Imaging Core Facility) |
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In reply to this post by Julio Vazquez
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Hello
Julio,
We can do both
phase contrast and DIC in our Leica TCS SP2 system with 40x 1.25 and 63x 1.32
oil immersion objectives. I'm curious about what exactly do you
mean with "DIC does not interfere with fluorescence". One of the polarizers
used for DIC is set between the sample and the detector, so it is somehow
interfering the fluorescence lightpath. At least in our system, using the same
laser and PMT settings, a fluorescence image got with that polarizer in will be
darker than with the polarizer out. That's the reason why when somebody wants to
get DIC and fluorescence images of a sample, we let that polarizer out
of the system, getting still a good "pseudo" DIC image (just empirical, I can't
explain why still a DIC image is formed without one of the filters needed to do
it)).
Bst
regards,
Xavi.
___________________________________ Xavier Sanjuan
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Search the CONFOCAL archive at
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Dear Xavier,
as the lasers are already polarized you don´t need the
polarizer (between scanhead and objective lens) in when taking DIC images
with your SP2 (but you still need the polarizer between condensor and
transmission detector). So you already do the right thing by leaving it out,
it´s not "pseudo" DIC. And I guess this is what Julio meant with "DIC does not
interfere with fluorescence" (Ok still the DIC prism is in, but this doesn´t
decrease the detected fluorescence intensity significantly).
regards,
Stefan
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In reply to this post by xavier Sanjuan
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The reason you can do DIC with only one polariser in the system is because the laser itself is polarised. But if you have a Wollaston prism between scan head and specimen when collecting a fluorescence image, it will be degraded, because you’ll be able to detect the prism interacting with the beam – the image is scattered, reducing resolution. Cheers, rosemary Dr Rosemary White [hidden email] CSIRO Plant Industry ph. 02-6246 5475 GPO Box 1600 fax. 02-6246 5334 Canberra, ACT 2601 Australia On 31/10/07 9:02 PM, "xavier Sanjuan" <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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Search the CONFOCAL archive at
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We also have problems with DIC on our SP2 which have never been fixed. In our case it is sometimes rings of light dark, and sometimes serious gradients across the field. Dave
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) On Oct 31, 2007, at 6:52 AM, Rosemary White wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have trouble doing this on our SP2, because the polarity of the lasers has never been stable, drives us crazy. So DIC images oscillate from light to dark and back again – within a single image. The engineers have never been able to fix it. |
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JOEL B. SHEFFIELD |
Re: transmited ligth and LSM
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi David, We have no trouble with our SP2, except with a LWD 40 dry objective. I think the issue of gradients is often one of a mismatch between the Wollaston prism and the objective. Also, check the settings of your condenser aperture. On our system, that aperture has a profound effect on the DIC images. I find that there is greater fluctuation with our Argon laser than with the HeNe green and red lasers. Since we often use sequential scan, I do the DIC image with the green laser source. Joel -------------- Original message --------------- Date sent: Wed, 31 Oct 2007 08:26:44 -0400 Send reply to: Confocal Microscopy List <[hidden email]> From: David Knecht <[hidden email]> Subject: Re: transmited ligth and LSM To: [hidden email] Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi- bin/wa?S1=confocal We also have problems with DIC on our SP2 which have never been fixed. In our case it is sometimes rings of light dark, and sometimes serious gradients across the field. Dave Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) On Oct 31, 2007, at 6:52 AM, Rosemary White wrote: 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 xavier Sanjuan
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal You don't need a polarizer with laser light, so you should remove it when capturing images. It is there only for observation prior to image capture. Judy Judy Trogadis Bio-Imaging Coordinator St. Michael's Hospital, 7Queen 30 Bond St. Toronto, ON M5B 1W8, Canada ph: 416-864-6060 x6337 pager: 416-685-9219 fax: 416-864-6043 [hidden email] >>> xavier Sanjuan <[hidden email]> 10/31/2007 6:02 AM >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello Julio, We can do both phase contrast and DIC in our Leica TCS SP2 system with 40x 1.25 and 63x 1.32 oil immersion objectives. I'm curious about what exactly do you mean with "DIC does not interfere with fluorescence". One of the polarizers used for DIC is set between the sample and the detector, so it is somehow interfering the fluorescence lightpath. At least in our system, using the same laser and PMT settings, a fluorescence image got with that polarizer in will be darker than with the polarizer out. That's the reason why when somebody wants to get DIC and fluorescence images of a sample, we let that polarizer out of the system, getting still a good "pseudo" DIC image (just empirical, I can't explain why still a DIC image is formed without one of the filters needed to do it)). Bst regards, Xavi. ___________________________________ Xavier Sanjuan Servei de Microscopia Confocal Departament de Ciencies Experimentals i de la Salut Universitat Pompeu Fabra Parc de Recerca Biomedica de Barcelona Doctor Aiguader, 88 - Sala 309 08003 Barcelona - Spain Tel.: + 34 93 316 08 64 Fax: + 34 93 316 09 01 E-mail: [hidden email] Web: http://www.upf.edu/cexs/sct -----Mensaje original----- De: Confocal Microscopy List [mailto:[hidden email]]En nombre de Julio Vazquez Enviado el: martes, 30 de octubre de 2007 19:03 Para: [hidden email] Asunto: Re: transmited ligth and LSM Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal - Hello Maria, I think transmitted light images (including DIC and Phase) on a confocal will look pretty much the same as they would on a normal microscope. The light path is reversed (illumination coming from the objective side, going though the sample, collected by the condenser, and detected on a PMT behind the condenser). These images will not be confocal. On our LSM, DIC is actually very nice, better than on some of our other scopes, but I think it has to do with different implementations by different vendors. We do not do phase on our confocals, so I can't comment much on that. I believe one can implement phase on a confocal, but I am not sure this is very widespread. Phase objectives have lower Numerical Apertures than their standard counterparts, and therefore are not ideal for fluorescence. DIC is more common, does not interfere with fluorescence imaging, and can substitute for Phase in most situations, except some special applications. One important issue is that you need to have very stable lasers, otherwise you may see streaks or shading in your image. We improve our transmitted light images by averaging several frames. Another issue is that confocals are generally used for the imaging of thick samples, while DIC and Phase work best with thin samples (such as cells). Transmitted light images of thick samples will generally not look very good, with Phase being the most sensitive probably. When imaging live cells and such on a confocal, however, you can have very nice DIC images in addition to your fluorescent channels. Phase and DIC all use differences in refractive index to enhance contrast. This means that it works better with some samples (such as live cells in medium, where there are nice RI gradients between water and cell membranes, for instance), and not too well for others (such as fixed cells mounted in glycerol or other medium, where RI is very homogeneous). What you can also do on a confocal, is reflected light, which uses the same confocal light path used for fluorescence (except that you detect the same wavelength band used for illumination). Works especially well for surfaces. You can find more on this and related topics here: http://micro.magnet.fsu.edu/primer/techniques/index.html and they have image galleries on various themes, such as DIC: http://micro.magnet.fsu.edu/primer/techniques/dic/dicgallery/index.html -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 http://www.fhcrc.org/ == On Oct 30, 2007, at 5:26 AM, Maria Jimena Ortega wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello everybody! I`d like to ask you what I should expect from my LSM when trying to get transmited images... Is somewhere in the web any image gallery you could recommend? How much will DIC, Ph, etc improve? Thanks a lot Maria |
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In reply to this post by Knecht, David
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have just gone through this same problem with our own SP2 system. The rings are there during brightfield observation at 10x and according to the Leica people, cannot be eliminated. If you zoom even a little, the rings will be out of the field of view. We decided not to answer questions at 10x brightfield ;-) The gradient issue could be due to misalignment of the fiber optic detector. Judy Judy Trogadis Bio-Imaging Coordinator St. Michael's Hospital, 7Queen 30 Bond St. Toronto, ON M5B 1W8, Canada ph: 416-864-6060 x6337 pager: 416-685-9219 fax: 416-864-6043 [hidden email] >>> David Knecht <[hidden email]> 10/31/2007 8:26 AM >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We also have problems with DIC on our SP2 which have never been fixed. In our case it is sometimes rings of light dark, and sometimes serious gradients across the field. Dave Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) On Oct 31, 2007, at 6:52 AM, Rosemary White wrote: > Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi- > bin/wa?S1=confocal We have trouble doing this on our SP2, because > the polarity of the lasers has never been stable, drives us crazy. > So DIC images oscillate from light to dark and back again * within > a single image. The engineers have never been able to fix it. > > The reason you can do DIC with only one polariser in the system is > because the laser itself is polarised. But if you have a Wollaston > prism between scan head and specimen when collecting a fluorescence > image, it will be degraded, because you*ll be able to detect the > prism interacting with the beam * the image is scattered, reducing > resolution. > > Cheers, > rosemary > > Dr Rosemary White [hidden email] > CSIRO Plant Industry ph. 02-6246 5475 > GPO Box 1600 fax. 02-6246 5334 > Canberra, ACT 2601 > Australia > > > On 31/10/07 9:02 PM, "xavier Sanjuan" <[hidden email]> > >> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/ >> cgi-bin/wa?S1=confocal >> Hello Julio, >> >> We can do both phase contrast and DIC in our Leica TCS SP2 system >> with 40x 1.25 and 63x 1.32 oil immersion objectives. I'm curious >> about what exactly do you mean with "DIC does not interfere with >> fluorescence". One of the polarizers used for DIC is set between >> the sample and the detector, so it is somehow interfering the >> fluorescence lightpath. At least in our system, using the same >> laser and PMT settings, a fluorescence image got with that >> polarizer in will be darker than with the polarizer out. That's >> the reason why when somebody wants to get DIC and fluorescence >> images of a sample, we let that polarizer out of the system, >> getting still a good "pseudo" DIC image (just empirical, I can't >> explain why still a DIC image is formed without one of the filters >> needed to do it)). >> >> Bst regards, >> >> Xavi. >> ___________________________________ >> >> Xavier Sanjuan >> Servei de Microscòpia Confocal >> Departament de Ciències Experimentals i de la Salut >> Universitat Pompeu Fabra >> Parc de Recerca Biomèdica de Barcelona >> Doctor Aiguader, 88 - Sala 309 >> 08003 Barcelona - Spain >> >> Tel.: + 34 93 316 08 64 >> Fax: + 34 93 316 09 01 >> E-mail: [hidden email] <mailto:[hidden email]> >> Web: http://www.upf.edu/cexs/sct <http://www.upf.edu/cexs/sct> >> >> >>> >>> -----Mensaje original----- >>> De: Confocal Microscopy List >>> [mailto:[hidden email]]En nombre de Julio Vazquez >>> Enviado el: martes, 30 de octubre de 2007 19:03 >>> Para: CON >>> Asunto: Re: transmited ligth and LSM >>> >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/ >>> cgi-bin/wa?S1=confocal - >>> Hello Maria, >>> >>> >>> >>> I think transmitted light images (including DIC and Phase) on a >>> confocal will look pretty much the same as they would on a >>> normal microscope. The light path is reversed (illumination >>> coming from the objective side, going though the sample, >>> collected by the condenser, and detected on a PMT behind the >>> condenser). These images will not be confocal. On our LSM, DIC is >>> actually very nice, better than on some of our other scopes, but >>> I think it has to do with different implementations by different >>> vendors. We do not do phase on our confocals, so I can't comment >>> much on that. I believe one can implement phase on a confocal, >>> but I am not sure this is very widespread. Phase objectives have >>> lower Numerical Apertures than their standard counterparts, and >>> therefore are not ideal for fluorescence. DIC is more common, >>> does not interfere with fluorescence imaging, and can substitute >>> for Phase in most situations, except some special applications. >>> >>> >>> >>> One important issue is that you need to have very stable lasers, >>> otherwise you may see streaks or shading in your image. We >>> improve our transmitted light images by averaging several frames. >>> >>> >>> >>> Another issue is that confocals are generally used for the >>> imaging of thick samples, while DIC and Phase work best with >>> thin samples (such as cells). Transmitted light images of thick >>> samples will generally not look very good, with Phase being the >>> most sensitive probably. When imaging live cells and such on a >>> confocal, however, you can have very nice DIC images in addition >>> to your fluorescent channels. >>> >>> >>> >>> Phase and DIC all use differences in refractive index to enhance >>> contrast. This means that it works better with some samples (such >>> as live cells in medium, where there are nice RI gradients >>> between water and cell membranes, for instance), and not too >>> well for others (such as fixed cells mounted in glycerol or >>> other medium, where RI is very homogeneous). >>> >>> >>> >>> What you can also do on a confocal, is reflected light, which >>> uses the same confocal light path used for fluorescence (except >>> that you detect the same wavelength band used for illumination). >>> Works especially well for surfaces. >>> >>> >>> >>> You can find more on this and related topics here: >>> >>> >>> >>> http://micro.magnet.fsu.edu/primer/techniques/index.html >>> >>> >>> >>> and they have image galleries on various themes, such as DIC: >>> >>> >>> >>> http://micro.magnet.fsu.edu/primer/techniques/dic/dicgallery/ >>> index.html >>> >>> >>> >>> >>> >>> >>> >>> >>> -- >>> >>> Julio Vazquez >>> >>> Fred Hutchinson Cancer Research Center >>> >>> Seattle, WA 98109-1024 >>> >>> >>> >>> http://www.fhcrc.org/ >>> >>> >>> >>> == >>> >>> >>> >>> >>> On Oct 30, 2007, at 5:26 AM, Maria Jimena Ortega wrote: >>> >>> >>>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/ >>>> cgi-bin/wa?S1=confocal >>>> Hello everybody! >>>> >>>> >>>> I`d like to ask you what I should expect from my LSM when >>>> trying to get transmited images... >>>> >>>> Is somewhere in the web any image gallery you could recommend? >>>> >>>> How much will DIC, Ph, etc improve? >>>> >>>> >>>> Thanks a lot >>>> >>>> >>>> Maria >>> >> > |
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Michael Weber-4 |
Re: AW: transmited ligth and LSM
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In reply to this post by Stefan Terjung
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Stefan, in fact the DIC prism does interfer significantly with the image, it has quite some impact at resolution. This can be easily observed with small beads, already 500nm ones will look quite scattered. On a Leica DMI6000 the DIC prisms can be located in an additional turrent, which means you can acquire DIC (with prism in) sequentially to fluorescence (with prism out). cheers, Michael Stefan Terjung wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Dear Xavier, > > as the lasers are already polarized you don´t need the polarizer > (between scanhead and objective lens) in when taking DIC images with > your SP2 (but you still need the polarizer between condensor and > transmission detector). So you already do the right thing by leaving it > out, it´s not "pseudo" DIC. And I guess this is what Julio meant with > "DIC does not interfere with fluorescence" (Ok still the DIC prism is > in, but this doesn´t decrease the detected fluorescence intensity > significantly). > > regards, > > Stefan > > > ------------------------ > Dr. Stefan Terjung > Advanced Light Microscopy Facility > EMBL Heidelberg > Meyerhofstr.1 > D - 69117 Heidelberg > Phone +49(0)6221 387-8467 > > > ------------------------------------------------------------------------ > *Von:* Confocal Microscopy List > [mailto:[hidden email]] *Im Auftrag von *xavier Sanjuan > *Gesendet:* Mittwoch, 31. Oktober 2007 11:03 > *An:* [hidden email] > *Betreff:* Re: transmited ligth and LSM > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Hello Julio, > > We can do both phase contrast and DIC in our Leica TCS SP2 system > with 40x 1.25 and 63x 1.32 oil immersion objectives. I'm curious > about what exactly do you mean with "DIC does not interfere with > fluorescence". One of the polarizers used for DIC is set between the > sample and the detector, so it is somehow interfering the > fluorescence lightpath. At least in our system, using the same laser > and PMT settings, a fluorescence image got with that polarizer in > will be darker than with the polarizer out. That's the reason why > when somebody wants to get DIC and fluorescence images of a sample, > we let that polarizer out of the system, getting still a good > "pseudo" DIC image (just empirical, I can't explain why still a DIC > image is formed without one of the filters needed to do it)). > > Bst regards, > > Xavi. > > /___________________________________/ > > ///Xavier Sanjuan// > //Servei de Microscòpia Confocal// > /Departament de Ciències Experimentals i de la Salut/ > /Universitat Pompeu Fabra / > /Parc de Recerca Biomèdica de Barcelona// > /Doctor Aiguader, 88 - Sala 309 > 08003 Barcelona - Spain > > Tel.: + 34 93 316 08 64 > Fax: + 34 93 316 09 01 > E-mail: //[hidden email]/ <mailto:[hidden email]> > /Web: //http://www.upf.edu/cexs/sct/ > > > > -----Mensaje original----- > *De:* Confocal Microscopy List > [mailto:[hidden email]]*En nombre de *Julio Vazquez > *Enviado el:* martes, 30 de octubre de 2007 19:03 > *Para:* [hidden email] > *Asunto:* Re: transmited ligth and LSM > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal - > Hello Maria, > > I think transmitted light images (including DIC and Phase) on a > confocal will look pretty much the same as they would on a > normal microscope. The light path is reversed (illumination > coming from the objective side, going though the sample, > collected by the condenser, and detected on a PMT behind the > condenser). These images will not be confocal. On our LSM, DIC > is actually very nice, better than on some of our other scopes, > but I think it has to do with different implementations by > different vendors. We do not do phase on our confocals, so I > can't comment much on that. I believe one can implement phase on > a confocal, but I am not sure this is very widespread. Phase > objectives have lower Numerical Apertures than their standard > counterparts, and therefore are not ideal for fluorescence. DIC > is more common, does not interfere with fluorescence imaging, > and can substitute for Phase in most situations, except some > special applications. > > One important issue is that you need to have very stable lasers, > otherwise you may see streaks or shading in your image. We > improve our transmitted light images by averaging several frames. > > Another issue is that confocals are generally used for the > imaging of thick samples, while DIC and Phase work best with > thin samples (such as cells). Transmitted light images of thick > samples will generally not look very good, with Phase being the > most sensitive probably. When imaging live cells and such on a > confocal, however, you can have very nice DIC images in addition > to your fluorescent channels. > > Phase and DIC all use differences in refractive index to enhance > contrast. This means that it works better with some samples > (such as live cells in medium, where there are nice RI gradients > between water and cell membranes, for instance), and not too > well for others (such as fixed cells mounted in glycerol or > other medium, where RI is very homogeneous). > > What you can also do on a confocal, is reflected light, which > uses the same confocal light path used for fluorescence (except > that you detect the same wavelength band used for illumination). > Works especially well for surfaces. > > You can find more on this and related topics here: > > http://micro.magnet.fsu.edu/primer/techniques/index.html > > and they have image galleries on various themes, such as DIC: > > http://micro.magnet.fsu.edu/primer/techniques/dic/dicgallery/index.html > > > > -- > Julio Vazquez > Fred Hutchinson Cancer Research Center > Seattle, WA 98109-1024 > > http://www.fhcrc.org/ > > == > > > On Oct 30, 2007, at 5:26 AM, Maria Jimena Ortega wrote: > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> Hello everybody! >> I`d like to ask you what I should expect from my LSM when >> trying to get transmited images... >> Is somewhere in the web any image gallery you could recommend? >> How much will DIC, Ph, etc improve? >> Thanks a lot >> Maria |
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