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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
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>>> xavier Sanjuan <[log in to unmask]> 10/31/2007 6:02 AM >>>
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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
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: [log in to unmask]
Web: http://www.upf.edu/cexs/sct
-----Mensaje original-----
De: Confocal Microscopy List [mailto:[log in to unmask]]En
nombre de Julio Vazquez
Enviado el: martes, 30 de octubre de 2007 19:03
Para: [log in to unmask]
Asunto: Re: transmited ligth and LSM
Search the CONFOCAL archive at
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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:
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:
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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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