Hi all,
I too think that DIC can be a great addition to confocal
fluorescence, especially as both CAN be collected without
interference.
As was noted on the list a while ago, the presence of the Wollaston
in the light path behind the objective produces two spots in the
specimen centered about half the "Rayleigh resolution" apart. This
occurs if the pol light from the laser (or fibre) is polarized at 45
deg to the two axes of the Wollaston as it should be for DIC. So this
slight blurring in the sheer direction may be a consideration. (This
blurring will not occur if the laser pol axis coincides with only one
of the two Wollaston axes and there is no other pol element between
the laser and the objective Wollaston. But then you can't do DIC.)
More important is the loss of fluorescence signal if a polarizing
element is present in the confocal light path between the dichroic
and the specimen. If such an element is present, it may reduce any
excitation light going towards the objective (perhaps almost to zero,
if its pol axis is crossed with the laser), and it will also absorb
about 70% of the returning fluorescent light. Bad news all around.
As others have mentioned, this should not be necessary as the laser
is already polarized. (Random pol lasers are actually constantly
switching their pol axes. They are unsuitable for scanning microscopy
because the different directions are likely to be reflected
differently from interference filters etc. and this causes the change
of axis becomes an unacceptable variation in brightness.)
However, just because laser light is polarized, it does not follow
that it is polarized at 45 deg to the Wollastons in your microscope
(both of them) as is required to create and then combine the two
separate, coherent, light beams needed for DIC.
There is usually no way to rotate the Wollastons.
To check if the light from your laser is in the right pol orientation,
- set up for DIC as normal (check your Koehler ill., polarizer and
analyzer crossed),
- switch to confocal (full laser power so you can see what is going on),
- remove what would normally be the Analyser (the pol element between
the objective and the occular),
- reflect any light emerging on the far side of what would normally
be the Polarizer (near the far side of the condenser) off a piece of
paper.
- If you now rotate what would normally be the Polarizer, the
intensity of the light hitting the paper should go through a null at
just the orientation at which it is normally be used for DIC.
If this occurs, you don't need to use what is normally the Analyser
to collect a good DIC image with a "confocal" (not really)
transmission detector on the far side of what is normally the
Polarizer.
However, remember that this is a coherent imaging layout and if your
detector collects only the transmitted light near the axis, the
entire DIC system will be operating at low NA and will produce only
low resolution results.
If the expected null does not occur, the pol direction of the laser
can be changed by inserting a half-wave plate anywhere along the
laser illumination path. Rotate this plate until the null does occur
when the Polarizer is in the standard position.
It doesn't help to mess with the pol or analyser orientations as then
they will not be set with the Wollastons.
That should confuse everyone....
Cheers,
Jim P.
>Jason Kirk wrote:
>
>>I want to make sure we aren't getting terms mixed up here in your
>>description of the problem. When you say you remove the "polarizer" are you
>>talking about the polarizer between the condenser and field aperture (called
>>Polarizer) or the polarizer between the excitation source and primary
>>Wollaston (called Analyzer)?
>>
>
>Jason,
>Actually this naming of "polarizer" and "analyzer" you give here is
>applicable to the lightpath for visual observation, where the light is
>first polarized in the "polarizer" and then analyzed in the "analyzer".
>In LSM transmission mode the light usually goes along the reverse
>direction, i.e. the detector is behind the condensor, not the light
>source. Consequently the roles of "polarizer" and "analyzer" are
>exchanged, and it matters, whether the "polarizer" is in the lightpath
>or not.
>Particularly, in our LSM 410 we have a DIC setup and used it for some
>time. Here, the (visual) analyzer is not in the laser-excitation
>lightpath but just in the path for visual observation (after the two are
>separated). The lasers are polarized correctly in relation to the
>Wollaston prism, so everything should be nice, and DIC by transmission
>laserscanning is possible. However, the images we take in this mode
>always have ugly (interference?) fringes and a gradient from the upper
>left to the lower right corner. Does anybody have an idea what could be
>the reason and how to get rid of it? We can do postprocessing in
>Photoshop/ImageJ, but of course it's nicer to have a good image to begin
>with. In visual observation there are no fringes.
>Now, we mostly take transmission images without any contrast technique
>(DIC, Phasecontrast), which come out very nicely. Actually I was
>wondering, why they are so nice, because by visual observation one
>doesn't see anything without some contrast technique. Any ideas?
>
>Joachim
>
>
>
>
>--
>------------------------------------------------------------------
> Joachim Walter, Dipl. Phys.
> Institut für Anthropologie und Humangenetik der LMU München
> AG Cremer
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>
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Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building, FAX 608-265-5315
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