Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
An even better reference - for adherent cells - is the confocal mode
reflection of the coverglass (or slide, if the cells are on that
side). Coverglass is brightest when in focus. With a 63x/1.4 NA lens,
488 nm excitation, optical slice thickness is ~0.7 um. As a bonus,
you for free the interference reflection contrast of the
cell-substratum adhesion (note to software manufacturers or graduate
students who need an open source software project: Sackmann has
published absolute distance measurements by dual wavelength IRM, see
for example PubMed 14995485, but their software may not be available
- would be nice to add this calculation to, say, ImageJ, Zeis LSM and
Leica confocal software).
Speaking of Barbara's statement that DIC has shallowest depth of
field, the only location in Z where the IRM image goes to its minimum
is when an object is effectively at 'zero distance' from the
coverglass, in cells these are focal adhesion sites or where
filopodia touch the glass. The literature suggests this 'zero
distance' is <5 nm, so this is much thinner than DIC. In the more
general case (no object dependency), TIRF is likely to have a thinner
depth of field than DIC. An exception with TIRF is that if an object
has high refractive index at the interface, the waves/rays/photons
can escape into the second medium (ex. at focal adhesions).
In the context of this thread, using DIC to focus is good if the user
is trying to image, say, "nucleus", but this is unlikely to result in
great reproducibility in acquiring single plane images. With the
coverglass reflection method, it is easy to focus consistently on the
coverglass, and have the focus motor acquire at that location and at,
for example, +2 um.
George
At 09:51 AM 9/11/2008, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Dick
>
>I applaud your using DIC as a reference for your fluorescence.
>
>Also, you've made an interesting comment re: DIC not being confocal.
>Of all techniques, DIC has the shallowest depth of focus, so it is
>very nearly confocal. You can test this using a simple cheek cell
>smear. Set with the condenser aperture wide open and the beam
>splitters on optimum shear (dove gray background). You can readily
>focus on the bacteria and folds on the top of a cheek cell, then
>focus on granules and structures within the cytoplasm, then on the
>folds where the cell adheres to the slide.
>
>For those of you who are interested in more of the details behind
>how DIC works, there is a scan of an old article from the April 1988
>American Lab on our website, www.MicroscopyEducation.com. Just go to
>The Library and Articles. They are listed in chronological order,
>with the most recent at the top.
>
>Good hunting!
>
>Best regards,
>Barbara Foster, President
>
>Microscopy/Microscopy Education
>7101 Royal Glen Trail, Suite A
>McKinney TX 75070
>P: (972)924-5310
>Skype: fostermme
>W: www.MicroscopyEducation.com
>
>NEWS! Visit the NEW and IMPROVED www.MicroscopyEducation.com! And
>don't forget: MME is now scheduling customized, on-site courses
>through Dec 2008. Call me for a free assessment and quote.
>
>
>
>
>
>At 09:23 PM 9/10/2008, you wrote:.
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>Graham<?xml:namespace prefix = o ns =
>>"urn:schemas-microsoft-com:office:office" />
>>
>>
>>
>>While this is not elegant, we collect all three fluorochromes at
>>the same time in a single z stack with the 510. With the single
>>track option (assuming no bleed through) this will take no more
>>time that a single z tack. The advantage is that you can sellect
>>one z section, with one fluorophore and know where that fits with
>>regard to all three fluorophores in the z stack. The only down
>>side is that the data files are larger.
>>
>>
>>
>>In fact we frequently add a DIC image to the Z stack just so we
>>have a historical record of cells. The DIC is not confocal but a
>>single DIC image is sometimes very useful, and it takes no more
>>time to add a DIC. For viewing, the DIC channel can turned off
>>when so only the other channels are seen.
>>
>>
>>
>>Dick Burry
>>
>>
>>----- Original Message -----
>>From: "Dr. Graham Wright" <[log in to unmask]>
>>Date: Wednesday, September 10, 2008 9:27 pm
>>Subject: Different Z setting per channel - possible on Zeiss/Leica?
>>To: [log in to unmask]
>>
>> > Search the CONFOCAL archive at
>> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>> >
>> > Hello,
>> >
>> > Here's question that I've been asked that doesn't seem possible
>> > to do, but I
>> > wonder if anyone has already done it? One of our users has a
>> > fixed sample
>> > with 3 fluorophores (for ease of explanation blue, green and
>> > red) and would
>> > like to image all three channels, but with different z setting
>> > depending on
>> > the channel. She would like a single medial optical section of
>> > the blue and
>> > red channels, but a full z-series from the green channel.
>> >
>> > We have both Zeiss (from the LSM 510 series) and Leica (SPE and
>> > SP5)
>> > confocals available to us.
>> >
>> > I know this is easy by doing 2 different scans: 1 2-channel scan
>> > of the blue
>> > and red, then a second scan of a z-series of the green, BUT is
>> > it possible
>> > to do this at the single click of a button to save time and user
>> > input. It
>> > is possible on the SP5 using the live data mode, but this scope
>> > is really
>> > reserved and heavily used for live cell imaging. Our SPE doesn't
>> > have live
>> > data mode and therefore applies any Z settings to all sequential
>> > scans
>> > (generating unnecessary quantities of data and taking too long).
>> >
>> > Any suggestions welcome,
>> > Thanks,
>> > Graham
>> >
>> >
>> > --
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>
>
>
>
>
>
>George McNamara, Ph.D.
>University of Miami, Miller School of Medicine
>Image Core
>Miami, FL 33010
>[log in to unmask]
>[log in to unmask]
>305-243-8436 office
>http://home.earthlink.net/~pubspectra/
>http://home.earthlink.net/~geomcnamara/
>http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc
>(Analytical Imaging Core Facility)
>
>
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