Re: a double dichroic in a widefield fluorescence microscope *COMMERCIAL REPLY*

[Jeffrey Carmichael]

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Hi Joel,

There are basically three different types of multi band configurations to
consider, depending on whether you have external filter wheel(s) or an
excitation slider as Glen mentioned.

        1) No external filter wheel (or slider).  Multi band excitation
filter, multi band emission filter and matching multi band dichroic
mounted in filter cube in microscope slider.  This allows for simultaneous
visualization through the oculars of each fluor.  This will not work with
a monochrome camera as there is no way to distinguish colors, and all
fluors will appear in every image. (69000 or 69002, depending on whether
TRITC or Texas Red-like).

        2) Excitation filter wheel (or slider).  This allows for the
arrangement that Glen describes below.  Individual, single band excitation
filters are housed in the filter wheel controlled by software, while the
filter cube houses the matching multi band dichroic and emission filter.
This allows for selective excitation, however there will often be
noticeable bleedthrough from the other fluors (and autofluorescence)
because the multi band emission filter will transmit any of their
respective fluorescence emission.  In some hands, this is negligible, in
others' it's problematic.  It often depends on sample processing,
autofluorescence, relative concentration of the various fluors, etc.

Something like the 89084 filter set mentioned below would work here, but
you can get a less expensive version with fewer excitation filters.  89084
is intended for FISH applications and has 3 individual single band
excitation filters, but also a dual band and a triple band excitation
filter.  You may only need the corresponding set with the 3 single band
ex. filters (Chroma 69300 or 69302, depending on whether TRITC or Texas
Red), although the dual and triple band excitation filter can be useful
when viewing live, providing a glimpse of potential colocalization when
evaluating a slide by eye.

        3)Excitation and emission filter wheel.  The best and most
versatile choice if you can afford it....... Allows for selective
excitation and detection with single band excitation and emission filters
housed in their respective filter wheels.  Matching multi band dichroic
housed in filter cube (Chroma 89013 or 89014, depending on whether TRITC
or Texas Red).

In addition to the more familiar filter wheels, 89 North (Chroma
subsidiary) offers compact OptoSpin filter wheels manufactured by Cairn
Research (U.K.).  They can spin continuously (10K rpm) enabling fast
imaging and tandem configuration.

Best Regards,

Jeff Carmichael
Technical and Product Marketing Mgr.
[hidden email]

Chroma Technology Corp.
an employee owned company
10 Imtec Lane
Bellows Falls, VT 05101
1 802 428 2528 Voice Direct
1 802 428 2525 Fax
1 800 824 7662 ext. 2528 Toll Free






-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Glen MacDonald
Sent: Friday, October 09, 2015 6:41 PM
To: [hidden email]
Subject: Re: a double dichroic in a widefield fluorescence microscope

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Hi Joel,
Our Diaphot has a slot for a slider in front of the xenon lamp's field
stop diaphragm.  You could get, or have made, a slider to accept 3 exciter
filters for a triple cube.The ancient Chroma 61002 triple set on our
Diaphot displays only modest bleedthrough with the 3 band exciter, but
depends on labels.  The 82000v2 Chroma set on our Marianas uses single
band exciters on a filterwheel and shows neglible bleedthrough with
Alexa488/Alexa568 or  Mitotracker Red.  that set has been replaced by the
89084 triple set and the spectra look much better than the old filter set.
Semrock probably has something similar.
 
Regards,
Glen MacDonald
        Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
        Cellular Morphology Core
Center on Human Development and Disability Box 357923 University of
Washington Seattle, WA 98195-7923  USA
[phone]
[hidden email]







On Oct 9, 2015, at 11:04 AM, JOEL B. SHEFFIELD <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your
posting.

> *****
>
> Hi,
>
> I realize that this might be off topic for this list, but I'm sure
> that someone here can advise me.  We have inherited an old Nikon
> optiphot2 microscope that has a fluorescence slider that can be used
> withonly two cubes at a time.  In these days of multiple labels, with
> the obligatory DAPI, I would like to be able to use the instrument for
> triple labeled samples, assuming roughly DAPI, FITC, RITC or TxRed.  I
> have thought of using a double dichroic that would work for DAPI and
> the red dyes, in order to minimize bleedthrough.  This way, we could
> work with three dyes, even with two positions.
>
> Any opinions?  Is there a specific filter set that you would recommend?
>
> Thank you very much in advance
>
> Joel
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail: [hidden email]
> URL:  *http://tinyurl.com/khbouft <http://tinyurl.com/khbouft>*