a double dichroic in a widefield fluorescence microscope

[JOEL B. SHEFFIELD]

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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>*

[Craig Brideau]

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Colour camera, or monochrome? Or eyepiece? If you use a color camera, you
sacrifice resolution but can then roughly get RGB separation at the
detector level. In this case you just need a pass dichroic that sends
everything longer than your excitation right to the color camera, then let
the camera sort out the mess. Same goes for the eye, unless your users are
colorblind. @:-)

Craig Brideau

On Fri, Oct 9, 2015 at 12:04 PM, 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>*
>

[Glen MacDonald-2]

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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>*

[George McNamara]

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Hi Joel,
See also other people's replies.

I suggest (and some product web page possibilities):

- inexpensive TWO triple pass cubes
* D/F/R or D/F/R/NIR (Semrock or Chroma, makes sense to get both cubes
from the same vendor)
* Semrock LED-CFP/YFP/mCherry *or Chroma equivalent) ...
       note: I have the LED-CFP/YFP/HcRed (no exciter) and wish the
/mCherry had been available when I bought it, since /mCherry has wider
emission band.

- inexpensive LED light source (time to ditch Hg arc lamp anyway!):
http://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=3836&pn=LED4D002#7181

- excitation and/or emission filter wheels ... manual or motorized
depending on how much money you want to spend:
http://www.thorlabs.com/search/thorsearch.cfm?search=filter wheel

when you retire the Optiphot, could move to a newer scope, so don't be
afraid to spend extra money for useful automation ... if motorize, ask
the vendor if it works with whatever software you use or want to use,
such as micro-manager or MetaMorph.

More questions for you to think about:

1. Is this going to redirect users to a scope that is of minimal use?
... i.e. should they just go sit down at a confocal or other advanced
microscope?

2. Is the Optiphot worth upgrading?
   - would the space be better utilized with an inverted microscope? ...
inverted would enable imaging dishes, flasks, etc.
   - if yes, could you find an underutilized inverted scope in a
colleague's lab, and swap the Optiphot for it? ... sweeten the trade by
buying LED lamps for both.
   - eBay, various microscope vendors, auctions, have lots of
inexpensive inverted scopes ... consider buying one, and if appropriate,
upgrade that (LED lamp etc).

enjoy,

George

On 10/9/2015 1:04 PM, JOEL B. SHEFFIELD 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>*
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42