fluorescent reference slides/beads for spinning disc

[Hanspeter Niederstrasser-3]

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We're testing new probes and filters for our microscope and amongst the
things we wanted to check was signal loss btw different filters as well
as intensity distribution across the view field (this is separate from
the filters of course).  I found Invitrogen's FluoSpheres as well as
Fluor-Ref fluorescent slides (Ted Pella being one vendor here:
<http://www.tedpella.com/histo_html/fluor.htm>).

The reference slides are obviously much more economical, but they don't
list their emission spectra, and since the filters we're testing have
fairly different OD spectra, we would like the references to be as
similar to our probes.  What are people's experiences and
recommendations with these references or others you have used?

Hanspeter

--
Hanspeter Niederstrasser, Ph.D.      Dept. of Microbiology
hn2157 at columbia dot edu           701 W. 168th St.
Chang Lab                            New York, NY 10032
Columbia University

[Boswell, Carl A - (cboswell)]

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Hi Hanspeter,
You  might consider assessing filter characteristics with a scanning
spectrophotometer.  Assuming you have access to one, you simply place the
filter in the light path and have the scanner produces a transmissiton vs.
wavelength plot.  It produces a very nice detailed spectral plot that would
be comparable for all components analyzed.  Repositioning the filter will
give you an idea of field uniformity.

Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]
----- Original Message -----
From: "Hanspeter Niederstrasser" <[hidden email]>
To: <[hidden email]>
Sent: Friday, February 01, 2008 11:26 AM
Subject: fluorescent reference slides/beads for spinning disc

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> We're testing new probes and filters for our microscope and amongst the
> things we wanted to check was signal loss btw different filters as well as
> intensity distribution across the view field (this is separate from the
> filters of course).  I found Invitrogen's FluoSpheres as well as Fluor-Ref
> fluorescent slides (Ted Pella being one vendor here:
> <http://www.tedpella.com/histo_html/fluor.htm>).
>
> The reference slides are obviously much more economical, but they don't
> list their emission spectra, and since the filters we're testing have
> fairly different OD spectra, we would like the references to be as similar
> to our probes.  What are people's experiences and recommendations with
> these references or others you have used?
>
> Hanspeter
>
> --
> Hanspeter Niederstrasser, Ph.D.      Dept. of Microbiology
> hn2157 at columbia dot edu           701 W. 168th St.
> Chang Lab                            New York, NY 10032
> Columbia University
>

[Judy Trogadis]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Whenever I want to test even signal intensity distribution across a field of view, I use translucent fluorescent plastic slides. At a given setting, ideally, all pixels should have the same intensity value. I once picked up a complimentary set of 4 different coloured ones from the Chroma booth at a meeting. They should also be useful to compare intensities between filters having similar ex/em specs.
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: [phone]
[hidden email]


>>> Hanspeter Niederstrasser <[hidden email]> 02/01/08 1:26 PM >>>
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 

We're testing new probes and filters for our microscope and amongst the
things we wanted to check was signal loss btw different filters as well
as intensity distribution across the view field (this is separate from
the filters of course).  I found Invitrogen's FluoSpheres as well as
Fluor-Ref fluorescent slides (Ted Pella being one vendor here:
<http://www.tedpella.com/histo_html/fluor.htm>).

The reference slides are obviously much more economical, but they don't
list their emission spectra, and since the filters we're testing have
fairly different OD spectra, we would like the references to be as
similar to our probes.  What are people's experiences and
recommendations with these references or others you have used?

Hanspeter

--
Hanspeter Niederstrasser, Ph.D.      Dept. of Microbiology
hn2157 at columbia dot edu           701 W. 168th St.
Chang Lab                            New York, NY 10032
Columbia University

[Ignatius, Mike]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Hanspeter,

One more choice for this application is our fluospheres preps, but
already mounted on slides.

F36909 FocalCheck(tm) fluorescence microscope test slide #1, for
alignment, intensity, and calibration.  

Two rows of five preps of dye stained beads showing a size and intensity
series - 10 total regions.  Intensity series is a relative series of
100%, 33%, 10%, 3%, .66%. Very photostabile.  We have some others for
spectral unmixing and such as well.

Call if you would like more info.

Mike Ignatius,

541 33-0414

Molecular Probes/Invitrogen

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Judy Trogadis
Sent: Friday, February 01, 2008 11:19 AM
To: [hidden email]
Subject: Re: fluorescent reference slides/beads for spinning disc

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Whenever I want to test even signal intensity distribution across a
field of view, I use translucent fluorescent plastic slides. At a given
setting, ideally, all pixels should have the same intensity value. I
once picked up a complimentary set of 4 different coloured ones from the
Chroma booth at a meeting. They should also be useful to compare
intensities between filters having similar ex/em specs.
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: [phone]
[hidden email]


>>> Hanspeter Niederstrasser <[hidden email]> 02/01/08 1:26 PM >>>
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 

We're testing new probes and filters for our microscope and amongst the
things we wanted to check was signal loss btw different filters as well
as intensity distribution across the view field (this is separate from
the filters of course).  I found Invitrogen's FluoSpheres as well as
Fluor-Ref fluorescent slides (Ted Pella being one vendor here:
<http://www.tedpella.com/histo_html/fluor.htm>).

The reference slides are obviously much more economical, but they don't
list their emission spectra, and since the filters we're testing have
fairly different OD spectra, we would like the references to be as
similar to our probes.  What are people's experiences and
recommendations with these references or others you have used?

Hanspeter

--
Hanspeter Niederstrasser, Ph.D.      Dept. of Microbiology
hn2157 at columbia dot edu           701 W. 168th St.
Chang Lab                            New York, NY 10032
Columbia University

[Peter Gabriel Pitrone]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello Hanspeter,

If you buy a test slide from Molecular Probes, make sure they made the sample with the right coverslip thickness. We've had a nightmare trying to sort out this problem to no avail. They used to sell 3 slides with 2 samples per slide (4 um, 2 um, 1 um, 0.5 um, 0.1 um, and mixed), but now the make it all on on slide in a line. Needless to say it's almost impossible to focus on the beads with a thick coverglass, but just trying to look at the beads closest to the right edge of the slide is impossible. All we want is the "original" 3 slide set which is unfortunately out of commission.

Good luck finding what you need.

Peter

On Feb 1, 2008, at 7:26 PM, Hanspeter Niederstrasser wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

We're testing new probes and filters for our microscope and amongst the things we wanted to check was signal loss btw different filters as well as intensity distribution across the view field (this is separate from the filters of course).  I found Invitrogen's FluoSpheres as well as Fluor-Ref fluorescent slides (Ted Pella being one vendor here: <http://www.tedpella.com/histo_html/fluor.htm>).

The reference slides are obviously much more economical, but they don't list their emission spectra, and since the filters we're testing have fairly different OD spectra, we would like the references to be as similar to our probes.  What are people's experiences and recommendations with these references or others you have used?

Hanspeter

-- 

Hanspeter Niederstrasser, Ph.D.      Dept. of Microbiology

hn2157 at columbia dot edu           701 W. 168th St.

Chang Lab                            New York, NY 10032

Columbia University

Peter Gabriel Pitrone

Light Microscopy Facility

Max Planck Institute for 

Cell Biology and Genetics

Pfotenhauerstrasse 108

01307 Dresden, Germany

E-Mail: [hidden email]