Fluorophore excitable with 405nm and 488nm lines

[Daniel Gitler]

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Dear Confocalists/microscopists,
I am in need of either beads or a fluorophore in solution which is exited consistently by both the 405nm and 488nm lines of a confocal microscope. What I really need is that the ratio of excitation should be constant, in which case two separate dyes are probably not a good choice, unless their molar ratio can be quite consistent (perhaps in beads?). I also need that the efficiency of excitation for both lines be quite decent (doesn't have to be maximal, just decent). Finally (it appears I have a lot of requisites) the dye/beads should be as insensitive as possible to environmental changes (i.e., pH etc). The idea is to have a good standard in order to calibrate the ratio of the power of these two lines when imaging a sample (I do not need to know the actual number, just a relative measure will do fine).
I would appreciate any advice,
Thanks,
Daniel


Daniel Gitler, Ph.D.
Department of Physiology and Neurobiology
Faculty of Health Sciences
Ben Gurion University of the Negev
Beer-Sheva 84105
Israel

Tel:  [phone]
Cell: [phone]
Fax: + 972-8-6477628
http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎

[Julio Vazquez]

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Quantum dots come to mind.

Otherwise, I would suggest a few other options:

1. Fluorescent plexiglas slides (such as offered by Ted Pella and others: http://www.tedpella.com/histo_html/fluor.htm ). These tend to come in sets of four for blue/green/orange and red fluorescence, but they do show some level of cross-excitation. Best of all, they will last a long time.

2. Get a set of two appropriate fluorescent dyes, mix them and mount the mix under a sealed coverslip. I suppose this may not be as stable over time as the plastic slide. Reproducibility may be an issue due to differential bleaching and other issues.

3. Just bounce the lasers off a mirror slide (or surface of a glass slide) and measure the reflected light. In this mode, you need to use a long pass or bandpass emission filter that includes the laser lines (or two separate filters). I use this method on point scanning confocal scopes.... it is easy and quite sensitive. Ideal for measuring laser power stability over time. Did not use it to measure illumination intensity ratios, but don't see why it wouldn't work.

4. Use a power meter and just measure the power at the objective (not as practical as (3).

5. Use a mix of fluorescence intensity calibration beads, such as the Inspek calibration kit from Invitrogen.

We use beads primarily for spatial calibration, i.e to measure image registration, focal shifts, etc, rather than for intensity calibrsations, but according to the product description, these should work (I would just measure enough beads).

If using an AOTF or other system to attenuate the laser intensity, you'll probably need to measure the intensity ratios at the actual settings, since it is not certain that the AOTF attenuation will be linear, and/or that it will be similar for both wavelengths. This should be easy to test though.

--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, USA.


http://www.fhcrc.org/




On Jun 2, 2011, at 11:59 AM, Daniel Gitler wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Confocalists/microscopists,
> I am in need of either beads or a fluorophore in solution which is exited consistently by both the 405nm and 488nm lines of a confocal microscope. What I really need is that the ratio of excitation should be constant, in which case two separate dyes are probably not a good choice, unless their molar ratio can be quite consistent (perhaps in beads?). I also need that the efficiency of excitation for both lines be quite decent (doesn't have to be maximal, just decent). Finally (it appears I have a lot of requisites) the dye/beads should be as insensitive as possible to environmental changes (i.e., pH etc). The idea is to have a good standard in order to calibrate the ratio of the power of these two lines when imaging a sample (I do not need to know the actual number, just a relative measure will do fine).
> I would appreciate any advice,
> Thanks,
> Daniel
>
>
> Daniel Gitler, Ph.D.
> Department of Physiology and Neurobiology
> Faculty of Health Sciences
> Ben Gurion University of the Negev
> Beer-Sheva 84105
> Israel
>
> Tel:  [phone]
> Cell: [phone]
> Fax: + 972-8-6477628
> http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎

[Martin Wessendorf-2]

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Dear Daniel--

On 6/2/2011 1:59 PM, Daniel Gitler wrote:

> I am in need of either beads or a fluorophore in solution which is exited consistently by both the 405nm and 488nm lines of a confocal microscope. What I really need is that the ratio of excitation should be constant, in which case two separate dyes are probably not a good choice, unless their molar ratio can be quite consistent (perhaps in beads?). I also need that the efficiency of excitation for both lines be quite decent (doesn't have to be maximal, just decent). Finally (it appears I have a lot of requisites) the dye/beads should be as insensitive as possible to environmental changes (i.e., pH etc). The idea is to have a good standard in order to calibrate the ratio of the power of these two lines when imaging a sample (I do not need to know the actual number, just a relative measure will do fine).

Based on a quick search, uranium glass looks like a possibility.  The
absorbance is perhaps 2x better at 488 than 405.  However, if you're
imaging with a confocal, you would probably need to use point-scanning,
since uranium has a loooong fluorescence time-constant (e.g. 170 usec in
solution).

Here are a few (mediocre) references--they might be a starting point:

http://pubs.acs.org/doi/abs/10.1021/ac00230a029
http://webhost.bridgew.edu/cnoda/research/uranyl/index.html
http://onlinelibrary.wiley.com/doi/10.1002/pssa.2211150136/pdf

Good luck!

Martin
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Boswell, Carl A - (cboswell)]

Hi Martin,
I just went through the effort of finding some uranyl glass to use as a fluorescent standard for a 2P imaging system.  The biggest difficulty was finding some.  After searching for and calling various commercial concerns, I managed to get a couple of pieces from scrap that the glassblowers in the Chemistry Dept. had stashed for special use.  Any idea on current sources of this material?  
Cheers,
C


Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
Univ. of Arizona
520-954-7053
FAX [phone]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Thursday, June 02, 2011 2:12 PM
To: [hidden email]
Subject: Re: Fluorophore excitable with 405nm and 488nm lines

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Dear Daniel--

On 6/2/2011 1:59 PM, Daniel Gitler wrote:

> I am in need of either beads or a fluorophore in solution which is exited consistently by both the 405nm and 488nm lines of a confocal microscope. What I really need is that the ratio of excitation should be constant, in which case two separate dyes are probably not a good choice, unless their molar ratio can be quite consistent (perhaps in beads?). I also need that the efficiency of excitation for both lines be quite decent (doesn't have to be maximal, just decent). Finally (it appears I have a lot of requisites) the dye/beads should be as insensitive as possible to environmental changes (i.e., pH etc). The idea is to have a good standard in order to calibrate the ratio of the power of these two lines when imaging a sample (I do not need to know the actual number, just a relative measure will do fine).

Based on a quick search, uranium glass looks like a possibility.  The absorbance is perhaps 2x better at 488 than 405.  However, if you're imaging with a confocal, you would probably need to use point-scanning, since uranium has a loooong fluorescence time-constant (e.g. 170 usec in solution).

Here are a few (mediocre) references--they might be a starting point:

http://pubs.acs.org/doi/abs/10.1021/ac00230a029
http://webhost.bridgew.edu/cnoda/research/uranyl/index.html
http://onlinelibrary.wiley.com/doi/10.1002/pssa.2211150136/pdf

Good luck!

Martin
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Martin Wessendorf-2]

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On 6/2/2011 5:16 PM, Boswell, Carl A - (cboswell) wrote:

> I just went through the effort of finding some uranyl glass to use as a fluorescent standard for a 2P imaging system.  The biggest difficulty was finding some.  After searching for and calling various commercial concerns, I managed to get a couple of pieces from scrap that the glassblowers in the Chemistry Dept. had stashed for special use.  Any idea on current sources of this material?

Other than an antique store (--uranium glass was used for its yellow or
green color in years past), no.  We have a uranium glass slide in our
facility but I hadn't thought about current availability--oops.

Perhaps Daniel should go with Julio's suggestions!

Martin

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Yuval Ebenstein]

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We routinely excite red emitting quantum dots with both 405 and 488.
choose the emission that suits you best and you can excite anywhere to
the blue of the emission. The 625nm are the brightest from my experience.
You can also embed them in polymer to make an everlasting calibration
sample and you can tune the concentration down to the single quantum dot
level for PSF measurements.
Hope this helps
Yuval

On 6/2/2011 11:59 AM, Daniel Gitler wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Confocalists/microscopists,
> I am in need of either beads or a fluorophore in solution which is exited consistently by both the 405nm and 488nm lines of a confocal microscope. What I really need is that the ratio of excitation should be constant, in which case two separate dyes are probably not a good choice, unless their molar ratio can be quite consistent (perhaps in beads?). I also need that the efficiency of excitation for both lines be quite decent (doesn't have to be maximal, just decent). Finally (it appears I have a lot of requisites) the dye/beads should be as insensitive as possible to environmental changes (i.e., pH etc). The idea is to have a good standard in order to calibrate the ratio of the power of these two lines when imaging a sample (I do not need to know the actual number, just a relative measure will do fine).
> I would appreciate any advice,
> Thanks,
> Daniel
>
>
> Daniel Gitler, Ph.D.
> Department of Physiology and Neurobiology
> Faculty of Health Sciences
> Ben Gurion University of the Negev
> Beer-Sheva 84105
> Israel
>
> Tel:  [phone]
> Cell: [phone]
> Fax: + 972-8-6477628
> http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎

--
--------------------------------------
Yuval Ebenstein
Department of Chemistry and Biochemistry, UCLA
Young Hall-2002, 607 Charles E. Young Drive East
Los Angeles, CA 90095-1569
Ph: [phone]; Fax: [phone]

[Guy Cox-2]

I just checked out a Chroma green fluorescent plastic slide - it seemed to fluoresce pretty well with both uv and blue LEDs so surely that should work well enough.  Doesn't have the ultra-long lifetime problem of uranyl glass.  Reproducible, robust and best of all free!  

                                   Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Friday, 3 June 2011 8:33 AM
To: [hidden email]
Subject: Re: Fluorophore excitable with 405nm and 488nm lines

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On 6/2/2011 5:16 PM, Boswell, Carl A - (cboswell) wrote:

> I just went through the effort of finding some uranyl glass to use as a fluorescent standard for a 2P imaging system.  The biggest difficulty was finding some.  After searching for and calling various commercial concerns, I managed to get a couple of pieces from scrap that the glassblowers in the Chemistry Dept. had stashed for special use.  Any idea on current sources of this material?

Other than an antique store (--uranium glass was used for its yellow or
green color in years past), no.  We have a uranium glass slide in our
facility but I hadn't thought about current availability--oops.

Perhaps Daniel should go with Julio's suggestions!

Martin

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

-----
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[mcammer]

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Depending on your tolerance, they may or may not be stable.  I use these all the time for aligning the microscope because they are great for this, although there may be an issue with refractive index mismatch especially with the newer TIRF objectives, but I'm not so sure as a reproducible sample.
Please see http://www.einstein.yu.edu/aif/instructions/fluor-ref-slides/01.htm and
http://www.einstein.yu.edu/aif/instructions/zeiss/liveduo/Zaxis_bleachingtest/index.htm
for two examples of bleaching.  
-Michael

_________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Guy Cox [[hidden email]]
Sent: Thursday, June 02, 2011 11:32 PM
To: [hidden email]
Subject: Re: Fluorophore excitable with 405nm and 488nm lines

I just checked out a Chroma green fluorescent plastic slide - it seemed to fluoresce pretty well with both uv and blue LEDs so surely that should work well enough.  Doesn't have the ultra-long lifetime problem of uranyl glass.  Reproducible, robust and best of all free!

                                   Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]

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[Barbara Foster]

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Hi, Daniel

A couple of quick comments:
Re: Uranyl glass ... because it emits low doses of radiation, it is
not considered safe here in the US.  We have some sources overseas,
but they have told us that they would probably be picked up as
terrorists if they started exporting to the US.

Re: "Use a power meter and measure the power at the objective" and
"not as practical":
Take a look a the new XP750 from Lumen Dynamics (formerly
EXFO).  This neat little power meter was specifically designed to
mimic a microscope slide so that it sits flat on the stage and
measures the intensity right where you want it: at the sample
plane.  You can use it on an inverted or upright and with any type of
illuminator (lamp, LED, laser).  For those of you who took part in
our Illumination Study in 2008, you might have noticed two sneaky
questions about this device.  The response were huge.. and now it's
available... for real.

For further information, you can visit their website (
http://www.ldgi-xcite.com/products-xr2100-xp750.php).  Also, I have
an article coming out next month (July) in BioPhotonics written
around interviews from users in three key labs (Jen Waters' NIC at
Harvard, the YIP lab in Canada, and LBL).  If you want to chat with
someone, get in touch with their Sr. Apps Scientist, Dr. Kavita
Aswant (P:  1 905 812-3342, E: [hidden email]).  I know she'd
welcome hearing from you.

Caveat: No commercial interest.

Good hunting!
Barbara Foster, President and Sr. Consultant

M icroscopy/Microscopy Education
P: [phone]
W: <http://www.microscopyeducation.com/>www.MicroscopyEducation.com


At 05:09 PM 6/2/2011, Martin Wessendorf wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear Daniel--
>
>On 6/2/2011 1:59 PM, Daniel Gitler wrote:
>
>>I am in need of either beads or a fluorophore in solution which is
>>exited consistently by both the 405nm and 488nm lines of a confocal
>>microscope. What I really need is that the ratio of excitation
>>should be constant, in which case two separate dyes are probably
>>not a good choice, unless their molar ratio can be quite consistent
>>(perhaps in beads?). I also need that the efficiency of excitation
>>for both lines be quite decent (doesn't have to be maximal, just
>>decent). Finally (it appears I have a lot of requisites) the
>>dye/beads should be as insensitive as possible to environmental
>>changes (i.e., pH etc). The idea is to have a good standard in
>>order to calibrate the ratio of the power of these two lines when
>>imaging a sample (I do not need to know the actual number, just a
>>relative measure will do fine).
>
>Based on a quick search, uranium glass looks like a
>possibility.  The absorbance is perhaps 2x better at 488 than
>405.  However, if you're imaging with a confocal, you would probably
>need to use point-scanning, since uranium has a loooong fluorescence
>time-constant (e.g. 170 usec in solution).
>
>Here are a few (mediocre) references--they might be a starting point:
>
>http://pubs.acs.org/doi/abs/10.1021/ac00230a029
>http://webhost.bridgew.edu/cnoda/research/uranyl/index.html
>http://onlinelibrary.wiley.com/doi/10.1002/pssa.2211150136/pdf
>
>Good luck!
>
>Martin
>--
>Martin Wessendorf, Ph.D.                   office: [phone]
>Assoc Prof, Dept Neuroscience                 lab: [phone]
>University of Minnesota             Preferred FAX: [phone]
>6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>Minneapolis, MN  55455                    e-mail: [hidden email]

[Craig Brideau]

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On Fri, Jun 3, 2011 at 11:59 AM, Barbara Foster <[hidden email]> wrote:

>
> Re: "Use a power meter and measure the power at the objective" and "not as
> practical":
> Take a look a the new XP750 from Lumen Dynamics (formerly EXFO).  This neat
> little power meter was specifically designed to mimic a microscope slide so
> that it sits flat on the stage and measures the intensity right where you
> want it: at the sample plane.  You can use it on an inverted or upright and
> with any type of illuminator (lamp, LED, laser).  For those of you who took
> part in our Illumination Study in 2008, you might have noticed two sneaky
> questions about this device.  The response were huge.. and now it's
> available... for real.
>

I actually got to evaluate this thing.  It was a good product, but EXFO
didn't have a good answer for me when I asked them what the meter does about
the high-angle rays from large NA lenses.  Since the face of the meter is
glass, the higher-angle rays from oil and water immersion lenses will tend
to be reflected and not reach the detecting element of the meter.
 Optimally, you'd want a sealed detector unit that you could put water or
oil on.  That way you could test your oil and water lenses with the power
meter.

Craig

>
> For further information, you can visit their website (
> http://www.ldgi-xcite.com/products-xr2100-xp750.php).  Also, I have an
> article coming out next month (July) in BioPhotonics written around
> interviews from users in three key labs (Jen Waters' NIC at Harvard, the YIP
> lab in Canada, and LBL).  If you want to chat with someone, get in touch
> with their Sr. Apps Scientist, Dr. Kavita Aswant (P:  1 905 812-3342, E:
> [hidden email]).  I know she'd welcome hearing from you.
>
> Caveat: No commercial interest.
>
> Good hunting!
> Barbara Foster, President and Sr. Consultant
>
> M icroscopy/Microscopy Education
> P: [phone]
> W: <http://www.microscopyeducation.com/>www.MicroscopyEducation.com
>
>
>
> At 05:09 PM 6/2/2011, Martin Wessendorf wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Daniel--
>>
>> On 6/2/2011 1:59 PM, Daniel Gitler wrote:
>>
>>  I am in need of either beads or a fluorophore in solution which is exited
>>> consistently by both the 405nm and 488nm lines of a confocal microscope.
>>> What I really need is that the ratio of excitation should be constant, in
>>> which case two separate dyes are probably not a good choice, unless their
>>> molar ratio can be quite consistent (perhaps in beads?). I also need that
>>> the efficiency of excitation for both lines be quite decent (doesn't have to
>>> be maximal, just decent). Finally (it appears I have a lot of requisites)
>>> the dye/beads should be as insensitive as possible to environmental changes
>>> (i.e., pH etc). The idea is to have a good standard in order to calibrate
>>> the ratio of the power of these two lines when imaging a sample (I do not
>>> need to know the actual number, just a relative measure will do fine).
>>>
>>
>> Based on a quick search, uranium glass looks like a possibility.  The
>> absorbance is perhaps 2x better at 488 than 405.  However, if you're imaging
>> with a confocal, you would probably need to use point-scanning, since
>> uranium has a loooong fluorescence time-constant (e.g. 170 usec in
>> solution).
>>
>> Here are a few (mediocre) references--they might be a starting point:
>>
>> http://pubs.acs.org/doi/abs/10.1021/ac00230a029
>> http://webhost.bridgew.edu/cnoda/research/uranyl/index.html
>> http://onlinelibrary.wiley.com/doi/10.1002/pssa.2211150136/pdf
>>
>> Good luck!
>>
>> Martin
>> --
>> Martin Wessendorf, Ph.D.                   office: [phone]
>> Assoc Prof, Dept Neuroscience                 lab: [phone]
>> University of Minnesota             Preferred FAX: [phone]
>> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>> Minneapolis, MN  55455                    e-mail: [hidden email]
>>
>

[Rosemary.White]

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Uranium glass is available from some suppliers of glass billets for glass-blowing, e.g. uranium green from Gaffer Glass in New Zealand - they now have a US branch.  I'm told by glassblowers that it's the real deal.
cheers,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Martin Wessendorf [[hidden email]]
Sent: Friday, 3 June 2011 8:33 a.m.
To: [hidden email]
Subject: Re: Fluorophore excitable with 405nm and 488nm lines

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On 6/2/2011 5:16 PM, Boswell, Carl A - (cboswell) wrote:

> I just went through the effort of finding some uranyl glass to use as a fluorescent standard for a 2P imaging system.  The biggest difficulty was finding some.  After searching for and calling various commercial concerns, I managed to get a couple of pieces from scrap that the glassblowers in the Chemistry Dept. had stashed for special use.  Any idea on current sources of this material?

Other than an antique store (--uranium glass was used for its yellow or
green color in years past), no.  We have a uranium glass slide in our
facility but I hadn't thought about current availability--oops.

Perhaps Daniel should go with Julio's suggestions!

Martin

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Guy Cox-2]

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Hang on, the requirement was for a sample which would measure the
RELATIVE intensities of 505 and 488nm lasers.  Mild bleaching shouldn't
affect that.  If a bit is seriously bleached, use another spot.  A quick
scan at very low power will reveal that.  You're doing pretty well if
you can bleach an entire slide in a lifetime - and it you do, the
replacement is free.  I've never succeeded in bleaching any part but I
have managed to laser-ablate spots on the surface!  As for RI mismatch -
well of course there is.  I'll bet there is with uranyl glass too (but
in the opposite direction).  But the killer with the U glass is that you
can't actually see it very well in a scanned image.  If you really want
a solid-state unbleachable sample get a slice of emerald.  I've never
actually tried it at 405nm but it's so bright at 488 I'm sure it would
work.  Unless you have a very generous lab budget I would recommend the
synthetic version over the natural one!

                                 Guy


Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Cammer, Michael
Sent: Friday, 3 June 2011 9:00 PM
To: [hidden email]
Subject: Re: Fluorophore excitable with 405nm and 488nm lines

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Depending on your tolerance, they may or may not be stable.  I use these
all the time for aligning the microscope because they are great for
this, although there may be an issue with refractive index mismatch
especially with the newer TIRF objectives, but I'm not so sure as a
reproducible sample.
Please see
http://www.einstein.yu.edu/aif/instructions/fluor-ref-slides/01.htm and
http://www.einstein.yu.edu/aif/instructions/zeiss/liveduo/Zaxis_bleachin
gtest/index.htm
for two examples of bleaching.  
-Michael

_________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] On
Behalf Of Guy Cox [[hidden email]]
Sent: Thursday, June 02, 2011 11:32 PM
To: [hidden email]
Subject: Re: Fluorophore excitable with 405nm and 488nm lines

I just checked out a Chroma green fluorescent plastic slide - it seemed
to fluoresce pretty well with both uv and blue LEDs so surely that
should work well enough.  Doesn't have the ultra-long lifetime problem
of uranyl glass.  Reproducible, robust and best of all free!

                                   Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]

------------------------------------------------------------
This email message, including any attachments, is for the sole use of
the intended recipient(s) and may contain information that is
proprietary, confidential, and exempt from disclosure under applicable
law. Any unauthorized review, use, disclosure, or distribution is
prohibited. If you have received this email in error please notify the
sender by return email and delete the original message. Please note, the
recipient should check this email and any attachments for the presence
of viruses. The organization accepts no liability for any damage caused
by any virus transmitted by this email.
=================================

-----
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Version: 10.0.1382 / Virus Database: 1511/3677 - Release Date: 06/03/11

[Craig Brideau]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

That's pretty neat Guy!  Where on earth would you find synthetic emerald
though?

Craig



On Sat, Jun 4, 2011 at 2:05 AM, Guy Cox <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hang on, the requirement was for a sample which would measure the
> RELATIVE intensities of 505 and 488nm lasers.  Mild bleaching shouldn't
> affect that.  If a bit is seriously bleached, use another spot.  A quick
> scan at very low power will reveal that.  You're doing pretty well if
> you can bleach an entire slide in a lifetime - and it you do, the
> replacement is free.  I've never succeeded in bleaching any part but I
> have managed to laser-ablate spots on the surface!  As for RI mismatch -
> well of course there is.  I'll bet there is with uranyl glass too (but
> in the opposite direction).  But the killer with the U glass is that you
> can't actually see it very well in a scanned image.  If you really want
> a solid-state unbleachable sample get a slice of emerald.  I've never
> actually tried it at 405nm but it's so bright at 488 I'm sure it would
> work.  Unless you have a very generous lab budget I would recommend the
> synthetic version over the natural one!
>
>                                 Guy
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Cammer, Michael
> Sent: Friday, 3 June 2011 9:00 PM
> To: [hidden email]
> Subject: Re: Fluorophore excitable with 405nm and 488nm lines
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Depending on your tolerance, they may or may not be stable.  I use these
> all the time for aligning the microscope because they are great for
> this, although there may be an issue with refractive index mismatch
> especially with the newer TIRF objectives, but I'm not so sure as a
> reproducible sample.
> Please see
> http://www.einstein.yu.edu/aif/instructions/fluor-ref-slides/01.htm and
> http://www.einstein.yu.edu/aif/instructions/zeiss/liveduo/Zaxis_bleachin
> gtest/index.htm
> for two examples of bleaching.
> -Michael
>
> _________________________________________
> Michael Cammer, Assistant Research Scientist
> Skirball Institute of Biomolecular Medicine
> Lab: [phone]  Cell: [phone]
>
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] On
> Behalf Of Guy Cox [[hidden email]]
> Sent: Thursday, June 02, 2011 11:32 PM
> To: [hidden email]
> Subject: Re: Fluorophore excitable with 405nm and 488nm lines
>
> I just checked out a Chroma green fluorescent plastic slide - it seemed
> to fluoresce pretty well with both uv and blue LEDs so surely that
> should work well enough.  Doesn't have the ultra-long lifetime problem
> of uranyl glass.  Reproducible, robust and best of all free!
>
>                                   Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of
> the intended recipient(s) and may contain information that is
> proprietary, confidential, and exempt from disclosure under applicable
> law. Any unauthorized review, use, disclosure, or distribution is
> prohibited. If you have received this email in error please notify the
> sender by return email and delete the original message. Please note, the
> recipient should check this email and any attachments for the presence
> of viruses. The organization accepts no liability for any damage caused
> by any virus transmitted by this email.
> =================================
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1382 / Virus Database: 1511/3677 - Release Date: 06/03/11
>

[Daniel Gitler]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I would like to thank everyone for their suggestions. I will try some out next week, and I'll let you know what worked for me.
Daniel

----- Original Message -----
From: Craig Brideau <[hidden email]>
Date: Monday, June 6, 2011 20:10
Subject: Re: Fluorophore excitable with 405nm and 488nm lines
To: [hidden email]

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> That's pretty neat Guy!  Where on earth would you find
> synthetic emerald
> though?
>
> Craig
>
>
>
> On Sat, Jun 4, 2011 at 2:05 AM, Guy Cox
> <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hang on, the requirement was for a sample which would measure the
> > RELATIVE intensities of 505 and 488nm lasers.  Mild
> bleaching shouldn't
> > affect that.  If a bit is seriously bleached, use another
> spot.  A quick
> > scan at very low power will reveal that.  You're doing
> pretty well if
> > you can bleach an entire slide in a lifetime - and it you do, the
> > replacement is free.  I've never succeeded in bleaching
> any part but I
> > have managed to laser-ablate spots on the surface!  As
> for RI mismatch -
> > well of course there is.  I'll bet there is with uranyl
> glass too (but
> > in the opposite direction).  But the killer with the U
> glass is that you
> > can't actually see it very well in a scanned image.  If
> you really want
> > a solid-state unbleachable sample get a slice of
> emerald.  I've never
> > actually tried it at 405nm but it's so bright at 488 I'm sure
> it would
> > work.  Unless you have a very generous lab budget I would
> recommend the
> > synthetic version over the natural one!
> >
> >                                 Guy
> >
> >
> > Optical Imaging Techniques in Cell Biology
> > by Guy Cox    CRC Press / Taylor & Francis
> >     http://www.guycox.com/optical.htm
> > ______________________________________________
> > Associate Professor Guy Cox, MA, DPhil(Oxon)
> > Australian Centre for Microscopy & Microanalysis,
> > Madsen Building F09, University of Sydney, NSW 2006
> >
> > Phone [phone]     Fax [phone]
> >             Mobile [phone]
> > ______________________________________________
> >      http://www.guycox.net
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> [mailto:[hidden email]]> On Behalf Of Cammer,
> Michael> Sent: Friday, 3 June 2011 9:00 PM
> > To: [hidden email]
> > Subject: Re: Fluorophore excitable with 405nm and 488nm lines
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Depending on your tolerance, they may or may not be
> stable.  I use these
> > all the time for aligning the microscope because they are
> great for
> > this, although there may be an issue with refractive index mismatch
> > especially with the newer TIRF objectives, but I'm not so sure
> as a
> > reproducible sample.
> > Please see
> > http://www.einstein.yu.edu/aif/instructions/fluor-ref-
> slides/01.htm and
> >
> http://www.einstein.yu.edu/aif/instructions/zeiss/liveduo/Zaxis_bleachin> gtest/index.htm
> > for two examples of bleaching.
> > -Michael
> >
> > _________________________________________
> > Michael Cammer, Assistant Research Scientist
> > Skirball Institute of Biomolecular Medicine
> > Lab: [phone]  Cell: [phone]
> >
> > ________________________________________
> > From: Confocal Microscopy List
> [[hidden email]] On
> > Behalf Of Guy Cox [[hidden email]]
> > Sent: Thursday, June 02, 2011 11:32 PM
> > To: [hidden email]
> > Subject: Re: Fluorophore excitable with 405nm and 488nm lines
> >
> > I just checked out a Chroma green fluorescent plastic slide -
> it seemed
> > to fluoresce pretty well with both uv and blue LEDs so surely that
> > should work well enough.  Doesn't have the ultra-long
> lifetime problem
> > of uranyl glass.  Reproducible, robust and best of all free!
> >
> >                                   Guy
> >
> > Optical Imaging Techniques in Cell Biology
> > by Guy Cox    CRC Press / Taylor & Francis
> >     http://www.guycox.com/optical.htm
> > ______________________________________________
> > Associate Professor Guy Cox, MA, DPhil(Oxon)
> > Australian Centre for Microscopy & Microanalysis,
> > Madsen Building F09, University of Sydney, NSW 2006
> >
> > Phone [phone]     Fax [phone]
> >             Mobile [phone]
> >
> > ------------------------------------------------------------
> > This email message, including any attachments, is for the sole
> use of
> > the intended recipient(s) and may contain information that is
> > proprietary, confidential, and exempt from disclosure under
> applicable> law. Any unauthorized review, use, disclosure, or
> distribution is
> > prohibited. If you have received this email in error please
> notify the
> > sender by return email and delete the original message. Please
> note, the
> > recipient should check this email and any attachments for the
> presence> of viruses. The organization accepts no liability for
> any damage caused
> > by any virus transmitted by this email.
> > =================================
> >
> > -----
> > No virus found in this message.
> > Checked by AVG - www.avg.com
> > Version: 10.0.1382 / Virus Database: 1511/3677 - Release Date:
> 06/03/11>
>

Daniel Gitler, Ph.D.
Department of Physiology and Neurobiology
Faculty of Health Sciences
Ben Gurion University of the Negev
Beer-Sheva 84105
Israel

Tel:  [phone]
Cell: [phone]
Fax: + 972-8-6477628
http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎

[Guy Cox-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Jewellers' supplies.  Or ask a friendly jeweller for a small piece.  Or
if you want a whole boule go to a manufacturer like Saint Gobain.  They
gave me a very large (10cm * 2.5 cm) piece of titanium sapphire for
nothing (it was a reject, of course, but fine for such purposes).  The
large emerald I played with came from someone whose research was on
gemstones - if it was natural it was worth a fortune but I assume it
wasn't!  

                                                    Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Craig Brideau
Sent: Tuesday, 7 June 2011 3:09 AM
To: [hidden email]
Subject: Re: Fluorophore excitable with 405nm and 488nm lines

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

That's pretty neat Guy!  Where on earth would you find synthetic emerald
though?

Craig



On Sat, Jun 4, 2011 at 2:05 AM, Guy Cox <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hang on, the requirement was for a sample which would measure the
> RELATIVE intensities of 505 and 488nm lasers.  Mild bleaching
shouldn't
> affect that.  If a bit is seriously bleached, use another spot.  A
quick
> scan at very low power will reveal that.  You're doing pretty well if
> you can bleach an entire slide in a lifetime - and it you do, the
> replacement is free.  I've never succeeded in bleaching any part but I
> have managed to laser-ablate spots on the surface!  As for RI mismatch
-
> well of course there is.  I'll bet there is with uranyl glass too (but
> in the opposite direction).  But the killer with the U glass is that
you
> can't actually see it very well in a scanned image.  If you really
want
> a solid-state unbleachable sample get a slice of emerald.  I've never
> actually tried it at 405nm but it's so bright at 488 I'm sure it would
> work.  Unless you have a very generous lab budget I would recommend
the

> synthetic version over the natural one!
>
>                                 Guy
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
> -----Original Message-----
> From: Confocal Microscopy List

[mailto:[hidden email]]

> On Behalf Of Cammer, Michael
> Sent: Friday, 3 June 2011 9:00 PM
> To: [hidden email]
> Subject: Re: Fluorophore excitable with 405nm and 488nm lines
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Depending on your tolerance, they may or may not be stable.  I use

these
> all the time for aligning the microscope because they are great for
> this, although there may be an issue with refractive index mismatch
> especially with the newer TIRF objectives, but I'm not so sure as a
> reproducible sample.
> Please see
> http://www.einstein.yu.edu/aif/instructions/fluor-ref-slides/01.htm
and
>
http://www.einstein.yu.edu/aif/instructions/zeiss/liveduo/Zaxis_bleachin

> gtest/index.htm
> for two examples of bleaching.
> -Michael
>
> _________________________________________
> Michael Cammer, Assistant Research Scientist
> Skirball Institute of Biomolecular Medicine
> Lab: [phone]  Cell: [phone]
>
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] On
> Behalf Of Guy Cox [[hidden email]]
> Sent: Thursday, June 02, 2011 11:32 PM
> To: [hidden email]
> Subject: Re: Fluorophore excitable with 405nm and 488nm lines
>
> I just checked out a Chroma green fluorescent plastic slide - it

seemed

> to fluoresce pretty well with both uv and blue LEDs so surely that
> should work well enough.  Doesn't have the ultra-long lifetime problem
> of uranyl glass.  Reproducible, robust and best of all free!
>
>                                   Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of
> the intended recipient(s) and may contain information that is
> proprietary, confidential, and exempt from disclosure under applicable
> law. Any unauthorized review, use, disclosure, or distribution is
> prohibited. If you have received this email in error please notify the
> sender by return email and delete the original message. Please note,

the
> recipient should check this email and any attachments for the presence
> of viruses. The organization accepts no liability for any damage
caused
> by any virus transmitted by this email.
> =================================
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1382 / Virus Database: 1511/3677 - Release Date:
06/03/11
>

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 10.0.1382 / Virus Database: 1511/3683 - Release Date: 06/05/11

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Invitrogen/Molecular Probes TetraSpeck beads.

With respect to Uranyl glass --  you can get Uranium crystals from any
TEM lab or cell biology lab that does a lot of TEM. And yes, sticking a
Geiger counter over an open bottle results in more clicks than elsewhere
in a lab. My thanks to Gregg Gundersen for demonstrating this.

George

On 6/2/2011 2:59 PM, Daniel Gitler wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Confocalists/microscopists,
> I am in need of either beads or a fluorophore in solution which is exited consistently by both the 405nm and 488nm lines of a confocal microscope. What I really need is that the ratio of excitation should be constant, in which case two separate dyes are probably not a good choice, unless their molar ratio can be quite consistent (perhaps in beads?). I also need that the efficiency of excitation for both lines be quite decent (doesn't have to be maximal, just decent). Finally (it appears I have a lot of requisites) the dye/beads should be as insensitive as possible to environmental changes (i.e., pH etc). The idea is to have a good standard in order to calibrate the ratio of the power of these two lines when imaging a sample (I do not need to know the actual number, just a relative measure will do fine).
> I would appreciate any advice,
> Thanks,
> Daniel
>
>
> Daniel Gitler, Ph.D.
> Department of Physiology and Neurobiology
> Faculty of Health Sciences
> Ben Gurion University of the Negev
> Beer-Sheva 84105
> Israel
>
> Tel:  [phone]
> Cell: [phone]
> Fax: + 972-8-6477628
> http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami