Fluorophores for 633nm laser

[Andrew Lindsay]

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Hi All,

I was wondering if people could recommend far-red fluorophores for
immunofluorescence that are excitable with a 633nm laser, I have tried
Alexa633 with little success. Have other people had more luck with secondary
antibodies conjugated to other fluorophores such as Cy5, DyLight649,
Alexa647.....?

Thanks in advance,
Andrew

[Tamara Howard]

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We've used DyLight 649 - works beautifully on our SP5.

Tamara
...................................................
Tamara Howard
Dept. of Cell Biology & Physiology
University of New Mexico
Albuquerque, NM


________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Andrew Lindsay [[hidden email]]
Sent: Friday, July 12, 2013 8:29 AM
To: [hidden email]
Subject: Fluorophores for 633nm laser

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Hi All,

I was wondering if people could recommend far-red fluorophores for
immunofluorescence that are excitable with a 633nm laser, I have tried
Alexa633 with little success. Have other people had more luck with secondary
antibodies conjugated to other fluorophores such as Cy5, DyLight649,
Alexa647.....?

Thanks in advance,
Andrew

[Mike Tighe]

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We have had luck with Alexa 647, Dylight649, as well as APC conjugates.
________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Andrew Lindsay <[hidden email]>
Sent: Friday, July 12, 2013 10:29 AM
To: [hidden email]
Subject: Fluorophores for 633nm laser

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Hi All,

I was wondering if people could recommend far-red fluorophores for
immunofluorescence that are excitable with a 633nm laser, I have tried
Alexa633 with little success. Have other people had more luck with secondary
antibodies conjugated to other fluorophores such as Cy5, DyLight649,
Alexa647.....?

Thanks in advance,
Andrew

[Gabriel Lapointe-4]

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Hi Andrew,
Personally, Alexa647 is one of my favorite fluorophore. It is very stable
and I haven't notice any significant cross talk with Alexa568. However, you
have to remember that any far red dye are very hard to see with the naked
eye (some say impossible) and that the quantum efficiency of a lot of
cameras and PMT is usually much lower at 700nm than at 500nm. However,
there is no auto-fluorescence in the far-red region and the 633nm is much
less toxic so it is usually not an issue to increase exposure time or
excitation power.

Sincerely

*Gabriel Lapointe, M.Sc.*
Lab Manager / Microscopy Specialist
Concordia University, Biology Department
7141 Sherbrooke St. West SP 534
Montréal QC H4B 1R6 Canada
Lab : [phone] x5988
Office : [phone] x3008
Fax : [phone]
Cell : [phone]
[hidden email]
cmac.concordia.ca
http://gabriellapointe.ca


2013/7/12 Andrew Lindsay <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi All,
>
> I was wondering if people could recommend far-red fluorophores for
> immunofluorescence that are excitable with a 633nm laser, I have tried
> Alexa633 with little success. Have other people had more luck with
> secondary
> antibodies conjugated to other fluorophores such as Cy5, DyLight649,
> Alexa647.....?
>
> Thanks in advance,
> Andrew
>

[Cedric Espenel]

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Hi Andrew,

You should try Atto647N.

Best

Cedric

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andrew Lindsay
Sent: vendredi 12 juillet 2013 10:30
To: [hidden email]
Subject: Fluorophores for 633nm laser

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Hi All,

I was wondering if people could recommend far-red fluorophores for
immunofluorescence that are excitable with a 633nm laser, I have tried
Alexa633 with little success. Have other people had more luck with secondary
antibodies conjugated to other fluorophores such as Cy5, DyLight649,
Alexa647.....?

Thanks in advance,
Andrew

[Rosemary.White]

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The Cy5 and Cy5.5 dyes are great and emission can be separated on spectral
confocals if both used together.
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
E [hidden email]


On 13/07/13 12:29 AM, "Andrew Lindsay" <[hidden email]> wrote:

>*****
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>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi All,
>
>I was wondering if people could recommend far-red fluorophores for
>immunofluorescence that are excitable with a 633nm laser, I have tried
>Alexa633 with little success. Have other people had more luck with
>secondary
>antibodies conjugated to other fluorophores such as Cy5, DyLight649,
>Alexa647.....?
>
>Thanks in advance,
>Andrew

[Franziska Curdt]

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Alexa should work, what filters do you use?
Atto 633 and 647 are more photostable, as well as Abberior STAR 635.
I cannot recommend Cy5.

You find a list of photostable dyes here.

http://nanobiophotonics.mpibpc.mpg.de/old/dyes/

Best regards,

F.Curdt


________________________________________
Von: Confocal Microscopy List [[hidden email]] im Auftrag von Andrew Lindsay [[hidden email]]
Gesendet: Freitag, 12. Juli 2013 16:29
An: [hidden email]
Betreff: Fluorophores for 633nm laser

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Hi All,

I was wondering if people could recommend far-red fluorophores for
immunofluorescence that are excitable with a 633nm laser, I have tried
Alexa633 with little success. Have other people had more luck with secondary
antibodies conjugated to other fluorophores such as Cy5, DyLight649,
Alexa647.....?

Thanks in advance,
Andrew

[Steffen Dietzel]

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Cy5 worked great for us in the past. But so did Alexa633. Both mounted
in Vectashield. As was pointed out, the human eye is a miserable
detector in that range, so expect to see nothing down the eye piece.

Steffen

On 12.07.2013 16:29, Andrew Lindsay wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi All,
>
> I was wondering if people could recommend far-red fluorophores for
> immunofluorescence that are excitable with a 633nm laser, I have tried
> Alexa633 with little success. Have other people had more luck with secondary
> antibodies conjugated to other fluorophores such as Cy5, DyLight649,
> Alexa647.....?
>
> Thanks in advance,
> Andrew
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[George McNamara]

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Dear listserv,

Lee et al 2013 reports that heavy water (D2O) doubes the quantum yield
for some fluorophores:
*
Brighter dyes in heavy water:* A simple and cost-effective method
increases the brightness of a whole class of commonly used red-emitting
fluorophores, including ATTO655, ATTO680, and ATTO700. Replacing water
(H_2 O) by heavy water (D_2 O) in the imaging buffer doubles the
fluorescence quantum yield of these dyes and significantly improves the
localization precision in super-resolution imaging.

I looked up some of the papers cited in their ref 4 (main text) and
found that Lucifer Yellow (always reminds me of the Rolling Stones song,
Sympathy for the Devil) is another that does doubles.

In the (free to access) supplemental file
http://onlinelibrary.wiley.com/doi/10.1002/anie.201302341/suppinfo
Their Table S4 shows a small increase for Alexa Fluor 647 (0.44 to 0.40)
as well as ATTO647N (0.51 to 0.66). Note that their precision
localization imaging experiments were done with ascorbic acid to induce
dark state(s). Other additives may be preferable for imaging for maximum
brightness.

I looked at Sigma-Aldrich.com for pricing - heavy water is about $1/gram
~$1/mL. Since a typical coverglass-slide preparation is 10 uL, or a
typical imaging dish's "imaging volume" is about 200 uL
(glassbottomdishes 20 mm diameter opening), price is trivial.

Enjoy,

George


On 7/15/2013 7:37 AM, Steffen Dietzel wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Cy5 worked great for us in the past. But so did Alexa633. Both mounted
> in Vectashield. As was pointed out, the human eye is a miserable
> detector in that range, so expect to see nothing down the eye piece.
>
> Steffen
>
> On 12.07.2013 16:29, Andrew Lindsay wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi All,
>>
>> I was wondering if people could recommend far-red fluorophores for
>> immunofluorescence that are excitable with a 633nm laser, I have tried
>> Alexa633 with little success. Have other people had more luck with
>> secondary
>> antibodies conjugated to other fluorophores such as Cy5, DyLight649,
>> Alexa647.....?
>>
>> Thanks in advance,
>> Andrew
>>
>
>

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
http://works.bepress.com/gmcnamara/26/

[JOEL B. SHEFFIELD]

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I've been following this discussion, and wondering if it would help for you
to specify the detection system that you will be using for the far red
signal.  As suggested by several people, the problems that you are having
might very well be due to that component of your optical system.

Joel


On Mon, Jul 15, 2013 at 8:37 AM, Steffen Dietzel <[hidden email]>wrote:

> *****
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> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> Cy5 worked great for us in the past. But so did Alexa633. Both mounted in
> Vectashield. As was pointed out, the human eye is a miserable detector in
> that range, so expect to see nothing down the eye piece.
>
> Steffen
>
> On 12.07.2013 16:29, Andrew Lindsay wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> *****
>>
>> Hi All,
>>
>> I was wondering if people could recommend far-red fluorophores for
>> immunofluorescence that are excitable with a 633nm laser, I have tried
>> Alexa633 with little success. Have other people had more luck with
>> secondary
>> antibodies conjugated to other fluorophores such as Cy5, DyLight649,
>> Alexa647.....?
>>
>> Thanks in advance,
>> Andrew
>>
>>
>
> --
> ------------------------------**------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
> Head of light microscopy
>
> Mail room:
> Marchioninistr. 15, D-81377 München
>
> Building location:
> Marchioninistr. 27,  München-Großhadern
>

--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs

[Andrew Lindsay]

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Hi All,

Thanks for all your replies and based on the advice I am leaning towards
Alexa647 or DyLight649. To clarify I will be using the Meta detector on a
Zeiss LSM510 to detect the far-red signal, and to further clarify I intend
to use the secondary antibody in combination with GFP and Cy3 to do
three-color immunofluorescence on mammalian cell lines.

Andrew

[Vladimir Zhukarev]

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Dear Andrew,
 
  You might find it really hard to use NIR emitting fluorophores no matter how 'bright' they are, specifically because your detector is of a LASM510 type. Segmented detectors, much more that regular PMT'es, are known to be not optimal for imaging in this spectrum range (check with Hamamatsu - source of all PMT'es for confocal microscopy). By the way this why the 'spectral scale on your machine ends at 710 nm as opposed to 800 nm with other spectral confocals.
But you probably noticed this already.
 
Best
 
Vladimir
 
Vladimir Zhukarev, Ph.D
 Advanced Microscopy Consulting
 Life Science and Bio-Medical Applications
 
 'From Image-to Discovery!'
 [hidden email]
 610.350.1510 www.from-image-to-discovery.com
--------- Original Message --------- Subject: Re: Fluorophores for 633nm laser
From: "Andrew Lindsay" <[hidden email]>
Date: 7/16/13 1:10 am
To: [hidden email]

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 Hi All,
 
 Thanks for all your replies and based on the advice I am leaning towards
 Alexa647 or DyLight649. To clarify I will be using the Meta detector on a
 Zeiss LSM510 to detect the far-red signal, and to further clarify I intend
 to use the secondary antibody in combination with GFP and Cy3 to do
 three-color immunofluorescence on mammalian cell lines.
 
 Andrew

[Vitaly Boyko]

We are looking for a highly sensitive EM-CCD camera but with
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Dear All,

We are looking for a highly sensitive EM-CCD camera but with small pixels (8 um) and the back-thinned one. 
I have already received endless suggestions around "scientific" CMOS cameras, but I am a bit stubborn, as you have probably already realized. And/or, is there anything cooler then CMOS? 
I like Canon 1D X and Nikon D800, and the latter seems to be a proper place for CMOS...

Cheers,

Vitaly

[Philippe clemenceau]

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Commercial response :

Hi Vitaly,

I don't think it exists unfortunately, and you might have to settle on
either small pixels or back thinned sensitivity if you stay with EMCCD.
I sell the FALCON EMCCD camera from Raptor Photonics. It is not back
thinned, it has 8 x 8 micron pixels, QE goes up to 65 % and it is a 1K x 1K
sensor. Sensitivity allows good performance for most applications and it is
a very cost effective camera.

If that is of interest to you, we can talk about it off the list !

Regards,

Philippe Clémenceau, Division Manager, MS in Optical Science

Imagine Optic Inc./Axiom Optics
Ph:[phone]
Cell: + 1 [phone]
1 Broadway, 14th floor
 Cambridge,  MA 02142
www.axiomoptics.com

Metrology, Adaptive Optics, Scientific Imaging, Lasers




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Vitaly Boyko
Sent: Tuesday, July 16, 2013 11:04 AM
To: [hidden email]
Subject: EM-CCD with 8 um/pixel back-thinned

We are looking for a highly sensitive EM-CCD camera but with
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Dear All,

We are looking for a highly sensitive EM-CCD camera but with small pixels (8
um) and the back-thinned one. I have already received endless suggestions
around "scientific" CMOS cameras, but I am a bit stubborn, as you have
probably already realized. And/or, is there anything cooler then CMOS? I
like Canon 1D X and Nikon D800, and the latter seems to be a proper place
for CMOS...

Cheers,

Vitaly

[James Pawley]

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>We are looking for a highly sensitive EM-CCD camera but with
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear All, We are looking for a highly sensitive
>EM-CCD camera but with small pixels (8 um) and
>the back-thinned one.  I have already received
>endless suggestions around "scientific" CMOS
>cameras, but I am a bit stubborn, as you have
>probably already realized. And/or, is there
>anything cooler then CMOS?  I like Canon 1D X
>and Nikon D800, and the latter seems to be a
>proper place for CMOS... Cheers, Vitaly

Dear Vitaliy,

I will be watching answers to your request with
some interest because I don't know of such a chip
(yet!).

I think that we need to keep a bit of geometry in
mind. Let's assume that the Si has been thinned
to 8µm. Photoelectrons created near the so-called
"back" (but actually now the front) surface will
see the attraction from the nearest, positive
charge-transfer electrode, about 8µm away.
However, if they are  created in the border
region where two electrodes are about
equidistant, there will be uncertainty as to
which one to go to. This uncertainty will
increase as the pixel dimensions are reduced. The
result is to reduce the "resolution" of the
sensor below that which one would assume from the
pixel count. (If you look carefully in the specs
of small-dimension CCDs, you will see that the
red resolution is lower than that for blue.)

In other words, just making smaller pixels may
not give you better resolution, just more
measurements to make in the same time (and hence,
higher read bandwidth and higher read noise).
This is specially true for red dyes where
otherwise, the BI-CCDs have a particular
advantage over PMTs etc.

Of course, this resolution loss also happens to
some extent in front-illuminated chips, but as
most photons are now absorbed pretty near the
charge transfer electrodes, it is less of a
problem.

Of course, you could thin more, but this would
reduce the QE in the red as red photons are
absorbed less strongly. The drop-off of BI-CCD
response in the red is largely due to this fact.

There may be a lot of other engineering
considerations but whatever they are, it seems
that BI CCDs usually have large pixels.

Hope I am wrong,

Jim Pawley
--
James and Christine Pawley, 5446 Burley Place (PO
Box 2348), Sechelt, BC, Canada, V0N3A0,
Phone [phone], email <[hidden email]>
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