Alexa 680 and 700 and Zeiss LSM510meta

[Michal Gdula-2]

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Dear Confocalists,

I am in desperate need for a new fuorophores for my multicolor FISH-
immunofluorescence experiments. I am  using at the moment DAPI, Alxea488, Cy3,
Texas Red and Cy5. I use for imaging Zeiss microscope LSM510meta.
I am thinking about purchasing antibodies conjugated with Alexa 680 or Alexa
700. According to olipmpus fluorophore tutorial web page my 633 nm laser should
be able to excite both although its quite far away from excitation maximum.
Has anybody used successfully any of these fluorophores with LSM510 meta?
Is it easy to separate them from Cy5 using meta detector?
Molecular mass of of these fluorophores is relatively high (1.2-1.4 kDa) compared
with the fluorophores which I am using right now, will it have effect on the
antibody penetration ?

Kind regards,

Michal Gdula
PhD student
Bradford University
Uk

[George McNamara]

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Hi Michal,

You have a META spectral detector - use the spectral unmixing
capability. If necessary, you can acquire sequentially with different
lasers and combine the images later.

PMT's have poor quantum efficiency in the far red, so even if Cy5.5,
AF680 or AF700 dye, or QD705 quantum dot conjugates work well at the
specimen level the signal might be weak. QD705 might have an advantage
in being excitable with the same laser as DAPI.

Tsurui et al published 7 color immunofluorescence eleven years ago:

    Tsurui H, Nishimura H, Hattori S, Hirose S, Okumura K, Shirai T.
    Seven-color fluorescence imaging of tissue samples based on Fourier
    spectroscopy and singular value decomposition. </pubmed/10769049> J
    Histochem Cytochem. 2000 May;48(5):653-62. PMID: 10769049

You might take a dye from them and add AF532 to your mix (AF546 is not
as useful a dye as AF555 or AF568). When you have an opportunity,
replacing Cy3 with any of Cy3B (B=Brighter), AF555 or AF568 should
increase brightness.

If any of your reagents are dim, you can use tyramide signal
amplification to boost the signal between 10x and 100x. PerkinElmer has
a limited TSA line-up, Invitrogen/Molecular probes has many fluorescent
TSA kits.

Anothre possibility (instead of NIRF or AF532) would be Lucifer Yellow
... AF430, excited with the same laser as DAPI.

If you need to see spectra of any of these, see
http://www.mcb.arizona.edu/ipc/fret/index.html
If you want the spectra data, see the XLSX file inside the PubSpectra
zip file at the top of
http://sylvester.org/research/shared-resources/laboratory-resources/analytical-imaging-core-facility/analytical-imaging-core-facility-links-forms/pubspectra-data 


Best wishes,

George
p.s. there are over 700 spectral karyotyping papers that use five
FISH/immuno reagents acquired simultaneously plus DAPI (acquired
separately with a DAPI filter set). The dye-detection reagents used
include DNA probes conjugated with Rhodamine 110 (green fluorophore),
spectrum orange, spectrum Red (Texas Red), biotin and digoxigenin, with
detection reagents Cy5-streptavidin and Cy5.5-anti-mouse/mouse anti-dig.
I will guess here that over 10,000 specimens have been acquired and
analyzed for clinical + research use. The META detector ought to be able
to handle five color unmxing as well as the SKY camera.


On 2/20/2011 9:01 AM, Michal Gdula wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Confocalists,
>
> I am in desperate need for a new fuorophores for my multicolor FISH-
> immunofluorescence experiments. I am  using at the moment DAPI, Alxea488, Cy3,
> Texas Red and Cy5. I use for imaging Zeiss microscope LSM510meta.
> I am thinking about purchasing antibodies conjugated with Alexa 680 or Alexa
> 700. According to olipmpus fluorophore tutorial web page my 633 nm laser should
> be able to excite both although its quite far away from excitation maximum.
> Has anybody used successfully any of these fluorophores with LSM510 meta?
> Is it easy to separate them from Cy5 using meta detector?
> Molecular mass of of these fluorophores is relatively high (1.2-1.4 kDa) compared
> with the fluorophores which I am using right now, will it have effect on the
> antibody penetration ?
>
> Kind regards,
>
> Michal Gdula
> PhD student
> Bradford University
> Uk
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[Julio Vazquez]

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Michal,

I have not tried those dyes. However, the range of the META detector  
on our system goes up to ~ 720 nm (according to the graphical display  
in the LMETA control panel in LSM software). According to this, Alexa  
680 (or Cy5.5) should be detected about twice as efficiently as Alexa  
700. Both Alexa 680, Cy5.5,  and 700 should be distinct enough from  
Cy5 for the META to separate them, again in theory, so my best bet  
would be 680 or Cy5.5, with 680 being possibly the brighter and more  
stable choice.

Also, as an alternative, you may consider using dyes with large  
stokes shift, i.e. separate your dyes based on excitation rather than  
emission, but I have no such dye in particular to recommend.
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024


http://www.fhcrc.org

==

On Feb 20, 2011, at 6:01 AM, Michal Gdula wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Confocalists,
>
> I am in desperate need for a new fuorophores for my multicolor FISH-
> immunofluorescence experiments. I am  using at the moment DAPI,  
> Alxea488, Cy3,
> Texas Red and Cy5. I use for imaging Zeiss microscope LSM510meta.
> I am thinking about purchasing antibodies conjugated with Alexa 680  
> or Alexa
> 700. According to olipmpus fluorophore tutorial web page my 633 nm  
> laser should
> be able to excite both although its quite far away from excitation  
> maximum.
> Has anybody used successfully any of these fluorophores with LSM510  
> meta?
> Is it easy to separate them from Cy5 using meta detector?
> Molecular mass of of these fluorophores is relatively high (1.2-1.4  
> kDa) compared
> with the fluorophores which I am using right now, will it have  
> effect on the
> antibody penetration ?
>
> Kind regards,
>
> Michal Gdula
> PhD student
> Bradford University
> Uk

[Michal Gdula-2]

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Thank you Julio for the answer,

I will probably use Alexa 680 as a best compromise between distance from
optimum of excitation ( my laser has 633 nm) and the distance from Cy5.
I am searching also on the other end of the spectrum - I can give up DAPI and
use Alexa 350, however my experience so far is that it gives weak signals. I am
restricted again with argon UV laser 360 nm (so unfortunatelly I can not use
DEAC)- any advice fro strong dye which would fit, or on succesful use of ALexa
350 for imaging of FISH signals with confocal microscope would be appreciated.

Michal

[Michal Gdula-2]

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Thank you George for such a comprehensive answer,

I have already tried spectral unmixing already and I got dissapointed.
Correct me if I am wrong but it works nicely only if you have objects stained
with overlapping dyes labelled with similar strength.
I tried to unmix Alexa 514 from Alexa 488 and Cy3. Alexa 514 was labelling
nucleoli and the staining was strong and visible in red and green channel. And
two other dyes labelled other structure which did not give such a strong signal
mostly because of their structure and quantity. I was not able to resolve these
dyes.
I am aware of the literarture on multicolor FISH with epifluorescent microscope,
however as far as I know many of dyes used there don't work well with
confocal microscope.

I am also considering the fluorophores used in the paper of Tsurui et al,
however its worth to mention that he used epifluorescent microscope for
imaging combined with spectral imager. I am limited to my LSM510 meta.

The Alexa 430 looks interesting - its emission seem to overlap with AF488 but
should not be excited almost at all with 488 nm laser ( at least according to
olimpus website
(http://www.olympusfluoview.com/java/dualprobes/index.html).

I am searching also for some blue fluorophore which would be bright, excited
efficiently with 364 nm laser and possibility to conjugate with dUTP.

Thank you one more time for your advice,

Michal

[Jacqueline Ross]

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Hi Michal,

Alexa 350 is quite a weak fluorophore, however we do have people here
using it successfully when used in high concentration (e.g. 1/50).  They
are using it for standard immunohistochemistry rather than FISH. You do
need to take extra care with regard to photobleaching however. For
example, don't spend a lot of time viewing it by eye down the microscope
prior to confocal imaging.

I think Mike Ignatius/Invitrogen has previously advised that Pacific
Blue is a better choice than Alexa 350 and if you check out the archives
you will probably find his email regarding the hierarchy of their UV
excitable fluorophores.

Kind regards,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: [phone] Ext 87438
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Michal Gdula
Sent: Monday, 21 February 2011 8:04 a.m.
To: [hidden email]
Subject: Re: Alexa 680 and 700 and Zeiss LSM510meta

*****
To join, leave or search the confocal microscopy listserv, go to:
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Thank you Julio for the answer,

I will probably use Alexa 680 as a best compromise between distance from

optimum of excitation ( my laser has 633 nm) and the distance from Cy5.
I am searching also on the other end of the spectrum - I can give up
DAPI and
use Alexa 350, however my experience so far is that it gives weak
signals. I am
restricted again with argon UV laser 360 nm (so unfortunatelly I can not
use
DEAC)- any advice fro strong dye which would fit, or on succesful use of
ALexa
350 for imaging of FISH signals with confocal microscope would be
appreciated.

Michal

[Ignatius, Mike-2]

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Hi Michael,

We have two other dyes that would work with that 360nm argon laser line, Marina Blue and Alexa Fluor 405.

Pac Blue recommended by Jacqui below, is not much for 360 ex (19%), preferring 405 ex much more.  And the archived statement on UV dye selection referred to won't have info comparing any of these to Alexa Fluor 350.  We didn't compare AF 350 to these others - sorry.  

While its name, AF405, implies otherwise, this dye absorbs both 360nm and 405nm lines quite well.  Nearly 60% for the 360 line. Try our spectraviewer to see the data for Alexa Fluor 405, as Olympus doesn't have this dye in their data base currently.

If you are viewing in PBS,  AF 405 is the better dye, while in Prolong Gold mounted samples, Marina is a bit better.

However your 360 laser, scan speeds, magnification, mountant choice and other imaging settings may well alter those predictions.  

All UV ex dyes fade very fast - except Qdots of course.  But no Qdot equivalent for this emission range.  

Obviously and especially for UV dyes, turn up detector gain, turn down the excitation power, dark adapt the eyes and focus /compose fast.  

Best of luck,

Mike Ignatius with help and data from Jason Kilgore

Molecular Probes, Life Technologies

PS. I changed the subject line for future archivists.  


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jacqui Ross
Sent: Sunday, February 20, 2011 2:44 PM
To: [hidden email]
Subject: Re: Alexa 680 and 700 and Zeiss LSM510meta

*****
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*****

Hi Michal,

Alexa 350 is quite a weak fluorophore, however we do have people here
using it successfully when used in high concentration (e.g. 1/50).  They
are using it for standard immunohistochemistry rather than FISH. You do
need to take extra care with regard to photobleaching however. For
example, don't spend a lot of time viewing it by eye down the microscope
prior to confocal imaging.

I think Mike Ignatius/Invitrogen has previously advised that Pacific
Blue is a better choice than Alexa 350 and if you check out the archives
you will probably find his email regarding the hierarchy of their UV
excitable fluorophores.

Kind regards,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: [phone] Ext 87438
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Michal Gdula
Sent: Monday, 21 February 2011 8:04 a.m.
To: [hidden email]
Subject: Re: Alexa 680 and 700 and Zeiss LSM510meta

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

Thank you Julio for the answer,

I will probably use Alexa 680 as a best compromise between distance from

optimum of excitation ( my laser has 633 nm) and the distance from Cy5.
I am searching also on the other end of the spectrum - I can give up
DAPI and
use Alexa 350, however my experience so far is that it gives weak
signals. I am
restricted again with argon UV laser 360 nm (so unfortunatelly I can not
use
DEAC)- any advice fro strong dye which would fit, or on succesful use of
ALexa
350 for imaging of FISH signals with confocal microscope would be
appreciated.

Michal