Nuclear Dyes at 405nm?

[Perry, Griffin M]

Dear Listserv,

 

I was wondering if anyone had better results with DAPI @ 405nm than I have.  It seems…off.  I know it is only getting excited @ 25%, but I know another group that gets good images at the same wavelength.  The staining has been uneven with DAPI, but with Topro-3 in the 638nm range, the staining has been beautiful with clearly defined nucleoli.  Does anybody use Hoechst 34580?    Are there any other alternatives to DAPI @ the 405nm range?

 

Thanks in advance,

 

-Griffin

[Rossi, Ann]

Griffin,

I have used Hoechst 34580 in live, freshly dissociated mouse skeletal muscle fibers (2.5ug/ml loaded 30 minutes at room temp). It was very bright with 405 laser excitation on the confocal.  Nuclear staining was beautiful.


-Ann Rossi
Postdoctoral Fellow
University of Chicago


-----Original Message-----
From: Confocal Microscopy List on behalf of Perry, Griffin M
Sent: Tue 6/23/2009 4:30 PM
To: [hidden email]
Subject: Nuclear Dyes at 405nm?
 
Dear Listserv,

 

I was wondering if anyone had better results with DAPI @ 405nm than I
have.  It seems...off.  I know it is only getting excited @ 25%, but I
know another group that gets good images at the same wavelength.  The
staining has been uneven with DAPI, but with Topro-3 in the 638nm range,
the staining has been beautiful with clearly defined nucleoli.  Does
anybody use Hoechst 34580?    Are there any other alternatives to DAPI @
the 405nm range?

 

Thanks in advance,

 

-Griffin

[Hong Zhou]

Please delist me, thanks a lot!
Hong

--------------------------------------------------
From: "Rossi, Ann" <[hidden email]>
Sent: Tuesday, June 23, 2009 2:32 PM
To: <[hidden email]>
Subject: Re: Nuclear Dyes at 405nm?

> Griffin,
>
> I have used Hoechst 34580 in live, freshly dissociated mouse skeletal
> muscle fibers (2.5ug/ml loaded 30 minutes at room temp). It was very
> bright with 405 laser excitation on the confocal.  Nuclear staining was
> beautiful.
>
>
> -Ann Rossi
> Postdoctoral Fellow
> University of Chicago
>
>
> -----Original Message-----
> From: Confocal Microscopy List on behalf of Perry, Griffin M
> Sent: Tue 6/23/2009 4:30 PM
> To: [hidden email]
> Subject: Nuclear Dyes at 405nm?
>
> Dear Listserv,
>
>
>
> I was wondering if anyone had better results with DAPI @ 405nm than I
> have.  It seems...off.  I know it is only getting excited @ 25%, but I
> know another group that gets good images at the same wavelength.  The
> staining has been uneven with DAPI, but with Topro-3 in the 638nm range,
> the staining has been beautiful with clearly defined nucleoli.  Does
> anybody use Hoechst 34580?    Are there any other alternatives to DAPI @
> the 405nm range?
>
>
>
> Thanks in advance,
>
>
>
> -Griffin
>

[David Basiji]

Griffin,

 

The 405 laser diodes can vary in wavelength from unit to unit and since you're exciting DAPI on the tail of it's excitation spectrum, this can give rise a big difference in efficiency from instrument to instrument. Per the Invitrogen spectrum viewer, the nominal excitation efficiency of DAPI at 405 is about 8%, but at 408 it's only 4% and at 402 it's about 11%. That's nearly a three-fold difference in effective excitation efficiency with just a +/-3 nm tolerance on laser wavelength. If feasible, you should check out a different 405 laser in your system.

 

-David

David Basiji, Ph.D.
President and CEO, Amnis Corporation
2505 Third Ave., Suite 210
Seattle, WA 98121
[phone] direct
[phone] mobile
[phone] fax
www.amnis.com

This email and any attachment contain information which is intended for the addressees only. If you have received this email in error, please notify the sender.

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Perry, Griffin M
Sent: Tuesday, June 23, 2009 1:31 PM
To: [hidden email]
Subject: Nuclear Dyes at 405nm?

Dear Listserv,

 

I was wondering if anyone had better results with DAPI @ 405nm than I have.  It seems…off.  I know it is only getting excited @ 25%, but I know another group that gets good images at the same wavelength.  The staining has been uneven with DAPI, but with Topro-3 in the 638nm range, the staining has been beautiful with clearly defined nucleoli.  Does anybody use Hoechst 34580?    Are there any other alternatives to DAPI @ the 405nm range?

 

Thanks in advance,

 

-Griffin

[Boswell, Carl A - (cboswell)]

Griffin,

 

We have excellent nuclear imaging with DAPI using our 405 laser, but it has not been without issues.  First off, I am curious as to the imaging system you are using, and has DAPI ever looked good on it?  I ask this because our Zeiss LSM 510 Meta has had troubles with 1) the AOM controlling the 405 input, 2) the electronics in the 405 system and 3) the laser itself.  Once each of these was dealt with (over the last 2 years), we returned to blissful DAPI imaging.  Currently all parts are holding up well and all users are quite happy with their very bright DAPI images.  

 

Aside from the above mentioned items, alignment is one of the first things to look at.

 

C

 

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

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Tuesday, June 23, 2009 1:30 PM

Subject: Nuclear Dyes at 405nm?

Dear Listserv,

 

I was wondering if anyone had better results with DAPI @ 405nm than I have.  It seems…off.  I know it is only getting excited @ 25%, but I know another group that gets good images at the same wavelength.  The staining has been uneven with DAPI, but with Topro-3 in the 638nm range, the staining has been beautiful with clearly defined nucleoli.  Does anybody use Hoechst 34580?    Are there any other alternatives to DAPI @ the 405nm range?

 

Thanks in advance,

 

-Griffin

[Keith Morris]

Hi Griffin,

 

Granted the DAPI excitation is at the tail of the excitation peak*** when using the 405nm laser line, but there’s a heck of a lot of labeled DNA in the nucleus to excite. The 405nm laser is essentially designed as a cheap replacement for an incredibly expensive water cooled uV laser [£50k+ including cooling system] and this 405nm line is still rarely used for anything other DAPI imaging. Hoechst is actually even worse than DAPI with the 405nm laser line, down from 11% to nearer 5% on the excitation peak tail.

 

In every case I have encountered where the 405nm line laser is consistently very poor at imaging it requires a long visit by the Zeiss or Leica engineer [for our German confocal systems]*. Fortunately our confocal systems are on maintenance contracts as generally an awful lot of DAPI associated hardware seems to been replaced during ‘repair’ [e.g. lasers, AOTF & other optics]. This assumes you have done the most a user can do, like align pinholes [for Zeiss 510s], etc… I always have a perfectly DAPI/FITC/TRITC labeled Invitrogen BPAE cell and kidney slice** to hand to either demonstrate to users that ‘the system is working perfectly and it’s their poor samples’ or, more rarely, that ‘we have a problem Huston’. The BPAE cells preps seem to store better than the kidney slice, where in the latter the fluorescence diffuses to ‘incorrect’ locations over time [it stays bright though]. These Invitrogen slides cost approx £80 and £130 respectively and last up to a year stored in the fridge/freezer.

 

As other threads have pointed out, DAPI labeling isn’t always perfectly straight-forward. I’d advise staining separately for DAPI and avoid the Likes of ‘Vectashield+DAPI’ to keep the background to the absolute minimum for top notch DAPI images- although use of the latter shouldn’t produce really naff ‘off’ DAPI images

 

Keith

 

*Assuming it was decent at installation – and with our Zeiss 510 and Leica SP2 confocals DAPI [and even Hoechst] imaging is excellent. We image at around 10% laser power [30mW 405nm laser].

 

**http://www.well.ox.ac.uk/cytogenetics/Zeiss.shtml see our Invitrogen slide images

***http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.reg.uk.html

 

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Perry, Griffin M
Sent: 23 June 2009 21:31
To: [hidden email]
Subject: Nuclear Dyes at 405nm?

 

Dear Listserv,

 

I was wondering if anyone had better results with DAPI @ 405nm than I have.  It seems…off.  I know it is only getting excited @ 25%, but I know another group that gets good images at the same wavelength.  The staining has been uneven with DAPI, but with Topro-3 in the 638nm range, the staining has been beautiful with clearly defined nucleoli.  Does anybody use Hoechst 34580?    Are there any other alternatives to DAPI @ the 405nm range?

 

Thanks in advance,

 

-Griffin

[Guy Cox]

Some manufacturers - including Nikon - supply VC (violet corrected) planapochromats designed to optimise the image from short wavelengths.  The improvement relative to a regular planapochromat is substantial.  If you are going to do a lot of 405nm work it's worth paying the (non trivial) cost of such an objective.

 

No commercial affiliation,

 

                                                    Guy Cox

 

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)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: Wednesday, 24 June 2009 7:18 PM
To: [hidden email]
Subject: Re: Nuclear Dyes at 405nm?

Hi Griffin,

 

Granted the DAPI excitation is at the tail of the excitation peak*** when using the 405nm laser line, but there’s a heck of a lot of labeled DNA in the nucleus to excite. The 405nm laser is essentially designed as a cheap replacement for an incredibly expensive water cooled uV laser [£50k+ including cooling system] and this 405nm line is still rarely used for anything other DAPI imaging. Hoechst is actually even worse than DAPI with the 405nm laser line, down from 11% to nearer 5% on the excitation peak tail.

 

In every case I have encountered where the 405nm line laser is consistently very poor at imaging it requires a long visit by the Zeiss or Leica engineer [for our German confocal systems]*. Fortunately our confocal systems are on maintenance contracts as generally an awful lot of DAPI associated hardware seems to been replaced during ‘repair’ [e.g. lasers, AOTF & other optics]. This assumes you have done the most a user can do, like align pinholes [for Zeiss 510s], etc… I always have a perfectly DAPI/FITC/TRITC labeled Invitrogen BPAE cell and kidney slice** to hand to either demonstrate to users that ‘the system is working perfectly and it’s their poor samples’ or, more rarely, that ‘we have a problem Huston’. The BPAE cells preps seem to store better than the kidney slice, where in the latter the fluorescence diffuses to ‘incorrect’ locations over time [it stays bright though]. These Invitrogen slides cost approx £80 and £130 respectively and last up to a year stored in the fridge/freezer.

 

As other threads have pointed out, DAPI labeling isn’t always perfectly straight-forward. I’d advise staining separately for DAPI and avoid the Likes of ‘Vectashield+DAPI’ to keep the background to the absolute minimum for top notch DAPI images- although use of the latter shouldn’t produce really naff ‘off’ DAPI images

 

Keith

 

*Assuming it was decent at installation – and with our Zeiss 510 and Leica SP2 confocals DAPI [and even Hoechst] imaging is excellent. We image at around 10% laser power [30mW 405nm laser].

 

**http://www.well.ox.ac.uk/cytogenetics/Zeiss.shtml see our Invitrogen slide images

***http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.reg.uk.html

 

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Perry, Griffin M
Sent: 23 June 2009 21:31
To: [hidden email]
Subject: Nuclear Dyes at 405nm?

 

Dear Listserv,

 

I was wondering if anyone had better results with DAPI @ 405nm than I have.  It seems…off.  I know it is only getting excited @ 25%, but I know another group that gets good images at the same wavelength.  The staining has been uneven with DAPI, but with Topro-3 in the 638nm range, the staining has been beautiful with clearly defined nucleoli.  Does anybody use Hoechst 34580?    Are there any other alternatives to DAPI @ the 405nm range?

 

Thanks in advance,

 

-Griffin

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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Hong Zhou
Sent: Tuesday, June 23, 2009 5:05 PM
To: [hidden email]
Subject: Re: Nuclear Dyes at 405nm?

Please delist me, thanks a lot!
Hong

--------------------------------------------------
From: "Rossi, Ann" <[hidden email]>
Sent: Tuesday, June 23, 2009 2:32 PM
To: <[hidden email]>
Subject: Re: Nuclear Dyes at 405nm?

> Griffin,
>
> I have used Hoechst 34580 in live, freshly dissociated mouse skeletal
> muscle fibers (2.5ug/ml loaded 30 minutes at room temp). It was very
> bright with 405 laser excitation on the confocal.  Nuclear staining was
> beautiful.
>
>
> -Ann Rossi
> Postdoctoral Fellow
> University of Chicago
>
>
> -----Original Message-----
> From: Confocal Microscopy List on behalf of Perry, Griffin M
> Sent: Tue 6/23/2009 4:30 PM
> To: [hidden email]
> Subject: Nuclear Dyes at 405nm?
>
> Dear Listserv,
>
>
>
> I was wondering if anyone had better results with DAPI @ 405nm than I
> have.  It seems...off.  I know it is only getting excited @ 25%, but I
> know another group that gets good images at the same wavelength.  The
> staining has been uneven with DAPI, but with Topro-3 in the 638nm range,
> the staining has been beautiful with clearly defined nucleoli.  Does
> anybody use Hoechst 34580?    Are there any other alternatives to DAPI @
> the 405nm range?
>
>
>
> Thanks in advance,
>
>
>
> -Griffin
>

[Perry, Griffin M]

Thanks for all the feedback from the listserv.  I have been using Vectashield+DAPI, and my next step was to use DAPI in the secondary AB procedures.  We have beautiful labeling with other nuclear markers in other channels.  I have checked the alignment, but I’m writing a How-To for the protocol binder before I leave, so I’ll do it again today.

 

Thanks for all the replies,

 

-Griffin Perry

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: Wednesday, June 24, 2009 4:18 AM
To: [hidden email]
Subject: Re: Nuclear Dyes at 405nm?

 

Hi Griffin,

 

Granted the DAPI excitation is at the tail of the excitation peak*** when using the 405nm laser line, but there’s a heck of a lot of labeled DNA in the nucleus to excite. The 405nm laser is essentially designed as a cheap replacement for an incredibly expensive water cooled uV laser [£50k+ including cooling system] and this 405nm line is still rarely used for anything other DAPI imaging. Hoechst is actually even worse than DAPI with the 405nm laser line, down from 11% to nearer 5% on the excitation peak tail.

 

In every case I have encountered where the 405nm line laser is consistently very poor at imaging it requires a long visit by the Zeiss or Leica engineer [for our German confocal systems]*. Fortunately our confocal systems are on maintenance contracts as generally an awful lot of DAPI associated hardware seems to been replaced during ‘repair’ [e.g. lasers, AOTF & other optics]. This assumes you have done the most a user can do, like align pinholes [for Zeiss 510s], etc… I always have a perfectly DAPI/FITC/TRITC labeled Invitrogen BPAE cell and kidney slice** to hand to either demonstrate to users that ‘the system is working perfectly and it’s their poor samples’ or, more rarely, that ‘we have a problem Huston’. The BPAE cells preps seem to store better than the kidney slice, where in the latter the fluorescence diffuses to ‘incorrect’ locations over time [it stays bright though]. These Invitrogen slides cost approx £80 and £130 respectively and last up to a year stored in the fridge/freezer.

 

As other threads have pointed out, DAPI labeling isn’t always perfectly straight-forward. I’d advise staining separately for DAPI and avoid the Likes of ‘Vectashield+DAPI’ to keep the background to the absolute minimum for top notch DAPI images- although use of the latter shouldn’t produce really naff ‘off’ DAPI images

 

Keith

 

*Assuming it was decent at installation – and with our Zeiss 510 and Leica SP2 confocals DAPI [and even Hoechst] imaging is excellent. We image at around 10% laser power [30mW 405nm laser].

 

**http://www.well.ox.ac.uk/cytogenetics/Zeiss.shtml see our Invitrogen slide images

***http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.reg.uk.html

 

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Perry, Griffin M
Sent: 23 June 2009 21:31
To: [hidden email]
Subject: Nuclear Dyes at 405nm?

 

Dear Listserv,

 

I was wondering if anyone had better results with DAPI @ 405nm than I have.  It seems…off.  I know it is only getting excited @ 25%, but I know another group that gets good images at the same wavelength.  The staining has been uneven with DAPI, but with Topro-3 in the 638nm range, the staining has been beautiful with clearly defined nucleoli.  Does anybody use Hoechst 34580?    Are there any other alternatives to DAPI @ the 405nm range?

 

Thanks in advance,

 

-Griffin

[Monique Vasseur]

Hello,

 

Also check if your objective lets pass UV or not.  Some objective don’t.

 

Monique Vasseur

Microscopie et imagerie

Département de biochimie

Université de Montréal

C.P. 6128, succursale Centre-ville

Montréal QC    H3C 3J7   Canada

tél. [phone] poste 5148

De : Confocal Microscopy List [mailto:[hidden email]] De la part de Carl Boswell
Envoyé : 23 juin 2009 19:00
À : [hidden email]
Objet : Re: Nuclear Dyes at 405nm?

 

Griffin,

 

We have excellent nuclear imaging with DAPI using our 405 laser, but it has not been without issues.  First off, I am curious as to the imaging system you are using, and has DAPI ever looked good on it?  I ask this because our Zeiss LSM 510 Meta has had troubles with 1) the AOM controlling the 405 input, 2) the electronics in the 405 system and 3) the laser itself.  Once each of these was dealt with (over the last 2 years), we returned to blissful DAPI imaging.  Currently all parts are holding up well and all users are quite happy with their very bright DAPI images.  

 

Aside from the above mentioned items, alignment is one of the first things to look at.

 

C

 

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

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Tuesday, June 23, 2009 1:30 PM

Subject: Nuclear Dyes at 405nm?

 

Dear Listserv,

 

I was wondering if anyone had better results with DAPI @ 405nm than I have.  It seems…off.  I know it is only getting excited @ 25%, but I know another group that gets good images at the same wavelength.  The staining has been uneven with DAPI, but with Topro-3 in the 638nm range, the staining has been beautiful with clearly defined nucleoli.  Does anybody use Hoechst 34580?    Are there any other alternatives to DAPI @ the 405nm range?

 

Thanks in advance,

 

-Griffin