Acceptof photobleaching FRET problems

[Michal Jarnik]

List,

I keep having problem with acceptor photobleaching of a CFP-YFP labeled protein. I am working with transiently transfected cells (the expression is not too uniform, but I am still able to find cells with pretty similar fluorescence) and the FRET is very high (around 30%) in supposedly FRETting and non-FRETting samples AND in the CFP only labeled cells. The cells were fixed with paraformaldehyde and mounted in glycerol. I am using 443 nm and 514 nm laser lines. Looks like "dark FRET" to me, but my experience in the technique is limited. Any hints what I am doing wrong and mainly, what can be done?

Thanks, Michael

[Janos Roszik]

I think first you should make sure that the error is not in the calculations.

Best regards,
Janos

[David Stanek-3]

We occasionally observe artificial increase when the stage is not stable and moves (mainly in z). But it's usually ~5-10% not 30%.  If you want you can contact me directly and I can send you samples that we use as positive and negative controls.

David

David Stanek, PhD.

Department of RNA biology

Institute of Molecular Genetics AS CR

Videnska 1083

142 20 Prague 4

Czech Republic

Tel.: [phone]

Fax: [phone]

email: [hidden email]

web: www.img.cas.cz

On Apr 27, 2009, at 11:20 AM, Jarnik, Michal wrote:

List,

I keep having problem with acceptor photobleaching of a CFP-YFP labeled protein. I am working with transiently transfected cells (the expression is not too uniform, but I am still able to find cells with pretty similar fluorescence) and the FRET is very high (around 30%) in supposedly FRETting and non-FRETting samples AND in the CFP only labeled cells. The cells were fixed with paraformaldehyde and mounted in glycerol. I am using 443 nm and 514 nm laser lines. Looks like "dark FRET" to me, but my experience in the technique is limited. Any hints what I am doing wrong and mainly, what can be done?

Thanks, Michael

[Miller, Jason]

Hi Michael-

 

Believe it or not, this exact problem has been posed to the confocal microscopy list several times in the past few years. I also experienced this exact same problem and found some comfort in the fact that others had noticed this phenomenon as well. Specifically, when applying acceptor photobleach to CFP only samples with a 514 laser line, I was seeing a post-bleach increase in CFP that was quite significant, especially compared to areas far-away from my bleach ROI. Among the possibilities discussed were photoconversion of CFP to a brighter variant and the elimination of dark acceptors. My gut feeling was that neither of these were the cause, but I honestly couldn't tell you what was causing the phenomenon. A few observations I made during this annoying period (before jettisoning acceptor photobleach altogether in favor of sensitized emission analysis):

 

1) The increase in CFP signal seemed to bleed slightly outside the actual bleach ROI. This made me strongly wonder whether the phenomenon I was seeing was an effect of thermal diffusion - i.e. diffusion of heat from the bleach ROI causing some sort of non-denaturing conversion of the surrounding CFP to a brighter variant.

 

2) I think (although I can't confirm because the experiments were a long time ago) that I also saw this phenomenon with GFP, although maybe to a lesser degree. It was because I saw the phenomenon with GFP (and therefore, my bleach laser was a much higher wavelength) that I felt dark acceptors was a less likely cause - unless there are dark acceptors in neurons with extraordinarily broad absorption spectra.

 

Anyhow, I never solved the problem and just ended up switching to sensitized emission analysis - which, I have to admit, is more annoying to do and inherently less accurate than properly performed acceptor photobleach. But my application was non-quantitiative, so I didn't need to get absolute FRET efficiency values.

 

Best-

Jason Miller

Gladstone Institute of Neurological Disease

University of California - San Francisco

 

 

-------------------

Medical Scientist Training Program (MD/PhD)

University of California- San Francisco

 

Home Address:

1434 Lakeshore Ave., Apt #8

Oakland, CA 94606

Home: [phone]

Cell: [phone]

E-mail: [hidden email]

 

A Few of Dave Barry's Pearls for Living:

1. Never, under any circumstances, take a sleeping pill and a laxative on the same night.
2. If you had to identify, in one word, the reason why the human race has not achieved and never will achieve its full potential, that word would be “meetings.”
3. There is a very fine line between “hobby” and “mental illness.”  
4. The one thing that unites all human beings, regardless of age, gender, religion, economic status or ethnic background, is that, deep down inside, we ALL believe that we are above average drivers.
5. Never be afraid to try something new. Remember that a lone amateur built the Ark. A large group of professionals built the Titanic.
6. Men are like fine wine. They start out as grapes, and it’s up to the women to stomp the crap out of them until they turn into something acceptable to have dinner with.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Stanek
Sent: Monday, April 27, 2009 4:06 AM
To: [hidden email]
Subject: Re: Acceptof photobleaching FRET problems

We occasionally observe artificial increase when the stage is not stable and moves (mainly in z). But it's usually ~5-10% not 30%.  If you want you can contact me directly and I can send you samples that we use as positive and negative controls.

David

David Stanek, PhD.

Department of RNA biology

Institute of Molecular Genetics AS CR

Videnska 1083

142 20 Prague 4

Czech Republic

Tel.: [phone]

Fax: [phone]

email: [hidden email]

web: www.img.cas.cz

On Apr 27, 2009, at 11:20 AM, Jarnik, Michal wrote:

List,

I keep having problem with acceptor photobleaching of a CFP-YFP labeled protein. I am working with transiently transfected cells (the expression is not too uniform, but I am still able to find cells with pretty similar fluorescence) and the FRET is very high (around 30%) in supposedly FRETting and non-FRETting samples AND in the CFP only labeled cells. The cells were fixed with paraformaldehyde and mounted in glycerol. I am using 443 nm and 514 nm laser lines. Looks like "dark FRET" to me, but my experience in the technique is limited. Any hints what I am doing wrong and mainly, what can be done?

Thanks, Michael

[Zoltan]

Hi Michael,

 

We talked about this a short while ago on this list. We also use 514 nm for yfp and 405 or 458 for cfp and we are aware of the potential artifact that comes from bleaching ypf and then exciting cfp with 405:

 

http://www.ncbi.nlm.nih.gov/pubmed/17481360?ordinalpos=11&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

 

We do get a very strong (artificial) signal in the donor channel when using 405 nm even in  a control prep where there is no donor at all!  It seems to be a conversion of the yfp molecules to a form that can be easily excited by 405, but not as much by 458. There was a paper about this around 2000 in Nature, I think. Some authors say they don't see this conversion, but we do.  Our recipe now is to excite cfp with 458, and we also mostly do sens. emission now..

  I hope this helps,

 

Zoltan

On Mon, Apr 27, 2009 at 8:14 PM, Miller, Jason <[hidden email]> wrote:

Hi Michael-

 

Believe it or not, this exact problem has been posed to the confocal microscopy list several times in the past few years. I also experienced this exact same problem and found some comfort in the fact that others had noticed this phenomenon as well. Specifically, when applying acceptor photobleach to CFP only samples with a 514 laser line, I was seeing a post-bleach increase in CFP that was quite significant, especially compared to areas far-away from my bleach ROI. Among the possibilities discussed were photoconversion of CFP to a brighter variant and the elimination of dark acceptors. My gut feeling was that neither of these were the cause, but I honestly couldn't tell you what was causing the phenomenon. A few observations I made during this annoying period (before jettisoning acceptor photobleach altogether in favor of sensitized emission analysis):

 

1) The increase in CFP signal seemed to bleed slightly outside the actual bleach ROI. This made me strongly wonder whether the phenomenon I was seeing was an effect of thermal diffusion - i.e. diffusion of heat from the bleach ROI causing some sort of non-denaturing conversion of the surrounding CFP to a brighter variant.

 

2) I think (although I can't confirm because the experiments were a long time ago) that I also saw this phenomenon with GFP, although maybe to a lesser degree. It was because I saw the phenomenon with GFP (and therefore, my bleach laser was a much higher wavelength) that I felt dark acceptors was a less likely cause - unless there are dark acceptors in neurons with extraordinarily broad absorption spectra.

 

Anyhow, I never solved the problem and just ended up switching to sensitized emission analysis - which, I have to admit, is more annoying to do and inherently less accurate than properly performed acceptor photobleach. But my application was non-quantitiative, so I didn't need to get absolute FRET efficiency values.

 

Best-

Jason Miller

Gladstone Institute of Neurological Disease

University of California - San Francisco

 

 

-------------------

Medical Scientist Training Program (MD/PhD)

University of California- San Francisco

 

Home Address:

1434 Lakeshore Ave., Apt #8

Oakland, CA 94606

Home: [phone]

Cell: [phone]

E-mail: [hidden email]

 

A Few of Dave Barry's Pearls for Living:

1. Never, under any circumstances, take a sleeping pill and a laxative on the same night.
2. If you had to identify, in one word, the reason why the human race has not achieved and never will achieve its full potential, that word would be “meetings.”
3. There is a very fine line between “hobby” and “mental illness.”  
4. The one thing that unites all human beings, regardless of age, gender, religion, economic status or ethnic background, is that, deep down inside, we ALL believe that we are above average drivers.
5. Never be afraid to try something new. Remember that a lone amateur built the Ark. A large group of professionals built the Titanic.
6. Men are like fine wine. They start out as grapes, and it’s up to the women to stomp the crap out of them until they turn into something acceptable to have dinner with.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Stanek
Sent: Monday, April 27, 2009 4:06 AM
To: [hidden email]
Subject: Re: Acceptof photobleaching FRET problems

We occasionally observe artificial increase when the stage is not stable and moves (mainly in z). But it's usually ~5-10% not 30%.  If you want you can contact me directly and I can send you samples that we use as positive and negative controls.

David

David Stanek, PhD.

Department of RNA biology

Institute of Molecular Genetics AS CR

Videnska 1083

142 20 Prague 4

Czech Republic

Tel.: [phone]

Fax: [phone]

email: [hidden email]

web: www.img.cas.cz

On Apr 27, 2009, at 11:20 AM, Jarnik, Michal wrote:

List,

I keep having problem with acceptor photobleaching of a CFP-YFP labeled protein. I am working with transiently transfected cells (the expression is not too uniform, but I am still able to find cells with pretty similar fluorescence) and the FRET is very high (around 30%) in supposedly FRETting and non-FRETting samples AND in the CFP only labeled cells. The cells were fixed with paraformaldehyde and mounted in glycerol. I am using 443 nm and 514 nm laser lines. Looks like "dark FRET" to me, but my experience in the technique is limited. Any hints what I am doing wrong and mainly, what can be done?

Thanks, Michael

--

Zoltan Cseresnyes
Facility manager, Imaging Suite

[Jeremy Adler-2]

Will not fixation alter the relative positions of the fluorophores ?

 

 

 

Dr Jeremy Adler

F451a

Cell Biologi

Wenner-Gren Inst.

The Arhenius Lab

Stockholm University

S-106 91 Stockholm

Sweden

tel [phone]

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jarnik, Michal
Sent: den 27 april 2009 10:21
To: [hidden email]
Subject: Acceptof photobleaching FRET problems

 

 

List,

I keep having problem with acceptor photobleaching of a CFP-YFP labeled protein. I am working with transiently transfected cells (the expression is not too uniform, but I am still able to find cells with pretty similar fluorescence) and the FRET is very high (around 30%) in supposedly FRETting and non-FRETting samples AND in the CFP only labeled cells. The cells were fixed with paraformaldehyde and mounted in glycerol. I am using 443 nm and 514 nm laser lines. Looks like "dark FRET" to me, but my experience in the technique is limited. Any hints what I am doing wrong and mainly, what can be done?

Thanks, Michael

[simona rodighiero]

I experienced the same problem in PFA fixed samples. Pay attention to the sealing agent: it would seem trivial, but avoid nail-polish!

Even if someone reported this "photoconversion" in live samples, I was not able to measure it in living cells (458nm cfp excitation). Moreover, without fixing, I measure the highest FRET signal of my positive control (CFP-link-YFP). For these reasons I usually work with living samples.

 

A different problem is the the fluorescence emission increase in the proximity of the photobleached area. This fenomenon seems energy dependent (becomes more evident increasing the bleaching power). I observed it in albumin-alexa 647 films on PBS bathed coverglasses (633nm bleaching) as well as in Corvallaria (514nm bleaching). Someone can explain this?

 

thank's

simona

 

 

 

2009/4/27 Jarnik, Michal <[hidden email]>

List,

I keep having problem with acceptor photobleaching of a CFP-YFP labeled protein. I am working with transiently transfected cells (the expression is not too uniform, but I am still able to find cells with pretty similar fluorescence) and the FRET is very high (around 30%) in supposedly FRETting and non-FRETting samples AND in the CFP only labeled cells. The cells were fixed with paraformaldehyde and mounted in glycerol. I am using 443 nm and 514 nm laser lines. Looks like "dark FRET" to me, but my experience in the technique is limited. Any hints what I am doing wrong and mainly, what can be done?

Thanks, Michael

--
Dr. Simona Rodighiero, Ph.D.

Fondazione Filarete
viale Ortles 22/24
Milano
tel. [phone]

CIMAINA
Università degli Studi di Milano
via Celoria, 26
20133 Milano

tel. [phone]/14876

[Jerry Sedgewick-2]

There are still some slots available in this year's Basic Confocal Workshop hosted by the University of South Carolina. This year's workshop will be from June 15-19, 2009 and will include a series of lectures on the theory and applications of confocal microscopy, specimen preparation, processing confocal images in Photoshop, and 3D reconstructions using AMIRA. Students will be able to process triple labeled samples (cell cultures and sections) on site or bring their own samples to the workshop.

Faculty will include Drs. Jay Jerome (Vanderbilt), Ralph Albrecht (Univ Wisconsin-Madison), John Mackenzie (North Carolina State Univ), Tom TrusK (Medical Univ South Carolina) and myself.

Instruments and applications experts from Leica, Nikon, Olympus, Perkin Elmer, Photometrics, and Zeiss will be available for hands on training and imaging of samples.

For those contemplating instrumentation proposals as part of the stimulus or other funding opportunities this is an excellent opportunity to see several systems side by side and to collect preliminary data on their instrument of choice.

For further information and registration go to:
http://dba.med.sc.edu/irf/price/irf/irf.htm or contact Anna McNeal
([hidden email] <mailto:[hidden email]>)

Bob

Bob Price

Research Professor

Dept Cell Biol and Anat

USC School of Medicine

6439 Garner's Ferry Road

Columbia, SC 29208

Tel: [phone]

Admin Tel: [phone]

Fax: [phone]



--
Jerry (Gerald) Sedgewick
Program Director, Biomedical Image Processing Lab (BIPL)
Department of Neuroscience, University of Minnesota
312 Church St. SE, 1-205 Hasselmo Hall
Minneapolis, MN  55455
[phone]
[hidden email]
http://www.bipl.umn.edu
Author: "Scientific Imaging with Photoshop: Methods, Measurement and Output."

Rawlight.com (dba Sedgewick Initiatives)
965 Cromwell Avenue
Saint Paul, MN  55114
[hidden email]
[phone]
http://www.quickphotoshop.com
http://www.heartFROMstone.com
http://www.rawlight.com




--- Get FREE High Speed Internet from USFamily.Net! -- http://www.usfamily.net/mkt-freepromo.html ---

[Cameron Nowell]

Hi List,

I think this has been discussed in the past but i have not been able to
find a definitive answer to the problem.

Basically if you have a sample stained with DAPI, after viewing it with
a DAPI filter, the signal can be then detected using a GFP/FITC filter.

I have tried this on samples with nothing but DAPI on them and it still
happens. Suggestions in the past have been to lower the concentration of
DAPI used and to scan/capture the other channels first and DAPI last.

But does anyone out there have any idea why this happens in the first
place? If you look at the spectra of DAPI it is a very good green
emitter (but needs to be excited in the UV-Blue range). It should not be
able to be excited by standard GFP type excitation at 480-490nm. So the
conversion that is happening is shifting the excitation spectra of DAPI
towards the red somehow.

Any ideas?


Cheers

Cam



Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: [phone]
Mobile: [phone]
Fax: [phone]
Facility Website



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Jerry Sedgewick
Sent: Tuesday, 5 May 2009 1:35 AM
To: [hidden email]
Subject: Re: Posting for Basic Confocal Workshop at U of South Carolina

There are still some slots available in this year's Basic Confocal
Workshop hosted by the University of South Carolina. This year's
workshop will be from June 15-19, 2009 and will include a series of
lectures on the theory and applications of confocal microscopy, specimen
preparation, processing confocal images in Photoshop, and 3D
reconstructions using AMIRA. Students will be able to process triple
labeled samples (cell cultures and sections) on site or bring their own
samples to the workshop.

Faculty will include Drs. Jay Jerome (Vanderbilt), Ralph Albrecht (Univ
Wisconsin-Madison), John Mackenzie (North Carolina State Univ), Tom
TrusK (Medical Univ South Carolina) and myself.

Instruments and applications experts from Leica, Nikon, Olympus, Perkin
Elmer, Photometrics, and Zeiss will be available for hands on training
and imaging of samples.

For those contemplating instrumentation proposals as part of the
stimulus or other funding opportunities this is an excellent opportunity
to see several systems side by side and to collect preliminary data on
their instrument of choice.

For further information and registration go to:
http://dba.med.sc.edu/irf/price/irf/irf.htm or contact Anna McNeal
([hidden email] <mailto:[hidden email]>)

Bob

Bob Price

Research Professor

Dept Cell Biol and Anat

USC School of Medicine

6439 Garner's Ferry Road

Columbia, SC 29208

Tel: [phone]

Admin Tel: [phone]

Fax: [phone]



--
Jerry (Gerald) Sedgewick
Program Director, Biomedical Image Processing Lab (BIPL)
Department of Neuroscience, University of Minnesota
312 Church St. SE, 1-205 Hasselmo Hall
Minneapolis, MN  55455
[phone]
[hidden email]
http://www.bipl.umn.edu
Author: "Scientific Imaging with Photoshop: Methods, Measurement and
Output."

Rawlight.com (dba Sedgewick Initiatives)
965 Cromwell Avenue
Saint Paul, MN  55114
[hidden email]
[phone]
http://www.quickphotoshop.com
http://www.heartFROMstone.com
http://www.rawlight.com




--- Get FREE High Speed Internet from USFamily.Net! --
http://www.usfamily.net/mkt-freepromo.html ---

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This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waiver any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

[Eric Scarfone]

Hi Cameron,

I suppose you looked at the obvious, ie the sharpness of your GFP excitation filters??

Eric

Eric Scarfone, PhD, CNRS,
Center for Hearing and communication Research
Department of Clinical Neuroscience
Karolinska Institutet

Postal Address:
CFH, M1:02
Karolinska Hospital,
SE-171 76 Stockholm, Sweden

Work: [phone],
Cell: [phone]
Fax: [phone]

email: [hidden email]
http://www.ki.se/cfh/


----- Original Message -----
From: Cameron Nowell <[hidden email]>
Date: Tuesday, May 5, 2009 2:40 am
Subject: Photoactivation/Conversion of DAPI
To: [hidden email]

> Hi List,
>
> I think this has been discussed in the past but i have not been
> able to
> find a definitive answer to the problem.
>
> Basically if you have a sample stained with DAPI, after viewing it
> witha DAPI filter, the signal can be then detected using a
> GFP/FITC filter.
>

> I have tried this on samples with nothing but DAPI on them and it
> stillhappens. Suggestions in the past have been to lower the
> concentration of
> DAPI used and to scan/capture the other channels first and DAPI last.
>
> But does anyone out there have any idea why this happens in the first
> place? If you look at the spectra of DAPI it is a very good green
> emitter (but needs to be excited in the UV-Blue range). It should
> not be
> able to be excited by standard GFP type excitation at 480-490nm.
> So the
> conversion that is happening is shifting the excitation spectra of
> DAPItowards the red somehow.
>
> Any ideas?
>
>
> Cheers
>
> Cam
>
>
>
> Cameron J. Nowell
> Microscopy Manager
> Centre for Advanced Microscopy
> Ludwig Institute for Cancer Research

> PO Box 2008

> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA
> Office: [phone]
> Mobile: [phone]
> Fax: [phone]
> Facility Website
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]]On Behalf Of Jerry Sedgewick
> Sent: Tuesday, 5 May 2009 1:35 AM
> To: [hidden email]
> Subject: Re: Posting for Basic Confocal Workshop at U of South
> Carolina
> There are still some slots available in this year's Basic Confocal
> Workshop hosted by the University of South Carolina. This year's
> workshop will be from June 15-19, 2009 and will include a series of
> lectures on the theory and applications of confocal microscopy,
> specimenpreparation, processing confocal images in Photoshop, and 3D

> reconstructions using AMIRA. Students will be able to process triple

> labeled samples (cell cultures and sections) on site or bring
> their own
> samples to the workshop.
>
> Faculty will include Drs. Jay Jerome (Vanderbilt), Ralph Albrecht
> (UnivWisconsin-Madison), John Mackenzie (North Carolina State
> Univ), Tom
> TrusK (Medical Univ South Carolina) and myself.
>
> Instruments and applications experts from Leica, Nikon, Olympus,
> PerkinElmer, Photometrics, and Zeiss will be available for hands
> on training
> and imaging of samples.
>
> For those contemplating instrumentation proposals as part of the
> stimulus or other funding opportunities this is an excellent
> opportunityto see several systems side by side and to collect
> preliminary data on
> their instrument of choice.
>

> For further information and registration go to:

> http://dba.med.sc.edu/irf/price/irf/irf.htm or contact Anna McNeal
> ([hidden email] <mailto:[hidden email]>)
>
> Bob
>
> Bob Price
>
> Research Professor
>
> Dept Cell Biol and Anat
>
> USC School of Medicine
>
> 6439 Garner's Ferry Road
>
> Columbia, SC 29208
>
> Tel: [phone]
>
> Admin Tel: [phone]
>
> Fax: [phone]
>
>
>
> --
> Jerry (Gerald) Sedgewick
> Program Director, Biomedical Image Processing Lab (BIPL)
> Department of Neuroscience, University of Minnesota
> 312 Church St. SE, 1-205 Hasselmo Hall
> Minneapolis, MN 55455
> [phone]
> [hidden email]
> http://www.bipl.umn.edu

> Author: "Scientific Imaging with Photoshop: Methods, Measurement and

> Output."
>
> Rawlight.com (dba Sedgewick Initiatives)
> 965 Cromwell Avenue
> Saint Paul, MN 55114
> [hidden email]
> [phone]
> http://www.quickphotoshop.com
> http://www.heartFROMstone.com
> http://www.rawlight.com
>
>
>
>
> --- Get FREE High Speed Internet from USFamily.Net! --
> http://www.usfamily.net/mkt-freepromo.html ---
>
> No virus found in this incoming message.
> Checked by AVG - www.avg.com
> Version: 8.5.320 / Virus Database: 270.12.15/2093 - Release Date:
> 05/04/09 17:51:00
>
>
> This communication is intended only for the named recipient and
> may contain information that is confidential, legally privileged
> or subject to copyright; the Ludwig Institute for Cancer Research

> Ltd does not waiver any rights if you have received this
> communication in error.
> The views expressed in this communication are those of the sender
> and do not necessarily reflect the views of the Ludwig Institute
> for Cancer Research Ltd.
>
>

[mmodel]

Have you tried to view the slide on another microscope?

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Cameron Nowell [[hidden email]]
Sent: Monday, May 04, 2009 8:40 PM
To: [hidden email]
Subject: Photoactivation/Conversion of DAPI

Hi List,

I think this has been discussed in the past but i have not been able to
find a definitive answer to the problem.

Basically if you have a sample stained with DAPI, after viewing it with
a DAPI filter, the signal can be then detected using a GFP/FITC filter.

I have tried this on samples with nothing but DAPI on them and it still
happens. Suggestions in the past have been to lower the concentration of
DAPI used and to scan/capture the other channels first and DAPI last.

But does anyone out there have any idea why this happens in the first
place? If you look at the spectra of DAPI it is a very good green
emitter (but needs to be excited in the UV-Blue range). It should not be
able to be excited by standard GFP type excitation at 480-490nm. So the
conversion that is happening is shifting the excitation spectra of DAPI
towards the red somehow.

Any ideas?


Cheers

Cam



Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: [phone]
Mobile: [phone]
Fax: [phone]
Facility Website



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Jerry Sedgewick
Sent: Tuesday, 5 May 2009 1:35 AM
To: [hidden email]
Subject: Re: Posting for Basic Confocal Workshop at U of South Carolina

There are still some slots available in this year's Basic Confocal
Workshop hosted by the University of South Carolina. This year's
workshop will be from June 15-19, 2009 and will include a series of
lectures on the theory and applications of confocal microscopy, specimen
preparation, processing confocal images in Photoshop, and 3D
reconstructions using AMIRA. Students will be able to process triple
labeled samples (cell cultures and sections) on site or bring their own
samples to the workshop.

Faculty will include Drs. Jay Jerome (Vanderbilt), Ralph Albrecht (Univ
Wisconsin-Madison), John Mackenzie (North Carolina State Univ), Tom
TrusK (Medical Univ South Carolina) and myself.

Instruments and applications experts from Leica, Nikon, Olympus, Perkin
Elmer, Photometrics, and Zeiss will be available for hands on training
and imaging of samples.

For those contemplating instrumentation proposals as part of the
stimulus or other funding opportunities this is an excellent opportunity
to see several systems side by side and to collect preliminary data on
their instrument of choice.

For further information and registration go to:
http://dba.med.sc.edu/irf/price/irf/irf.htm or contact Anna McNeal
([hidden email] <mailto:[hidden email]>)

Bob

Bob Price

Research Professor

Dept Cell Biol and Anat

USC School of Medicine

6439 Garner's Ferry Road

Columbia, SC 29208

Tel: [phone]

Admin Tel: [phone]

Fax: [phone]



--
Jerry (Gerald) Sedgewick
Program Director, Biomedical Image Processing Lab (BIPL)
Department of Neuroscience, University of Minnesota
312 Church St. SE, 1-205 Hasselmo Hall
Minneapolis, MN  55455
[phone]
[hidden email]
http://www.bipl.umn.edu
Author: "Scientific Imaging with Photoshop: Methods, Measurement and
Output."

Rawlight.com (dba Sedgewick Initiatives)
965 Cromwell Avenue
Saint Paul, MN  55114
[hidden email]
[phone]
http://www.quickphotoshop.com
http://www.heartFROMstone.com
http://www.rawlight.com




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This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waiver any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

[Keith Morris]

Hi Cameron,

 

I am almost a bit disappointed to have only ever seen this sort of thing when a user has messed up their filter settings on the confocal, and once the correct filter settings are applied it vanishes. I can’t reproduce it with our wide-field and confocal microscopes and Vectashield+DAPI & FITC samples [which presumably have excess DAPI stain].

 

DAPI can be excited by visible violet light - we use a 405nm line laser, and the DAPI excitation spectra goes up to just beyond ~430nm, and there is generally loads of DAPI labelled DNA to excite. However in our confocal DAPI emission can be excluded from the FITC channel by applying a 420nm-480nm emission filter, although naturally this won’t work if DAPI is bleeding though into the FITC when imaging the FITC. I wonder if your filter sets are less than perfect or perhaps the odd filter is incorrect [old Zeiss filter sets for example never had any writing on them and so are easy to mix up] – did you fit the filters and so trust them, or are they a sealed cube and thus presumably OK?. A dodgy filter set would seem a reasonable hypothesis except that you don’t see this DAPI ‘bleedthrough’ into the FITC channel at all if you image with FITC first, then the DAPI [is that correct?]. I assume that you are using a standard fluorescence microscope with wide band DAPI/FITC dichroic filter sets, and aren’t using a confocal like the Zeiss 510 Metahead where you have far more control over excitation and emission filters. If you try your samples with a modern confocal and select the narrow band emission filter options for the DAPI and FITC, do you see the same effect?

 

I have seen similar effects a few years ago on our Leica SP2 confocal when the LCS scanning software had a bug during scanning in sequential line mode, the DAPI laser wasn’t switching off [or any of them] as it should have been during FITC capture. It was capturing in simultaneous mode by mistake [or ‘single track’ as Zeiss call it for no apparent reason]. Capturing in sequential [multi-track] frame mode corrected this and eliminated the bleedthrough, and even though the bug was fixed with a software patch I’ve been suspicious of line scanning mode ever since [I always check it with a sequential frame mode capture as well]. Plus with the 510 [and 710] Meta you have the ‘advantage’ of the spectral ‘fingerprinting’ and ‘un-mixing’ modules should bleedthrough be a problem. But again swapping the imaging capture order shouldn’t have corrected the problem. So perhaps it is specimen chemistry and not optics.

 

Keith

---------------------------------------------------------------------------
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 Eric Scarfone
Sent: 05 May 2009 08:59
To: [hidden email]
Subject: Re: Photoactivation/Conversion of DAPI

 

Hi Cameron,

I suppose you looked at the obvious, ie the sharpness of your GFP excitation filters??

Eric

Eric Scarfone, PhD, CNRS,
Center for Hearing and communication Research
Department of Clinical Neuroscience
Karolinska Institutet

Postal Address:
CFH, M1:02
Karolinska Hospital,
SE-171 76 Stockholm, Sweden

Work: [phone],
Cell: [phone]
Fax: [phone]

email: [hidden email]
http://www.ki.se/cfh/


----- Original Message -----
From: Cameron Nowell <[hidden email]>
Date: Tuesday, May 5, 2009 2:40 am
Subject: Photoactivation/Conversion of DAPI
To: [hidden email]

> Hi List,
>
> I think this has been discussed in the past but i have not been
> able to
> find a definitive answer to the problem.
>
> Basically if you have a sample stained with DAPI, after viewing it
> witha DAPI filter, the signal can be then detected using a
> GFP/FITC filter.
>
> I have tried this on samples with nothing but DAPI on them and it
> stillhappens. Suggestions in the past have been to lower the
> concentration of
> DAPI used and to scan/capture the other channels first and DAPI last.
>
> But does anyone out there have any idea why this happens in the first
> place? If you look at the spectra of DAPI it is a very good green
> emitter (but needs to be excited in the UV-Blue range). It should
> not be
> able to be excited by standard GFP type excitation at 480-490nm.
> So the
> conversion that is happening is shifting the excitation spectra of
> DAPItowards the red somehow.
>
> Any ideas?
>
>
> Cheers
>
> Cam
>
>
>
> Cameron J. Nowell
> Microscopy Manager
> Centre for Advanced Microscopy
> Ludwig Institute for Cancer Research
> PO Box 2008
> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA
> Office: [phone]
> Mobile: [phone]
> Fax: [phone]
> Facility Website
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]]On Behalf Of Jerry Sedgewick
> Sent: Tuesday, 5 May 2009 1:35 AM
> To: [hidden email]
> Subject: Re: Posting for Basic Confocal Workshop at U of South
> Carolina
> There are still some slots available in this year's Basic Confocal
> Workshop hosted by the University of South Carolina. This year's
> workshop will be from June 15-19, 2009 and will include a series of
> lectures on the theory and applications of confocal microscopy,
> specimenpreparation, processing confocal images in Photoshop, and 3D
> reconstructions using AMIRA. Students will be able to process triple
> labeled samples (cell cultures and sections) on site or bring
> their own
> samples to the workshop.
>
> Faculty will include Drs. Jay Jerome (Vanderbilt), Ralph Albrecht
> (UnivWisconsin-Madison), John Mackenzie (North Carolina State
> Univ), Tom
> TrusK (Medical Univ South Carolina) and myself.
>
> Instruments and applications experts from Leica, Nikon, Olympus,
> PerkinElmer, Photometrics, and Zeiss will be available for hands
> on training
> and imaging of samples.
>
> For those contemplating instrumentation proposals as part of the
> stimulus or other funding opportunities this is an excellent
> opportunityto see several systems side by side and to collect
> preliminary data on
> their instrument of choice.
>
> For further information and registration go to:
> http://dba.med.sc.edu/irf/price/irf/irf.htm or contact Anna McNeal
> ([hidden email] <mailto:[hidden email]>)
>
> Bob
>
> Bob Price
>
> Research Professor
>
> Dept Cell Biol and Anat
>
> USC School of Medicine
>
> 6439 Garner's Ferry Road
>
> Columbia, SC 29208
>
> Tel: [phone]
>
> Admin Tel: [phone]
>
> Fax: [phone]
>
>
>
> --
> Jerry (Gerald) Sedgewick
> Program Director, Biomedical Image Processing Lab (BIPL)
> Department of Neuroscience, University of Minnesota
> 312 Church St. SE, 1-205 Hasselmo Hall
> Minneapolis, MN 55455
> [phone]
> [hidden email]
> http://www.bipl.umn.edu
> Author: "Scientific Imaging with Photoshop: Methods, Measurement and
> Output."
>
> Rawlight.com (dba Sedgewick Initiatives)
> 965 Cromwell Avenue
> Saint Paul, MN 55114
> [hidden email]
> [phone]
> http://www.quickphotoshop.com
> http://www.heartFROMstone.com
> http://www.rawlight.com
>
>
>
>
> --- Get FREE High Speed Internet from USFamily.Net! --
> http://www.usfamily.net/mkt-freepromo.html ---
>
> No virus found in this incoming message.
> Checked by AVG - www.avg.com
> Version: 8.5.320 / Virus Database: 270.12.15/2093 - Release Date:
> 05/04/09 17:51:00
>
>
> This communication is intended only for the named recipient and
> may contain information that is confidential, legally privileged
> or subject to copyright; the Ludwig Institute for Cancer Research
> Ltd does not waiver any rights if you have received this
> communication in error.
> The views expressed in this communication are those of the sender
> and do not necessarily reflect the views of the Ludwig Institute
> for Cancer Research Ltd.
>
>

[Aryeh Weiss]

I can confirm this apparent photoconversion with DAPI. We took the DAPI sample
and image it *first* with the FITC filter set. We  saw nothing other that some
slight cytoplasmic autofluorescence. We then imaged with the DAPI filter set
(near UV excitation). Following this we saw a strong nuclear signal using the
FITC cube in the nucleous (exactly where we saw the DAPI). I played with the
DAPI cube exposure times, and more exposure definitely led to a stronger signal
using the FITC cube. I also repeated the exercise a few times, to be sure I was
not imagining it. I did not further quantify it, because I was  debugging
someone else's experiment, but there is no doubt that exposure to the UV caused
the DAPI to become excitable with the blue FITC excitation.

I cannot say if the molecule that was emitting the green light was different
than DAPI, because an FITC cube will only transmit the green emission.
However, something in the excitation spectrum is definitely changing. This
should concern anyone who is using DAPI together with another FITC-like dye.

--aryeh

--aryeh

Keith Morris wrote:

> Hi Cameron,
>
>  
>
> I am almost a bit disappointed to have only ever seen this sort of thing
> when a user has messed up their filter settings on the confocal, and
> once the correct filter settings are applied it vanishes. I can’t
> reproduce it with our wide-field and confocal microscopes and
> Vectashield+DAPI & FITC samples [which presumably have excess DAPI stain].
>
>  
>
> DAPI can be excited by visible violet light - we use a 405nm line laser,
> and the DAPI excitation spectra goes up to just beyond ~430nm, and there
> is generally loads of DAPI labelled DNA to excite. However in our
> confocal DAPI emission can be excluded from the FITC channel by applying
> a 420nm-480nm emission filter, although naturally this won’t work if
> DAPI is bleeding though into the FITC when imaging the FITC. I wonder if
> your filter sets are less than perfect or perhaps the odd filter is
> incorrect [old Zeiss filter sets for example never had any writing on
> them and so are easy to mix up] – did you fit the filters and so trust
> them, or are they a sealed cube and thus presumably OK?. A dodgy filter
> set would seem a reasonable hypothesis except that you don’t see this
> DAPI ‘bleedthrough’ into the FITC channel at all if you image with FITC
> first, then the DAPI [is that correct?]. I assume that you are using a
> standard fluorescence microscope with wide band DAPI/FITC dichroic
> filter sets, and aren’t using a confocal like the Zeiss 510 Metahead
> where you have far more control over excitation and emission filters. If
> you try your samples with a modern confocal and select the narrow band
> emission filter options for the DAPI and FITC, do you see the same effect?
>
>  
>
> I have seen similar effects a few years ago on our Leica SP2 confocal
> when the LCS scanning software had a bug during scanning in sequential
> line mode, the DAPI laser wasn’t switching off [or any of them] as it
> should have been during FITC capture. It was capturing in simultaneous
> mode by mistake [or ‘single track’ as Zeiss call it for no apparent
> reason]. Capturing in sequential [multi-track] frame mode corrected this
> and eliminated the bleedthrough, and even though the bug was fixed with
> a software patch I’ve been suspicious of line scanning mode ever since
> [I always check it with a sequential frame mode capture as well]. Plus
> with the 510 [and 710] Meta you have the ‘advantage’ of the spectral
> ‘fingerprinting’ and ‘un-mixing’ modules should bleedthrough be a
> problem. But again swapping the imaging capture order shouldn’t have
> corrected the problem. So perhaps it is specimen chemistry and not optics.
>
>  
>
> Keith
>
> ---------------------------------------------------------------------------
> 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 *Eric Scarfone
> *Sent:* 05 May 2009 08:59
> *To:* [hidden email]
> *Subject:* Re: Photoactivation/Conversion of DAPI
>
>  
>
> Hi Cameron,
>
> I suppose you looked at the obvious, ie the sharpness of your GFP
> excitation filters??
>
> Eric
>
> Eric Scarfone, PhD, CNRS,
> Center for Hearing and communication Research
> Department of Clinical Neuroscience
> Karolinska Institutet
>
> Postal Address:
> CFH, M1:02
> Karolinska Hospital,
> SE-171 76 Stockholm, Sweden
>
> Work: [phone],
> Cell: [phone]
> Fax: [phone]
>
> email: [hidden email]
> http://www.ki.se/cfh/
>
>
> ----- Original Message -----
> From: Cameron Nowell <[hidden email]>
> Date: Tuesday, May 5, 2009 2:40 am
> Subject: Photoactivation/Conversion of DAPI
> To: [hidden email]
>
>>  Hi List,
>>
>>  I think this has been discussed in the past but i have not been
>>  able to
>>  find a definitive answer to the problem.
>>
>>  Basically if you have a sample stained with DAPI, after viewing it
>>  witha DAPI filter, the signal can be then detected using a
>>  GFP/FITC filter.
>>
>>  I have tried this on samples with nothing but DAPI on them and it
>>  stillhappens. Suggestions in the past have been to lower the
>>  concentration of
>>  DAPI used and to scan/capture the other channels first and DAPI last.
>>
>>  But does anyone out there have any idea why this happens in the first
>>  place? If you look at the spectra of DAPI it is a very good green
>>  emitter (but needs to be excited in the UV-Blue range). It should
>>  not be
>>  able to be excited by standard GFP type excitation at 480-490nm.
>>  So the
>>  conversion that is happening is shifting the excitation spectra of
>>  DAPItowards the red somehow.
>>
>>  Any ideas?
>>
>>
>>  Cheers
>>
>>  Cam
>>
>>
>>
>>  Cameron J. Nowell
>>  Microscopy Manager
>>  Centre for Advanced Microscopy
>>  Ludwig Institute for Cancer Research
>>  PO Box 2008
>>  Royal Melbourne Hospital
>>  Victoria, 3050
>>  AUSTRALIA
>>  Office: [phone]
>>  Mobile: [phone]
>>  Fax: [phone]
>>  Facility Website
>>
>>
>>
>>  -----Original Message-----
>>  From: Confocal Microscopy List
>>  [mailto:[hidden email]]On Behalf Of Jerry Sedgewick
>>  Sent: Tuesday, 5 May 2009 1:35 AM
>>  To: [hidden email]
>>  Subject: Re: Posting for Basic Confocal Workshop at U of South
>>  Carolina
>>  There are still some slots available in this year's Basic Confocal
>>  Workshop hosted by the University of South Carolina. This year's
>>  workshop will be from June 15-19, 2009 and will include a series of
>>  lectures on the theory and applications of confocal microscopy,
>>  specimenpreparation, processing confocal images in Photoshop, and 3D
>>  reconstructions using AMIRA. Students will be able to process triple
>>  labeled samples (cell cultures and sections) on site or bring
>>  their own
>>  samples to the workshop.
>>
>>  Faculty will include Drs. Jay Jerome (Vanderbilt), Ralph Albrecht
>>  (UnivWisconsin-Madison), John Mackenzie (North Carolina State
>>  Univ), Tom
>>  TrusK (Medical Univ South Carolina) and myself.
>>
>>  Instruments and applications experts from Leica, Nikon, Olympus,
>>  PerkinElmer, Photometrics, and Zeiss will be available for hands
>>  on training
>>  and imaging of samples.
>>
>>  For those contemplating instrumentation proposals as part of the
>>  stimulus or other funding opportunities this is an excellent
>>  opportunityto see several systems side by side and to collect
>>  preliminary data on
>>  their instrument of choice.
>>
>>  For further information and registration go to:
>>  http://dba.med.sc.edu/irf/price/irf/irf.htm or contact Anna McNeal
>>  ([hidden email] <mailto:[hidden email]>)
>>
>>  Bob
>>
>>  Bob Price
>>
>>  Research Professor
>>
>>  Dept Cell Biol and Anat
>>
>>  USC School of Medicine
>>
>>  6439 Garner's Ferry Road
>>
>>  Columbia, SC 29208
>>
>>  Tel: [phone]
>>
>>  Admin Tel: [phone]
>>
>>  Fax: [phone]
>>
>>
>>
>>  --
>>  Jerry (Gerald) Sedgewick
>>  Program Director, Biomedical Image Processing Lab (BIPL)
>>  Department of Neuroscience, University of Minnesota
>>  312 Church St. SE, 1-205 Hasselmo Hall
>>  Minneapolis, MN 55455
>>  [phone]
>>  [hidden email]
>>  http://www.bipl.umn.edu
>>  Author: "Scientific Imaging with Photoshop: Methods, Measurement and
>>  Output."
>>
>>  Rawlight.com (dba Sedgewick Initiatives)
>>  965 Cromwell Avenue
>>  Saint Paul, MN 55114
>>  [hidden email]
>>  [phone]
>>  http://www.quickphotoshop.com
>>  http://www.heartFROMstone.com
>>  http://www.rawlight.com
>>
>>

--
Aryeh Weiss
School of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

Israel Society for Microscopy 2009 meeting website:
http://materials.technion.ac.il/ism/ISM2009.html

[Keith Morris]

I'll look out for in future. I suppose most of our FITC & TRITC stains are
completely co-localised to the DAPI being highly specific to regions of the
DNA [being cytogenetics probes or chromosome paints]. So I guess I'd never
notice it on those samples.

Generally though I'm used to seeing a dark hole where the DAPI nucleus is,
viewing in the FITC channel, when using immuno-fluorescence stains. That
said, I've seen strange things happen to the fluorescence labels after long
term storage [months in the fridge], not a drop in brightness, more the
fluorescence regions moving about. I've tended to ignore such samples and
bin them, as they clearly aren't right anymore, but they may have shown this
sort of thing.  

Keith

---------------------------------------------------------------------------
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/
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Aryeh Weiss
Sent: 05 May 2009 19:39
To: [hidden email]
Subject: Re: Photoactivation/Conversion of DAPI

I can confirm this apparent photoconversion with DAPI. We took the DAPI
sample
and image it *first* with the FITC filter set. We  saw nothing other that
some
slight cytoplasmic autofluorescence. We then imaged with the DAPI filter set

(near UV excitation). Following this we saw a strong nuclear signal using
the
FITC cube in the nucleous (exactly where we saw the DAPI). I played with the

DAPI cube exposure times, and more exposure definitely led to a stronger
signal
using the FITC cube. I also repeated the exercise a few times, to be sure I
was
not imagining it. I did not further quantify it, because I was  debugging
someone else's experiment, but there is no doubt that exposure to the UV
caused
the DAPI to become excitable with the blue FITC excitation.

I cannot say if the molecule that was emitting the green light was different

than DAPI, because an FITC cube will only transmit the green emission.
However, something in the excitation spectrum is definitely changing. This
should concern anyone who is using DAPI together with another FITC-like dye.

--aryeh

--aryeh

Keith Morris wrote:

> Hi Cameron,
>
>  
>
> I am almost a bit disappointed to have only ever seen this sort of thing
> when a user has messed up their filter settings on the confocal, and
> once the correct filter settings are applied it vanishes. I can't
> reproduce it with our wide-field and confocal microscopes and
> Vectashield+DAPI & FITC samples [which presumably have excess DAPI stain].
>
>  
>
> DAPI can be excited by visible violet light - we use a 405nm line laser,
> and the DAPI excitation spectra goes up to just beyond ~430nm, and there
> is generally loads of DAPI labelled DNA to excite. However in our
> confocal DAPI emission can be excluded from the FITC channel by applying
> a 420nm-480nm emission filter, although naturally this won't work if
> DAPI is bleeding though into the FITC when imaging the FITC. I wonder if
> your filter sets are less than perfect or perhaps the odd filter is
> incorrect [old Zeiss filter sets for example never had any writing on
> them and so are easy to mix up] - did you fit the filters and so trust
> them, or are they a sealed cube and thus presumably OK?. A dodgy filter
> set would seem a reasonable hypothesis except that you don't see this
> DAPI 'bleedthrough' into the FITC channel at all if you image with FITC
> first, then the DAPI [is that correct?]. I assume that you are using a
> standard fluorescence microscope with wide band DAPI/FITC dichroic
> filter sets, and aren't using a confocal like the Zeiss 510 Metahead
> where you have far more control over excitation and emission filters. If
> you try your samples with a modern confocal and select the narrow band
> emission filter options for the DAPI and FITC, do you see the same effect?
>
>  
>
> I have seen similar effects a few years ago on our Leica SP2 confocal
> when the LCS scanning software had a bug during scanning in sequential
> line mode, the DAPI laser wasn't switching off [or any of them] as it
> should have been during FITC capture. It was capturing in simultaneous
> mode by mistake [or 'single track' as Zeiss call it for no apparent
> reason]. Capturing in sequential [multi-track] frame mode corrected this
> and eliminated the bleedthrough, and even though the bug was fixed with
> a software patch I've been suspicious of line scanning mode ever since
> [I always check it with a sequential frame mode capture as well]. Plus
> with the 510 [and 710] Meta you have the 'advantage' of the spectral
> 'fingerprinting' and 'un-mixing' modules should bleedthrough be a
> problem. But again swapping the imaging capture order shouldn't have
> corrected the problem. So perhaps it is specimen chemistry and not optics.
>
>  
>
> Keith
>
>

---------------------------------------------------------------------------

> 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 *Eric Scarfone
> *Sent:* 05 May 2009 08:59
> *To:* [hidden email]
> *Subject:* Re: Photoactivation/Conversion of DAPI
>
>  
>
> Hi Cameron,
>
> I suppose you looked at the obvious, ie the sharpness of your GFP
> excitation filters??
>
> Eric
>
> Eric Scarfone, PhD, CNRS,
> Center for Hearing and communication Research
> Department of Clinical Neuroscience
> Karolinska Institutet
>
> Postal Address:
> CFH, M1:02
> Karolinska Hospital,
> SE-171 76 Stockholm, Sweden
>
> Work: [phone],
> Cell: [phone]
> Fax: [phone]
>
> email: [hidden email]
> http://www.ki.se/cfh/
>
>
> ----- Original Message -----
> From: Cameron Nowell <[hidden email]>
> Date: Tuesday, May 5, 2009 2:40 am
> Subject: Photoactivation/Conversion of DAPI
> To: [hidden email]
>
>>  Hi List,
>>
>>  I think this has been discussed in the past but i have not been
>>  able to
>>  find a definitive answer to the problem.
>>
>>  Basically if you have a sample stained with DAPI, after viewing it
>>  witha DAPI filter, the signal can be then detected using a
>>  GFP/FITC filter.
>>
>>  I have tried this on samples with nothing but DAPI on them and it
>>  stillhappens. Suggestions in the past have been to lower the
>>  concentration of
>>  DAPI used and to scan/capture the other channels first and DAPI last.
>>
>>  But does anyone out there have any idea why this happens in the first
>>  place? If you look at the spectra of DAPI it is a very good green
>>  emitter (but needs to be excited in the UV-Blue range). It should
>>  not be
>>  able to be excited by standard GFP type excitation at 480-490nm.
>>  So the
>>  conversion that is happening is shifting the excitation spectra of
>>  DAPItowards the red somehow.
>>
>>  Any ideas?
>>
>>
>>  Cheers
>>
>>  Cam
>>
>>
>>
>>  Cameron J. Nowell
>>  Microscopy Manager
>>  Centre for Advanced Microscopy
>>  Ludwig Institute for Cancer Research
>>  PO Box 2008
>>  Royal Melbourne Hospital
>>  Victoria, 3050
>>  AUSTRALIA
>>  Office: [phone]
>>  Mobile: [phone]
>>  Fax: [phone]
>>  Facility Website
>>
>>
>>
>>  -----Original Message-----
>>  From: Confocal Microscopy List
>>  [mailto:[hidden email]]On Behalf Of Jerry Sedgewick
>>  Sent: Tuesday, 5 May 2009 1:35 AM
>>  To: [hidden email]
>>  Subject: Re: Posting for Basic Confocal Workshop at U of South
>>  Carolina
>>  There are still some slots available in this year's Basic Confocal
>>  Workshop hosted by the University of South Carolina. This year's
>>  workshop will be from June 15-19, 2009 and will include a series of
>>  lectures on the theory and applications of confocal microscopy,
>>  specimenpreparation, processing confocal images in Photoshop, and 3D
>>  reconstructions using AMIRA. Students will be able to process triple
>>  labeled samples (cell cultures and sections) on site or bring
>>  their own
>>  samples to the workshop.
>>
>>  Faculty will include Drs. Jay Jerome (Vanderbilt), Ralph Albrecht
>>  (UnivWisconsin-Madison), John Mackenzie (North Carolina State
>>  Univ), Tom
>>  TrusK (Medical Univ South Carolina) and myself.
>>
>>  Instruments and applications experts from Leica, Nikon, Olympus,
>>  PerkinElmer, Photometrics, and Zeiss will be available for hands
>>  on training
>>  and imaging of samples.
>>
>>  For those contemplating instrumentation proposals as part of the
>>  stimulus or other funding opportunities this is an excellent
>>  opportunityto see several systems side by side and to collect
>>  preliminary data on
>>  their instrument of choice.
>>
>>  For further information and registration go to:
>>  http://dba.med.sc.edu/irf/price/irf/irf.htm or contact Anna McNeal
>>  ([hidden email] <mailto:[hidden email]>)
>>
>>  Bob
>>
>>  Bob Price
>>
>>  Research Professor
>>
>>  Dept Cell Biol and Anat
>>
>>  USC School of Medicine
>>
>>  6439 Garner's Ferry Road
>>
>>  Columbia, SC 29208
>>
>>  Tel: [phone]
>>
>>  Admin Tel: [phone]
>>
>>  Fax: [phone]
>>
>>
>>
>>  --
>>  Jerry (Gerald) Sedgewick
>>  Program Director, Biomedical Image Processing Lab (BIPL)
>>  Department of Neuroscience, University of Minnesota
>>  312 Church St. SE, 1-205 Hasselmo Hall
>>  Minneapolis, MN 55455
>>  [phone]
>>  [hidden email]
>>  http://www.bipl.umn.edu
>>  Author: "Scientific Imaging with Photoshop: Methods, Measurement and
>>  Output."
>>
>>  Rawlight.com (dba Sedgewick Initiatives)
>>  965 Cromwell Avenue
>>  Saint Paul, MN 55114
>>  [hidden email]
>>  [phone]
>>  http://www.quickphotoshop.com
>>  http://www.heartFROMstone.com
>>  http://www.rawlight.com
>>
>>

--
Aryeh Weiss
School of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

Israel Society for Microscopy 2009 meeting website:
http://materials.technion.ac.il/ism/ISM2009.html

[Keith Morris]

Just a final thought or two on 'Photo-activation' of DAPI.

We regularly capture mFISH karyotypes of metaphase spread chromosomes here
and we always capture with DAPI last - after a complex fluorescence image
acquisition sequence of Spectrum Orange, Far Red [Cy5], Texas Red, Aqua
[DEAC], and FITC fluorescence filter sets. For mFISH capture we always
search for metaphases using the Spectrum Gold filter, and always capture
with DAPI last in the sequence, as the DAPI illumination is known to
seriously bleach the other dyes [according to mFISH manufacturer's Vysis*
and Applied Imaging]. During mFISH capture the FITC signal is particularly
weak**, possibly because it is the last non-DAPI fluorochrome captured [i.e.
bleached]. Typically, the DAPI is always bright, although if we hang around
during capture the other fluorochromes can be seriously 'dimmed' even before
DAPI capture**.

Likewise I've always repeated the dogma to new users that the published
excitation and emission spectra of any fluorochrome can only be taken as a
guide to that actually found in a specimen. The chemistry/biochemistry of
the tissue + added chemicals may affect the spectra measured in any given
sample. Possibly the energy provided by the various illumination lights
might do something to the spectra as well.

Try as I might though I can't easily fit the observed 'photoactivation' of
DAPI into the above two statements [largely because the effect is reversed
by capture sequence]. But I do wonder 'if, by chance, they are related?'

I've tried to reproduce the DAPI 'photo-activation & capture sequence' on a
few of my bright DAPI/FITC samples, and not succeeded so far.
 
Keith    

*Vysis is actually an ex-manufacturer of mFISH paints

**All the chromosomes have a reasonable 'FITC, DEAC etc..' signal, just the
targeted chromosomes aren't quite as 'significantly brighter than the FITC,
DEAC etc.. background' as they should be.

---------------------------------------------------------------------------
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/
 
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Cameron Nowell
Sent: 05 May 2009 01:40
To: [hidden email]
Subject: Photoactivation/Conversion of DAPI

Hi List,

I think this has been discussed in the past but i have not been able to
find a definitive answer to the problem.

Basically if you have a sample stained with DAPI, after viewing it with
a DAPI filter, the signal can be then detected using a GFP/FITC filter.

I have tried this on samples with nothing but DAPI on them and it still
happens. Suggestions in the past have been to lower the concentration of
DAPI used and to scan/capture the other channels first and DAPI last.

But does anyone out there have any idea why this happens in the first
place? If you look at the spectra of DAPI it is a very good green
emitter (but needs to be excited in the UV-Blue range). It should not be
able to be excited by standard GFP type excitation at 480-490nm. So the
conversion that is happening is shifting the excitation spectra of DAPI
towards the red somehow.

Any ideas?


Cheers

Cam



Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: [phone]
Mobile: [phone]
Fax: [phone]
Facility Website



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Jerry Sedgewick
Sent: Tuesday, 5 May 2009 1:35 AM
To: [hidden email]
Subject: Re: Posting for Basic Confocal Workshop at U of South Carolina

There are still some slots available in this year's Basic Confocal
Workshop hosted by the University of South Carolina. This year's
workshop will be from June 15-19, 2009 and will include a series of
lectures on the theory and applications of confocal microscopy, specimen
preparation, processing confocal images in Photoshop, and 3D
reconstructions using AMIRA. Students will be able to process triple
labeled samples (cell cultures and sections) on site or bring their own
samples to the workshop.

Faculty will include Drs. Jay Jerome (Vanderbilt), Ralph Albrecht (Univ
Wisconsin-Madison), John Mackenzie (North Carolina State Univ), Tom
TrusK (Medical Univ South Carolina) and myself.

Instruments and applications experts from Leica, Nikon, Olympus, Perkin
Elmer, Photometrics, and Zeiss will be available for hands on training
and imaging of samples.

For those contemplating instrumentation proposals as part of the
stimulus or other funding opportunities this is an excellent opportunity
to see several systems side by side and to collect preliminary data on
their instrument of choice.

For further information and registration go to:
http://dba.med.sc.edu/irf/price/irf/irf.htm or contact Anna McNeal
([hidden email] <mailto:[hidden email]>)

Bob

Bob Price

Research Professor

Dept Cell Biol and Anat

USC School of Medicine

6439 Garner's Ferry Road

Columbia, SC 29208

Tel: [phone]

Admin Tel: [phone]

Fax: [phone]



--
Jerry (Gerald) Sedgewick
Program Director, Biomedical Image Processing Lab (BIPL)
Department of Neuroscience, University of Minnesota
312 Church St. SE, 1-205 Hasselmo Hall
Minneapolis, MN  55455
[phone]
[hidden email]
http://www.bipl.umn.edu
Author: "Scientific Imaging with Photoshop: Methods, Measurement and
Output."

Rawlight.com (dba Sedgewick Initiatives)
965 Cromwell Avenue
Saint Paul, MN  55114
[hidden email]
[phone]
http://www.quickphotoshop.com
http://www.heartFROMstone.com
http://www.rawlight.com




--- Get FREE High Speed Internet from USFamily.Net! --
http://www.usfamily.net/mkt-freepromo.html ---

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 8.5.320 / Virus Database: 270.12.15/2093 - Release Date:
05/04/09 17:51:00


This communication is intended only for the named recipient and may contain
information that is confidential, legally privileged or subject to
copyright; the Ludwig Institute for Cancer Research Ltd does not waiver any
rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not
necessarily reflect the views of the Ludwig Institute for Cancer Research
Ltd.

[Stephen Cody-2]

Dear Cameron and list,

 

I have seen this and done similar tests as Aryeh to confirm that it seems to be a photoactivation. But at the time it seemed to be with just one persons slides. I did not see this effect with other preparations. I suspect it had something to do with the mounting media or the batch of DAPI.

 

Cheers

Stephen Cody

2009/5/18 Keith Morris <[hidden email]>

Just a final thought or two on 'Photo-activation' of DAPI.

We regularly capture mFISH karyotypes of metaphase spread chromosomes here
and we always capture with DAPI last - after a complex fluorescence image
acquisition sequence of Spectrum Orange, Far Red [Cy5], Texas Red, Aqua
[DEAC], and FITC fluorescence filter sets. For mFISH capture we always
search for metaphases using the Spectrum Gold filter, and always capture
with DAPI last in the sequence, as the DAPI illumination is known to
seriously bleach the other dyes [according to mFISH manufacturer's Vysis*
and Applied Imaging]. During mFISH capture the FITC signal is particularly
weak**, possibly because it is the last non-DAPI fluorochrome captured [i.e.
bleached]. Typically, the DAPI is always bright, although if we hang around
during capture the other fluorochromes can be seriously 'dimmed' even before
DAPI capture**.

Likewise I've always repeated the dogma to new users that the published
excitation and emission spectra of any fluorochrome can only be taken as a
guide to that actually found in a specimen. The chemistry/biochemistry of
the tissue + added chemicals may affect the spectra measured in any given
sample. Possibly the energy provided by the various illumination lights
might do something to the spectra as well.

Try as I might though I can't easily fit the observed 'photoactivation' of
DAPI into the above two statements [largely because the effect is reversed
by capture sequence]. But I do wonder 'if, by chance, they are related?'

I've tried to reproduce the DAPI 'photo-activation & capture sequence' on a
few of my bright DAPI/FITC samples, and not succeeded so far.

Keith

*Vysis is actually an ex-manufacturer of mFISH paints

**All the chromosomes have a reasonable 'FITC, DEAC etc..' signal, just the
targeted chromosomes aren't quite as 'significantly brighter than the FITC,
DEAC etc.. background' as they should be.

---------------------------------------------------------------------------
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/

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On

Behalf Of Cameron Nowell
Sent: 05 May 2009 01:40
To: [hidden email]

Subject: Photoactivation/Conversion of DAPI

Hi List,

I think this has been discussed in the past but i have not been able to
find a definitive answer to the problem.

Basically if you have a sample stained with DAPI, after viewing it with
a DAPI filter, the signal can be then detected using a GFP/FITC filter.

I have tried this on samples with nothing but DAPI on them and it still
happens. Suggestions in the past have been to lower the concentration of
DAPI used and to scan/capture the other channels first and DAPI last.

But does anyone out there have any idea why this happens in the first
place? If you look at the spectra of DAPI it is a very good green
emitter (but needs to be excited in the UV-Blue range). It should not be
able to be excited by standard GFP type excitation at 480-490nm. So the
conversion that is happening is shifting the excitation spectra of DAPI
towards the red somehow.

Any ideas?


Cheers

Cam



Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: [phone]
Mobile: [phone]
Fax: [phone]
Facility Website



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Jerry Sedgewick
Sent: Tuesday, 5 May 2009 1:35 AM
To: [hidden email]
Subject: Re: Posting for Basic Confocal Workshop at U of South Carolina

There are still some slots available in this year's Basic Confocal
Workshop hosted by the University of South Carolina. This year's
workshop will be from June 15-19, 2009 and will include a series of
lectures on the theory and applications of confocal microscopy, specimen
preparation, processing confocal images in Photoshop, and 3D
reconstructions using AMIRA. Students will be able to process triple
labeled samples (cell cultures and sections) on site or bring their own
samples to the workshop.

Faculty will include Drs. Jay Jerome (Vanderbilt), Ralph Albrecht (Univ
Wisconsin-Madison), John Mackenzie (North Carolina State Univ), Tom
TrusK (Medical Univ South Carolina) and myself.

Instruments and applications experts from Leica, Nikon, Olympus, Perkin
Elmer, Photometrics, and Zeiss will be available for hands on training
and imaging of samples.

For those contemplating instrumentation proposals as part of the
stimulus or other funding opportunities this is an excellent opportunity
to see several systems side by side and to collect preliminary data on
their instrument of choice.

For further information and registration go to:
http://dba.med.sc.edu/irf/price/irf/irf.htm or contact Anna McNeal
([hidden email] <mailto:[hidden email]>)

Bob

Bob Price

Research Professor

Dept Cell Biol and Anat

USC School of Medicine

6439 Garner's Ferry Road

Columbia, SC 29208

Tel: [phone]

Admin Tel: [phone]

Fax: [phone]



--
Jerry (Gerald) Sedgewick
Program Director, Biomedical Image Processing Lab (BIPL)
Department of Neuroscience, University of Minnesota
312 Church St. SE, 1-205 Hasselmo Hall
Minneapolis, MN  55455
[phone]
[hidden email]
http://www.bipl.umn.edu
Author: "Scientific Imaging with Photoshop: Methods, Measurement and
Output."

Rawlight.com (dba Sedgewick Initiatives)
965 Cromwell Avenue
Saint Paul, MN  55114
[hidden email]
[phone]
http://www.quickphotoshop.com
http://www.heartFROMstone.com
http://www.rawlight.com




--- Get FREE High Speed Internet from USFamily.Net! --
http://www.usfamily.net/mkt-freepromo.html ---

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 8.5.320 / Virus Database: 270.12.15/2093 - Release Date:
05/04/09 17:51:00


This communication is intended only for the named recipient and may contain
information that is confidential, legally privileged or subject to
copyright; the Ludwig Institute for Cancer Research Ltd does not waiver any
rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not
necessarily reflect the views of the Ludwig Institute for Cancer Research
Ltd.

--
Stephen H. Cody