wasabi freeware

[Deborah van der List]

Hello all,

Several years ago we installed the wasabi freeware to capture images from our fluorescent scope. It served our purposes very well. I need to reinstall the program and for the life of me, I can’t find it on the web. Does anyone have a link?

Thanks in advance

Deborah van der List

 

Department of Neurobiology, Physiology & Behavior
College of Biological Sciences
University of California, Davis
1 Shields Avenue
Davis, CA  95616
Phone: [phone]
Fax: [phone]
email: [hidden email]

 

[Frohlich, Victoria Centonze]

Talk to your Hamamatsu rep about Wasabi.  They should be able to help.

 

Victoria Centonze Frohlich, Ph.D.
Associate Director, Core Optical Imaging Facility &
Director of Institutional Research Cores

Mail Code 7762
University of Texas Health Science Center at San Antonio

7703 Floyd Curl Drive
San Antonio, TX. 78229-3900 

Office: 2.514 U  Main Campus

Office Phone: [phone]
Fax: [phone]
Email: <a href="BLOCKED::mailto:[e-mail]" title="mailto:[e-mail]">frohlich@...

Core Optical Imaging Facility
<a href="BLOCKED::http://www.uthscsa.edu/csb/imaging.asp" title="http://www.uthscsa.edu/csb/imaging.asp">http://www.uthscsa.edu/csb/imaging.asp

Office of the Vice President for Research
http://www.uthscsa.edu/research/ 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Deborah van der List
Sent: Monday, November 24, 2008 4:34 PM
To: [hidden email]
Subject: wasabi freeware

 

Hello all,

Several years ago we installed the wasabi freeware to capture images from our fluorescent scope. It served our purposes very well. I need to reinstall the program and for the life of me, I can’t find it on the web. Does anyone have a link?

Thanks in advance

Deborah van der List

 

Department of Neurobiology, Physiology & Behavior
College of Biological Sciences
University of California, Davis
1 Shields Avenue
Davis, CA  95616
Phone: [phone]
Fax: [phone]
email: [hidden email]

 

[mahogny]

or check if www.micro-manager.org does the work. also free, possibly
with more features

/Johan

Frohlich, Victoria wrote:

> Talk to your Hamamatsu rep about Wasabi.  They should be able to help.
>
>  
>
>
> Victoria Centonze Frohlich, Ph.D.
> Associate Director, Core Optical Imaging Facility &
> Director of Institutional Research Cores
>
> Mail Code 7762
> University of Texas Health Science Center at San Antonio
>
> 7703 Floyd Curl Drive
> San Antonio, TX. 78229-3900
>
>
> Office: 2.514 U  Main Campus
>
> Office Phone: [phone]
> Fax: [phone]
> Email: [hidden email] <BLOCKED::mailto:[hidden email]>  
>
> Core Optical Imaging Facility
> http://www.uthscsa.edu/csb/imaging.asp
> <BLOCKED::http://www.uthscsa.edu/csb/imaging.asp>  
>
> Office of the Vice President for Research
> http://www.uthscsa.edu/research/ <http://www.uthscsa.edu/research/>  
>
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Deborah van der List
> Sent: Monday, November 24, 2008 4:34 PM
> To: [hidden email]
> Subject: wasabi freeware
>
>  
>
> Hello all,
>
> Several years ago we installed the wasabi freeware to capture images
> from our fluorescent scope. It served our purposes very well. I need to
> reinstall the program and for the life of me, I can't find it on the
> web. Does anyone have a link?
>
> Thanks in advance
>
> Deborah van der List
>
>  
>
>
> Department of Neurobiology, Physiology & Behavior
> College of Biological Sciences
> University of California, Davis
> 1 Shields Avenue
> Davis, CA  95616
> Phone: [phone]
> Fax: [phone]
> email: [hidden email]
>
>  
>
>
>  

--
--
------------------------------------------------
Johan Henriksson
MSc Engineering
PhD student, Karolinska Institutet
http://mahogny.areta.org http://www.endrov.net

[Mayandi Sivaguru]

Hi all, One of our client is using a cell line expressing GFP bleaches very fast (Andor Revolution SDC system), despite using low excitation setting at the 488 line laser (set at 30 at the AOTF and approximately 240 microW on the 100x Olympus UPLANSAPO objective), EM Gain in combination with high amplifier gain. She wants to get two frames per second under two channels she is getting it but the cell bleaches within couple of minutes. Using live cell antioxidants such as Trolox is not an option, I would greatly appreciate any insights in this regard. Will neutral density filters help?

Thanking you in advance
Shiv

Mayandi Sivaguru, PhD, PhD
Microscopy Facility Manager
8, Institute for Genomic Biology
University of Illinois at Urbana-Champaign
1206 West Gregory Dr.
Urbana, IL 61801 USA

Office: 217.333.1214
Fax: [phone]
[hidden email]
http://core.igb.uiuc.edu

[Armstrong, Brian]

I believe that Hamamatsu bought Compix and that they now offer HC Image
with their cameras instead of Wasabi. You may want to get HC Image and
use that to run your camera. It is basically a stripped version of
Compix. Compix is pretty nice and HC Image is also good and real easy to
use. I do not know about backward compatibility, but I'll bet Hamamatsu
may have drivers for their own cameras! HC Image is free. Wasabi as you
know was not so great anyway. Cheers,

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
ing/Pages/default.aspx
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Johan Henriksson
Sent: Monday, November 24, 2008 2:54 PM
To: [hidden email]
Subject: Re: wasabi freeware

or check if www.micro-manager.org does the work. also free, possibly
with more features

/Johan

Frohlich, Victoria wrote:

> Talk to your Hamamatsu rep about Wasabi.  They should be able to help.
>
>  
>
>
> Victoria Centonze Frohlich, Ph.D.
> Associate Director, Core Optical Imaging Facility &
> Director of Institutional Research Cores
>
> Mail Code 7762
> University of Texas Health Science Center at San Antonio
>
> 7703 Floyd Curl Drive
> San Antonio, TX. 78229-3900
>
>
> Office: 2.514 U  Main Campus
>
> Office Phone: [phone]
> Fax: [phone]
> Email: [hidden email] <BLOCKED::mailto:[hidden email]>  
>
> Core Optical Imaging Facility
> http://www.uthscsa.edu/csb/imaging.asp
> <BLOCKED::http://www.uthscsa.edu/csb/imaging.asp>  
>
> Office of the Vice President for Research
> http://www.uthscsa.edu/research/ <http://www.uthscsa.edu/research/>  
>
> From: Confocal Microscopy List

[mailto:[hidden email]]

> On Behalf Of Deborah van der List
> Sent: Monday, November 24, 2008 4:34 PM
> To: [hidden email]
> Subject: wasabi freeware
>
>  
>
> Hello all,
>
> Several years ago we installed the wasabi freeware to capture images
> from our fluorescent scope. It served our purposes very well. I need

to

> reinstall the program and for the life of me, I can't find it on the
> web. Does anyone have a link?
>
> Thanks in advance
>
> Deborah van der List
>
>  
>
>
> Department of Neurobiology, Physiology & Behavior
> College of Biological Sciences
> University of California, Davis
> 1 Shields Avenue
> Davis, CA  95616
> Phone: [phone]
> Fax: [phone]
> email: [hidden email]
>
>  
>
>
>  

--
--
------------------------------------------------
Johan Henriksson
MSc Engineering
PhD student, Karolinska Institutet
http://mahogny.areta.org http://www.endrov.net


---------------------------------------------------------------------
 
SECURITY/CONFIDENTIALITY WARNING:  
This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender.  
---------------------------------------------------------------------

[Sam's Mail]

Here's some potentially unconventional suggestions for you, as I don't know the resolution needed for your client's story to be told.  However, if you'd like to get more light through the SDC system, take away some of the elements that are preventing light from travelling through the optics.  You may not need to perfectly match the pinhole size with your objective to gather the essential data from your live cell imaging experiment.  How about trying a lower power objective with less glass elements in it?  You will effectively gain some useable signal with each of those choices, while potentially losing some via NA, depending on the objectives in your quiver.

Perhaps if you shared with the list what the client was trying to image, then perhaps that might yield more insights.

Cheers,

--
Samuel A. Connell
Director of Light Microscopy
Cell & Tissue Imaging Center
St. Jude Children's Research Hospital
262 Danny Thomas Place
Memphis, TN 38105-3678
Office [phone]
Cell [phone]
[hidden email]




On 11/24/08 5:08 PM, "Mayandi Sivaguru" <[hidden email]> wrote:


Hi all, One of our client is using a cell line expressing GFP bleaches very fast (Andor Revolution SDC system), despite using low excitation setting at the 488 line laser (set at 30 at the AOTF and approximately 240 microW on the 100x Olympus UPLANSAPO objective), EM Gain in combination with high amplifier gain. She wants to get two frames per second under two channels she is getting it but the cell bleaches within couple of minutes. Using live cell antioxidants such as Trolox is not an option, I would greatly appreciate any insights in this regard. Will neutral density filters help?

Thanking you in advance
Shiv




Mayandi Sivaguru, PhD, PhD
Microscopy Facility Manager
8, Institute for Genomic Biology
University of Illinois at Urbana-Champaign
1206 West Gregory Dr.
Urbana, IL 61801 USA

Office: 217.333.1214
Fax: [phone]
[hidden email]
http://core.igb.uiuc.edu

 <http://core.igb.uiuc.edu/>

[George Peeters-2]

Dear Mayandi:

I'm not familiar enough with the "Revolution" to know what 30 means. Most AOTFs can be controlled all the way from essentially off (1/10,000), to some maximum power level. So neutral density filters shouldn't be needed. 240 uW is still quite a bit  of light and I can understand why bleaching may be occurring.  Is "30" as low as you can go?  if you have a CSU-10b it will have an ND built in if you have a CSUX it may or may not have an ND position. What is the other channel (561nm ?).

George

George A. Peeters MD, MS

President,  Solamere Technology Group Inc

1427 Perry Ave

Salt Lake City, UT 84103

www.solameretech.com

801 322-2645 office          801 322-2645 fax

Hi all, One of our client is using a cell line expressing GFP bleaches very fast (Andor Revolution SDC system), despite using low excitation setting at the 488 line laser (set at 30 at the AOTF and approximately 240 microW on the 100x Olympus UPLANSAPO objective), EM Gain in combination with high amplifier gain. She wants to get two frames per second under two channels she is getting it but the cell bleaches within couple of minutes. Using live cell antioxidants such as Trolox is not an option, I would greatly appreciate any insights in this regard. Will neutral density filters help?

Thanking you in advance
Shiv

Mayandi Sivaguru, PhD, PhD
Microscopy Facility Manager
8, Institute for Genomic Biology
University of Illinois at Urbana-Champaign
1206 West Gregory Dr.
Urbana, IL 61801 USA

Office: 217.333.1214
Fax: [phone]
[hidden email]
http://core.igb.uiuc.edu

[Gary Laevsky-2]

Hi Shiv,

 

A few options that may or may not have been tried;

 

Turning the EM gain off.  Depending on the intensity of the fluor, you actually may be adding noise.  If you turn the EM gain off, you may get a “better” image, and therefore you may be able to reduce the exposure/laser power.

 

Make sure you have optimized the filters in the path. 

 

Also, I believe you may have additional optics in the emission path that are stealing valuable photons.

 

As stated, “30” (FYI this is in a non-linear scale to 100) is not a very informative number to determine intensity at the sample as it depends size of laser and throughput i.e. optics in the excitation path (this is with a CSU22).  Your measurement of 240um, however, is perfectly objective (excuse the mild pun).  And, again as stated, is very reasonable.

 

Talk soon.

 

 

 

Best,

 

Gary Laevsky, Ph.D.

Imaging Application Specialist

 

Andor Technology

discover new ways of seeing

 

[hidden email]

Cell         (774) 291 - 9992

Office       (860) 290 - 9211 x219

Fax          (860) 290 - 9566

Web:       www.andor.com

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mayandi Sivaguru
Sent: Monday, November 24, 2008 6:09 PM
To: [hidden email]
Subject: Photobleaching of GFP in Spinning Disk Confocal

 

Hi all, One of our client is using a cell line expressing GFP bleaches very fast (Andor Revolution SDC system), despite using low excitation setting at the 488 line laser (set at 30 at the AOTF and approximately 240 microW on the 100x Olympus UPLANSAPO objective), EM Gain in combination with high amplifier gain. She wants to get two frames per second under two channels she is getting it but the cell bleaches within couple of minutes. Using live cell antioxidants such as Trolox is not an option, I would greatly appreciate any insights in this regard. Will neutral density filters help?

Thanking you in advance
Shiv

Mayandi Sivaguru, PhD, PhD
Microscopy Facility Manager
8, Institute for Genomic Biology
University of Illinois at Urbana-Champaign
1206 West Gregory Dr.
Urbana, IL 61801 USA

Office: 217.333.1214
Fax: [phone]
[hidden email]
http://core.igb.uiuc.edu

[Knecht, David]

I would have to respectfully disagree with Gary on this one.  If you are photobleaching GFP, then keep turning down the laser power until it stops.  There is no reason you should see rapid GFP photobleaching with an EM camera.   I have found very little benefit in turning down the EM gain. All that does is decrease the signal without affecting the noise much.  I find that it is far better to turn down the laser (I go as low as 1% on the AOTF for bright GFP cells) and use a 2x or 4x average if the image is noisy.  The EM camera is amazingly sensitive, but noisy.  You gain very little by having high laser power as that camera is designed to detect faint signals and benefits little from a strong signal.  You also have to be careful not to be misled by the autocontrasting the software does to display the 16 bit image.  If you have an intensity that is 100-200 over background, the conversion can make the screen image look poor, but adjusting the brightness/contrast manually can get you a nice looking image.  I go by the relative pixel intensity of cell vs. background rather than the subjective image quality to set up an experiment that is challenging.  Dave

On Nov 25, 2008, at 8:34 AM, Gary Laevsky wrote:

Hi Shiv,

 

A few options that may or may not have been tried;

 

Turning the EM gain off.  Depending on the intensity of the fluor, you actually may be adding noise.  If you turn the EM gain off, you may get a “better” image, and therefore you may be able to reduce the exposure/laser power.

 

Make sure you have optimized the filters in the path. 

 

Also, I believe you may have additional optics in the emission path that are stealing valuable photons.

 

As stated, “30” (FYI this is in a non-linear scale to 100) is not a very informative number to determine intensity at the sample as it depends size of laser and throughput i.e. optics in the excitation path (this is with a CSU22).  Your measurement of 240um, however, is perfectly objective (excuse the mild pun).  And, again as stated, is very reasonable.

 

Talk soon.

 

 

 

Best,

 

Gary Laevsky, Ph.D.

Imaging Application Specialist

 

Andor Technology

discover new ways of seeing

 

[hidden email]

Cell         (774) 291 - 9992

Office       (860) 290 - 9211 x219

Fax          (860) 290 - 9566

Web:       www.andor.com

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of Mayandi Sivaguru
Sent: Monday, November 24, 2008 6:09 PM
To: [hidden email]
Subject: Photobleaching of GFP in Spinning Disk Confocal

 

Hi all, One of our client is using a cell line expressing GFP bleaches very fast (Andor Revolution SDC system), despite using low excitation setting at the 488 line laser (set at 30 at the AOTF and approximately 240 microW on the 100x Olympus UPLANSAPO objective), EM Gain in combination with high amplifier gain. She wants to get two frames per second under two channels she is getting it but the cell bleaches within couple of minutes. Using live cell antioxidants such as Trolox is not an option, I would greatly appreciate any insights in this regard. Will neutral density filters help?

Thanking you in advance
Shiv

Mayandi Sivaguru, PhD, PhD
Microscopy Facility Manager
8, Institute for Genomic Biology
University of Illinois at Urbana-Champaign
1206 West Gregory Dr.
Urbana, IL 61801 USA

Office: 217.333.1214
Fax: [phone]
[hidden email]
http://core.igb.uiuc.edu

Dr. David Knecht    

Department of Molecular and Cell Biology

Co-head Flow Cytometry and Confocal Microscopy Facility

U-3125

91 N. Eagleville Rd.

University of Connecticut

Storrs, CT 06269

860-486-2200

860-486-4331 (fax)

[Julio Vazquez]

Hi Shiv, 

Anything that reduces illumination intensity at the sample will help. If you don't need ultimate resolution, you could try binning or using a lower power objective. If your samples are in water, you may try using a water lens if available to reduce spherical aberration, and hence increase detection efficiency. Also, there may be other trivial reasons for the bleaching, such as suboptimal pH in the sample/medium, and, of course, GFP is very sensitive to solvents (nail polish, etc...).  You mention Trolox is not an option. How about Oxyrase:

http://www.oxyrase.com/oxyfluor.html

There was a thread on this list a while ago about the merits of Catalase/Glucose Oxydase

10mM -mercaptoethanol, 2.5 mg/ml glucose, 20 g/ml catalase, 0.1

mg/ml glucose oxydase

The concentrations above are from Chabrillat et al, Molec Biol of the

Cell 16, 1640-1650 (2005).

I think there is still some debate as to whether this actually works...

Julio.

--

Julio Vazquez, 

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org/

===

On Nov 24, 2008, at 3:08 PM, Mayandi Sivaguru wrote:

Hi all, One of our client is using a cell line expressing GFP bleaches very fast (Andor Revolution SDC system), despite using low excitation setting at the 488 line laser (set at 30 at the AOTF and approximately 240 microW on the 100x Olympus UPLANSAPO objective), EM Gain in combination with high amplifier gain. She wants to get two frames per second under two channels she is getting it but the cell bleaches within couple of minutes. Using live cell antioxidants such as Trolox is not an option, I would greatly appreciate any insights in this regard. Will neutral density filters help?

Thanking you in advance
Shiv

Mayandi Sivaguru, PhD, PhD
Microscopy Facility Manager
8, Institute for Genomic Biology
University of Illinois at Urbana-Champaign
1206 West Gregory Dr.
Urbana, IL 61801 USA

Office: 217.333.1214
Fax: [phone]
[hidden email]
http://core.igb.uiuc.edu

[Beat Ludin]

I actually found that EGFP "bleaches" about 20x faster in the absence
of oxygen (e.g. in the presence of Oxyrase)! And it recovers slowly
once oxygen is present again. I made this observation (and tried to
publish it in vain) before the photoconversion properties of GFP
became known so, without having done any further tests, I would now
assume that what I observed back then wasn't bleaching in the true sense.
TCLSS, make sure there is oxygen around when you want to image EGFP
(I don't know about other variants). If you just put a wet coverslip
with cultured cells on a slide, the cells will metabolize the oxygen
in no time.

Cheers,  Beat

At 18:49 25-11-2008, you wrote:

>Hi Shiv,
>
>Anything that reduces illumination intensity at the sample will
>help. If you don't need ultimate resolution, you could try binning
>or using a lower power objective. If your samples are in water, you
>may try using a water lens if available to reduce spherical
>aberration, and hence increase detection efficiency. Also, there may
>be other trivial reasons for the bleaching, such as suboptimal pH in
>the sample/medium, and, of course, GFP is very sensitive to solvents
>(nail polish, etc...).  You mention Trolox is not an option. How about Oxyrase:
>
><http://www.oxyrase.com/oxyfluor.html>http://www.oxyrase.com/oxyfluor.html
>
>
>There was a thread on this list a while ago about the merits of
>Catalase/Glucose Oxydase
>
>>10mM -mercaptoethanol, 2.5 mg/ml glucose, 20 g/ml catalase, 0.1
>>mg/ml glucose oxydase
>>
>>The concentrations above are from Chabrillat et al, Molec Biol of the
>>Cell 16, 1640-1650 (2005).
>
>I think there is still some debate as to whether this actually works...
>
>Julio.
>
>
>--
>Julio Vazquez,
>Fred Hutchinson Cancer Research Center
>Seattle, WA 98109-1024
>
>
><http://www.fhcrc.org>http://www.fhcrc.org/
>
>===
>
>
>On Nov 24, 2008, at 3:08 PM, Mayandi Sivaguru wrote:
>
>>
>>Hi all, One of our client is using a cell line expressing GFP
>>bleaches very fast (Andor Revolution SDC system), despite using low
>>excitation setting at the 488 line laser (set at 30 at the AOTF and
>>approximately 240 microW on the 100x Olympus UPLANSAPO objective),
>>EM Gain in combination with high amplifier gain. She wants to get
>>two frames per second under two channels she is getting it but the
>>cell bleaches within couple of minutes. Using live cell
>>antioxidants such as Trolox is not an option, I would greatly
>>appreciate any insights in this regard. Will neutral density filters help?
>>
>>Thanking you in advance
>>Shiv
>>
>>
>>
>>Mayandi Sivaguru, PhD, PhD
>>Microscopy Facility Manager
>>8, Institute for Genomic Biology
>>University of Illinois at Urbana-Champaign
>>1206 West Gregory Dr.
>>Urbana, IL 61801 USA
>>
>>Office: 217.333.1214
>>Fax: [phone]
>><mailto:[hidden email]>[hidden email]
>>http://core.igb.uiuc.edu

[Knecht, David]

Interesting observation about Oxyrase.  We found that Oxyrase increased the resistance of cells to phototoxicity when imaging live GFP expressing cells at low intensity where photobleaching was not an issue.  Dictyostelium is very sensitive to light exposure and round up quickly when exposed to fluorescent excitation.  They tolerated the light better in the presence of Oxyrase.  Obviously this whole issue is very complicated!  Dave

On Nov 25, 2008, at 6:04 PM, Beat Ludin wrote:

I actually found that EGFP "bleaches" about 20x faster in the absence of oxygen (e.g. in the presence of Oxyrase)! And it recovers slowly once oxygen is present again. I made this observation (and tried to publish it in vain) before the photoconversion properties of GFP became known so, without having done any further tests, I would now assume that what I observed back then wasn't bleaching in the true sense.
TCLSS, make sure there is oxygen around when you want to image EGFP (I don't know about other variants). If you just put a wet coverslip with cultured cells on a slide, the cells will metabolize the oxygen in no time.

Cheers,  Beat

At 18:49 25-11-2008, you wrote:

Hi Shiv,

Anything that reduces illumination intensity at the sample will help. If you don't need ultimate resolution, you could try binning or using a lower power objective. If your samples are in water, you may try using a water lens if available to reduce spherical aberration, and hence increase detection efficiency. Also, there may be other trivial reasons for the bleaching, such as suboptimal pH in the sample/medium, and, of course, GFP is very sensitive to solvents (nail polish, etc...).  You mention Trolox is not an option. How about Oxyrase:

<http://www.oxyrase.com/oxyfluor.html>http://www.oxyrase.com/oxyfluor.html

There was a thread on this list a while ago about the merits of Catalase/Glucose Oxydase

10mM -mercaptoethanol, 2.5 mg/ml glucose, 20 g/ml catalase, 0.1

mg/ml glucose oxydase

The concentrations above are from Chabrillat et al, Molec Biol of the

Cell 16, 1640-1650 (2005).

I think there is still some debate as to whether this actually works...

Julio.

--

Julio Vazquez,

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

<http://www.fhcrc.org>http://www.fhcrc.org/

===

On Nov 24, 2008, at 3:08 PM, Mayandi Sivaguru wrote:

Hi all, One of our client is using a cell line expressing GFP bleaches very fast (Andor Revolution SDC system), despite using low excitation setting at the 488 line laser (set at 30 at the AOTF and approximately 240 microW on the 100x Olympus UPLANSAPO objective), EM Gain in combination with high amplifier gain. She wants to get two frames per second under two channels she is getting it but the cell bleaches within couple of minutes. Using live cell antioxidants such as Trolox is not an option, I would greatly appreciate any insights in this regard. Will neutral density filters help?

Thanking you in advance

Shiv

Mayandi Sivaguru, PhD, PhD

Microscopy Facility Manager

8, Institute for Genomic Biology

University of Illinois at Urbana-Champaign

1206 West Gregory Dr.

Urbana, IL 61801 USA

Office: 217.333.1214

Fax: [phone]

<[hidden email]>[hidden email]

http://core.igb.uiuc.edu

Dr. David Knecht    

Department of Molecular and Cell Biology

Co-head Flow Cytometry and Confocal Microscopy Facility

U-3125

91 N. Eagleville Rd.

University of Connecticut

Storrs, CT 06269

860-486-2200

860-486-4331 (fax)

[Gary Laevsky-2]

Shiv,

 

I have been informed I neglected to mention a very important issue.

 

Not only should you turn the EM gain off, but you should also switch to the conventional register.

 

The EM register, because of it’s amplification characteristics adds a tremendous amount of noise to the image.

 

Some EMCCDs allow you to be able to switch to the conventional register.  Your noise will be greatly reduced, and you can then possibly turn down the laser.

 

 

Best,

 

Gary Laevsky, Ph.D.

Imaging Application Specialist

 

Andor Technology

discover new ways of seeing

 

[hidden email]

Cell         (774) 291 - 9992

Office       (860) 290 - 9211 x219

Fax          (860) 290 - 9566

Web:       www.andor.com

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mayandi Sivaguru
Sent: Monday, November 24, 2008 6:09 PM
To: [hidden email]
Subject: Photobleaching of GFP in Spinning Disk Confocal

 

Hi all, One of our client is using a cell line expressing GFP bleaches very fast (Andor Revolution SDC system), despite using low excitation setting at the 488 line laser (set at 30 at the AOTF and approximately 240 microW on the 100x Olympus UPLANSAPO objective), EM Gain in combination with high amplifier gain. She wants to get two frames per second under two channels she is getting it but the cell bleaches within couple of minutes. Using live cell antioxidants such as Trolox is not an option, I would greatly appreciate any insights in this regard. Will neutral density filters help?

Thanking you in advance
Shiv

Mayandi Sivaguru, PhD, PhD
Microscopy Facility Manager
8, Institute for Genomic Biology
University of Illinois at Urbana-Champaign
1206 West Gregory Dr.
Urbana, IL 61801 USA

Office: 217.333.1214
Fax: [phone]
[hidden email]
http://core.igb.uiuc.edu