Re: Spinning Disk Exposure Times (shameless self-promotion)

[Jason Swedlow]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear All,

All modes of light microscopy have their limitations (and advantages, obviously).  We recently reported a comparison of three standard approaches to fluorescence microscopy (WFM, SDCM, and LSCM) where we were aiming to measure achieved SNR for a given light dose (what we referred to as exposure index) with the goal of definitively comparing different modes of microscopy, especially in live fluorescence imaging.  John Murray did a heroic job on this, analysing many different microscope systems across some two years of data collection.  The paper is available here:

http://www.ingentaconnect.com/content/bsc/jms/2007/00000228/00000003/art00015

When we published it, it was open-access, but something has happened-- we'll track that down (darn publishers)!!!!

Anyway, as part of this study, John Murray developed a very nice test sample-- dual color fluorescent beads in a sea of fluorophore of defined concentration-- that can be used to reveal all sorts of interesting sources of error and noise in a digital microscope system (including the artifacts discussed on this thread). 

As Claire said, it pays to pay attention to detail.

Cheers,

Jason

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On Fri, May 16, 2008 at 8:08 PM, Steve Baxter <[hidden email]> wrote:

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[Commercial Response]

Hi,

With the CSU / DSU approaches, it is vital to synchronise the exposure time of the camera to a whole number of disk segments. If this is not done then you will see curved scanline artefacts on the image (particularly at short exposure times). The UltraVIEW does this by synchronising the CSU disk speed, the camera exposure and the laser exposure together. This has to be done accurately, any jitter in the camera exposure time or mismatch in the disk speed will result in scanlines appearing again.

If anyone is interested in further technical detail of this, there is some in this PerkinElmer patent:

http://v3.espacenet.com/textdoc?DB=EPODOC&IDX=DE60312717T

Cheers,

Steve

On 29 Apr 2008, at 23:09, Farid Jalali wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Thank you Claire for this information. I have reported to Olympus a few times, with images, in the past year and a half or so regarding disk artifacts. I am happily using their Disk Scanning Unit (DSU) on an IX81 frame. Although I rarely get use the spinning disk at such low exposures, even with an EM-CCD, I can actually see the horizontal and vertical slit appertures that make up the Olympus DSU spinning disk. Its made more much more apparent during acquisition by exaggerating the image contrast. After acquisition and 3D deconvolution, the artifact becomes more robust and very obvious when setting thresholds for image segmentation; the disk lines can actually be problematic when trying to segment nuclei for example.  Our disk operates at about 3000rpm, and most of the time its not a problem. Shifting the speed to 5000 rpm only made things worse. I do not think I have been given notice of a fix as yet.

Cheers
Farid

On Tue, Apr 29, 2008 at 4:24 PM, Claire Brown <[hidden email]> wrote:
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Just a note to those of you using spinning disk confocal microscopes.

I have recently been testing out many different spinning disk confocal
microscopes and one of my worries was artifacts in the images due to

Variable sampling in different pixels due to the spinning nature of the
disk. Most of the commercial companies talk about the importance of making
sure

Your camera and your disk are synchronized. However, what I have found using
a uniform sample (Chroma Technology green fluorescent plastic slide) is that
it is also important to synchronize your exposure time. We found with a disk
spinning at 2500 rpms and an exposure time of 10 ms on an EM-CCD camera we
see lines in the images due to uneven sampling of pixels within the image.
However, if we went with 8, 12 or 16 ms (any multiple of 4 will be on the
same frequency as 2500 rpms or 400 us/spin) these line artifacts disappeared
because our exposure time was in sync with the disk and the camera.


These artifacts are not apparent by eye when a heterogeneous cellular sample
is in place because they are very subtle, but they will certainly be
important for quantitative imaging. So it is very important to use the
appropriate exposure times.


Sincerely,


Claire



_________________________________________________________________
Claire M. Brown, PhD
Life Sciences Complex Imaging Facility Director
McGill University Department of Biochemistry
http://www.lifesciencescomplex.mcgill.ca/imaging




--
Farid Jalali MSc
Senior Research Technician/ Lab Manager
Dr. Robert Bristow Lab
Applied Molecular Oncology
Princess Margaret Hospital
Toronto, Canada
416-946-4501 X4351 (Princess Margaret Hospital)
416-581-7754 STTARR at MaRS Building
416-581-7791 STTARR Microscopy Suite

Steve Baxter
R&D Leader
Improvision, a PerkinElmer Company
[hidden email]
[hidden email]
[phone]

Image Processing and Vision Company Limited (trading as Improvision) is a company registered in England and Wales with company number 2505778.  VAT number GB536188722.

[Adrian Smith-6]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

On 26/05/2008, at 10:42 AM, Jason Swedlow wrote:

http://www.ingentaconnect.com/content/bsc/jms/2007/00000228/00000003/art00015

When we published it, it was open-access, but something has happened-- we'll track that down (darn publishers)!!!!

Does this link provide open-access?

<http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-2818.2007.01861.x>

I'm not really sure because we have institutional access but the text on the page implies it is is open-access (and I wasn't offered free access from the ingentaconnect link).

Adrian

[Loralei Dewe-2]

Yes, it does, thank you for the link Jason!!

 

Lorie

 

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Adrian Smith
Sent: Sunday, May 25, 2008 5:24 PM
To: [hidden email]
Subject: Re: Spinning Disk Exposure Times (shameless self-promotion)

 

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

 

On 26/05/2008, at 10:42 AM, Jason Swedlow wrote:

http://www.ingentaconnect.com/content/bsc/jms/2007/00000228/00000003/art00015

When we published it, it was open-access, but something has happened-- we'll track that down (darn publishers)!!!!

 

 

Does this link provide open-access?

 

<http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-2818.2007.01861.x>

 

I'm not really sure because we have institutional access but the text on the page implies it is is open-access (and I wasn't offered free access from the ingentaconnect link).

 

Adrian

 

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[Jason Swedlow]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Thanks Adrian!!!!

Interesting that the publisher charges for articles that are open access.  Hmmmm....

Anyway, enjoy the beads!!!

Cheers,

Jason

On Mon, May 26, 2008 at 2:43 AM, Loralei Dewe <[hidden email]> wrote:

Yes, it does, thank you for the link Jason!!

 

Lorie

 

Loralei Dewe

Applications Specialist

5916 Sunnybrook Lane

Dixon, Ca 95620

Phone: [phone]

Fax: [phone]  *51

Cell:  707-301-0604

 

 MAG Biosystems     |     Photometrics     |     QImaging     |     Media Cybernetics     |     Gatan   
www.MAGbiosystems.com      www.photomet.com      www.qimaging.com            www.mediacy.com               www.gatan.com   

Microimaging Applications Group (MAG)

 

The Microimaging Applications Group comprises five imaging leaders: Photometrics, Gatan, Media Cybernetics,
QImaging, and MAG Biosystems. These technology partners work independently as well as in synergy to offer an
unparalleled range of solutions for microimaging applications.


This message and any attachments are solely for the use of intended recipients. They may contain privileged and/or confidential information.
If you are not the intended recipient, you are hereby notified that you received this email in error, and that any review, dissemination, distribution
or copying of this email and any attachment is strictly prohibited. If you receive this email in error please contact the sender and delete the
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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Adrian Smith
Sent: Sunday, May 25, 2008 5:24 PM
To: [hidden email]
Subject: Re: Spinning Disk Exposure Times (shameless self-promotion)

 

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

 

On 26/05/2008, at 10:42 AM, Jason Swedlow wrote:

http://www.ingentaconnect.com/content/bsc/jms/2007/00000228/00000003/art00015

When we published it, it was open-access, but something has happened-- we'll track that down (darn publishers)!!!!

 

 

Does this link provide open-access?

 

<http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-2818.2007.01861.x>

 

I'm not really sure because we have institutional access but the text on the page implies it is is open-access (and I wasn't offered free access from the ingentaconnect link).

 

Adrian

 

No virus found in this incoming message.
Checked by AVG.
Version: 7.5.524 / Virus Database: 269.24.1/1465 - Release Date: 5/25/2008 1:22 PM

No virus found in this outgoing message.
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--
**************************
Wellcome Trust Centre for Gene Regulation & Expression
College of Life Sciences
MSI/WTB/JBC Complex
University of Dundee
Dow Street
Dundee DD1 5EH
United Kingdom

phone [phone]
Intl phone: [phone]
FAX [phone]
email: [hidden email]

Lab Page: http://www.dundee.ac.uk/lifesciences/swedlow/
Open Microscopy Environment: http://openmicroscopy.org
**************************

[Rosemary.White]

Search the CONFOCAL archive at
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Dear all,

Has anyone used fluorescent tracers/dyes/probes successfully to quantify low
(sub-millimolar to maybe 20 micromolar) concentrations of sodium?  ...in the
presence of potassium?

thanks,
Rosemary

Rosemary White                    [hidden email]
CSIRO Plant Industry            ph.     61 (0)2-6246 5475
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Canberra, ACT 2601
Australia

[Rosemary.White]

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Sorry, I meant sub-millimolar to maybe 20 millimolar.....


On 29/5/08 5:46 PM, "Rosemary White" <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear all,
>
> Has anyone used fluorescent tracers/dyes/probes successfully to quantify low
> (sub-millimolar to maybe 20 micromolar) concentrations of sodium?  ...in the
> presence of potassium?
>
> thanks,
> Rosemary
>
> Rosemary White                    [hidden email]
> CSIRO Plant Industry            ph.     61 (0)2-6246 5475
> GPO Box 1600                       fax.     [phone]
> Canberra, ACT 2601
> Australia