Linear unmixing

[Samrina Aslam]

Hi,

I am currently using linear unmixing on the the Zeiss 510 Meta confocal
microscope to unmix the background autofluorescence from specific
fluorescence in parrafin sections.

I am using the 488nm laser, and the results are very varied depending on the
tissue type I am imaging.

Has anybody tried linear unmixing using the 488nm laser and produced good
images? Is there anything I can do to acquire better images?

Any help would be very much appreciated, thanks!

[mahogny]

samrina aslam wrote:

> Hi,
>
> I am currently using linear unmixing on the the Zeiss 510 Meta confocal
> microscope to unmix the background autofluorescence from specific
> fluorescence in parrafin sections.
>
> I am using the 488nm laser, and the results are very varied depending on the
> tissue type I am imaging.
>
> Has anybody tried linear unmixing using the 488nm laser and produced good
> images? Is there anything I can do to acquire better images?
>
> Any help would be very much appreciated, thanks!
>  

different tissues might/will have different profiles. have you tuned it
for each tissue type?

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

[Daniël]

You really should make good reference spectra from the different samples
you use: so take samples without fluorescence and make a background
autofluorescence spectrum for each tissue type.



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of samrina aslam
Sent: donderdag 13 november 2008 13:12
To: [hidden email]
Subject: Linear unmixing


Hi,

I am currently using linear unmixing on the the Zeiss 510 Meta confocal
microscope to unmix the background autofluorescence from specific
fluorescence in parrafin sections.

I am using the 488nm laser, and the results are very varied depending on
the
tissue type I am imaging.

Has anybody tried linear unmixing using the 488nm laser and produced
good
images? Is there anything I can do to acquire better images?

Any help would be very much appreciated, thanks!
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[Armstrong, Brian]

Yes, you must have controls for each group/sample type. I have never
been able to get very "pleasing" images myself from the unmixing. I
however see Linear Unmixing as a quantitative tool and not so much a
pretty picture tool.

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 samrina aslam
Sent: Thursday, November 13, 2008 4:12 AM
To: [hidden email]
Subject: Linear unmixing

Hi,

I am currently using linear unmixing on the the Zeiss 510 Meta confocal
microscope to unmix the background autofluorescence from specific
fluorescence in parrafin sections.

I am using the 488nm laser, and the results are very varied depending on
the
tissue type I am imaging.

Has anybody tried linear unmixing using the 488nm laser and produced
good
images? Is there anything I can do to acquire better images?

Any help would be very much appreciated, thanks!


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[Jeremy Adler]

Another problem is the quality of the images - you need good images to accurately unmix.

It is the usual problem with lack of emitted photons, noise and with PMTs their low quantum efficiency.
with a quantum efficiency of 20% and 20 photons reaching the PMT per pixel, the recorded count will have an average of 4.
But has range from 0 to 20. Admittedly 20 will not occur often, but there are a lot of pixels.
To unmix we have photons coming from at least 2  fluorophores and therefore unmixing can get messy.

As was mentioned earlier, autofluorescence arises from different fluorophores and will differ between tissues and within tissues.
So it maynot  have a single spectra, even in a single specimen-use the Meta software on autofluorescence only images to find how many different spectra you can find from whole cells and tissues and subregions (nuclei, cytoplasm etc)

Overall I would try to use fluorophores that work outside the autofluorescent range or try to alter fixation protocols to minimise autofluorescence, before resorting to linear unmixing.


Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories F5
Stockholm University
Stockholm 106 91
Sweden



-----Original Message-----
From: Confocal Microscopy List on behalf of Armstrong, Brian
Sent: Thu 13-Nov-08 18:13
To: [hidden email]
Subject: Re: Linear unmixing
 
Yes, you must have controls for each group/sample type. I have never
been able to get very "pleasing" images myself from the unmixing. I
however see Linear Unmixing as a quantitative tool and not so much a
pretty picture tool.

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 samrina aslam
Sent: Thursday, November 13, 2008 4:12 AM
To: [hidden email]
Subject: Linear unmixing

Hi,

I am currently using linear unmixing on the the Zeiss 510 Meta confocal
microscope to unmix the background autofluorescence from specific
fluorescence in parrafin sections.

I am using the 488nm laser, and the results are very varied depending on
the
tissue type I am imaging.

Has anybody tried linear unmixing using the 488nm laser and produced
good
images? Is there anything I can do to acquire better images?

Any help would be very much appreciated, thanks!


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[Neil Kad]

Hi Everyone,

We are currently advertising for a facility manager in Biological Sciences:

+++++++++

A university-funded post for a person to manage a multi-user and well established bioimaging facility in the area of Molecular medicine. The appointee is expected to be familiar with advanced cytometry, microscopy techniques, experimental protocols and analytical software. The facility is well equipped and includes a Beckton Dickinson FACSCalibur Flow Cytometer a Bio-Rad Radiance 2000 Confocal Microscope, Single particle fluorescence imaging high sensitivity fluorescence microscopy, wide-field microscopy, and a newly developed TIRF-equipped microscope. Routine maintenance of the instrumentation and infrastructure is expected. Collaborative research will be strongly encouraged.

A PhD or equivalent, in an appropriate scientific field, good interpersonal skills and experience in the management of multiple projects is required. Productive work experience in the form of peer-reviewed publications is also a requirement. An interest in computational modelling and image mining would be desirable. An opportunity in training on some aspects of bioimaging will also be available.
Please use the link below for further details about this job and how to make an application.  Visit our website: http://www.essex.ac.uk/ for information about the University of Essex.  If you have a disability and would like information in a different format, please telephone [phone]. 


www.jobs.ac.uk/jobs/YB112/

or
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+++++++++


Feel free to reply to this email for an informal discussion.


Thanks
Neil

=================================
Neil M. Kad PhD.,
Lecturer in Biological Sciences,
University of Essex,
Colchester,
Essex CO4 3SQ,
United Kingdom.

Tel: [phone]
Fax: [phone]

http://www.essex.ac.uk/bs/motor_protein/
=================================

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