Re: DIC/phase through spinning disk

Posted by Shalin Mehta on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DIC-phase-through-spinning-disk-tp591738p591751.html

Hi everyone,
Since DIC manipulates polarization of two sheared beams to achieve contrast based on phase gradient in specimen, it is unlikely to work with spinning disk systems. I tend to think that diffraction effects due to plenty of small obstructions (slit/pin-holes) will reduce the orthogonality of polarizations of sheared beams coming from condenser side wollaston prism. Similarly sample induced polarization variation in the sheared beams will be spoiled on the way out to objective side wollaston prism.

For qualitative work, one could use asymmetric illumination (by placing blocks in condenser BFP) that does give bas-relief effect but not as good a contrast as true DIC. We have started using this method with cells grown on plastic (plastic ruins polarization as well) and results seem encouraging. There is an interesting paper in science by Bechara Kachar in 1985 (http://www.sciencemag.org/cgi/content/abstract/227/4688/766) and recently by Jerome Mertz- http://www.opticsexpress.org/abstract.cfm?id=90277.

Cheers
Shalin

On Jan 29, 2008 8:32 AM, Stephen Cody <[hidden email]> wrote:

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

Sorry,

 

I should have added that: I have no commercial affiliation with iatia. We did share a fruitful academic collaboration with the company.

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008 Royal Melbourne Hospital
Victoria,      3050
Australia
Tel: [phone]    Fax: [phone]
email: [hidden email]
www.ludwig.edu.au/labs/confocal.html
www.ludwig.edu.au/confocal

Tip: Learn how to receive reminders about you microscope booking:
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-----Original Message-----
From: Stephen Cody
Sent: Tuesday, 29 January 2008 11:30 AM
To: 'Confocal Microscopy List'
Subject: RE: DIC/phase through spinning disk

 

G'day David,

 

While I have not tried the following technique with spinning disk confocals, I have used it with brightfield images collected with "conventional point scanning" confocals, I think it should also work well with spinning disk systems.

 

If you have a "through focus" series of brightfield images you can calculate the DIC images (or phase, Hoffman etc, etc) using software called QPm (Or QPI) Developed by Keith A. Nugent et al., and supplied by iatia. See list of papers at   www.iatia.com . You should be able to do this with any archival data you have saved to disk, provided it is a z-series of brightfield images.

 

You can see my paper specifically relating to QPm and confocal:

Cody, S.H., Xiang, S.D., Layton, M.J., Handman, E., Lam, M.H.C., Nice, E.C., and Heath, J.K. A simple method allows DIC imaging in conjunction with confocal microscopy. J. Microscopy.  217: 265-274 (2005).

http://dx.doi.org/10.1111/j.1365-2818.2005.01452.x

 

Application note:

http://www.iatia.com.au/technology/applicationNotes/qpmApplicationWithConfocalMicroscopes.pdf

 

Other application notes:

http://www.iatia.com.au/technology/applicationNotes.asp

 

Cheers

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008 Royal Melbourne Hospital
Victoria,      3050
Australia
Tel: [phone]    Fax: [phone]
email: [hidden email]
www.ludwig.edu.au/labs/confocal.html
www.ludwig.edu.au/confocal

Tip: Learn how to receive reminders about you microscope booking:
www.ludwig.edu.au/confocal/Local/Booking_Hint.htm 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht
Sent: Thursday, 10 January 2008 11:58 PM
To: [hidden email]
Subject: DIC/phase through spinning disk

 

We are in the process of putting together a spinning disk confocal system.  We have heard mixed things about how acceptable it is to do transmitted light microscopy (DIC or Phase) through the spinning disk, as opposed to running a separate camera through a separate microscope port.  What have others found?  

 

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)

 

 

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--
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Shalin Mehta
mobile: [phone]
blog: shalin.wordpress.com
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Bioimaging Lab, Block-E3A, #7-10                
Div of Bioengineering, NUS Singapore 117574  
website: http://www.bioeng.nus.edu.sg/optbioimaging/colin/index.html

Liver Cancer Functional Genomics Lab, #6-05
National Cancer Centre, Singapore 169610
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