Re: DIC/phase through spinning disk

Posted by Stephen Cody on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DIC-phase-through-spinning-disk-tp591738p591747.html

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Dear Urs,

 

With the technique you describe, using the transmitted light detector (TLD) on a point scanning confocal and collecting (non-confocal) brightfield (BF) images, without any DIC or phase elements, you can get variable results.

 

If the cells are quite thick or rounded up and especially if grown on plastic the results can be quite pleasing. Unfortunately if the cells are flat, grown on a coverslip and you are using a water immersion lens (ie ideal conditions for confocal microscopy) the BF images can be very disappointing. The cell can be all but invisible. Hence contrast enhancing techniques such as phase, DIC, and QPm come into their own.

 

The opposite applies too. If the specimen is quite thick like a 6 day old zebrafish embryo, parts of the specimen may have too much contrast rendering parts of the BF image black and hence obscuring details. Using the software QPm you can overcome these limitations. You can even separate absorption signals such as that cause by minerals or melanin, from the phase signals. I have used this technique to image zebrafish embryos with melanin pigment, and removed the absorption contribution of the image so that all is left is the pure DIC or Phase image. This is not possible with pure optical methods where the absorption component of the image normally makes up about 30% of a DIC signal.

 

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]
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Urs Utzinger
Sent: Wednesday, 30 January 2008 2:41 PM
To: [hidden email]
Subject: Re: DIC/phase through spinning disk

 

Collecting transmitted light without a condenser using a point scanning system or spinning disk most likely results in “phase contrast like” images.

There are no software, phase rings or prisms required.

But Cody’s images in his publication seem to have better phase contrast then what I get with the method above.

 

Urs Utzinger

University of Arizona

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Tuesday, January 29, 2008 5:56 AM
To: [hidden email]
Subject: Re: DIC/phase through spinning disk

 

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal But isn't the transmitted light imaging non-confocal in a common confocal setup ? There is a detector and no pinhole on the condenser side, so the transmitted image is non-confocal, right ?

Christophe

On Jan 29, 2008 12:13 PM, Guy Cox <[hidden email]> wrote:

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

Well, I'm baffled. The QPm approach uses defocus contrast to calculate

 phase shift.  Confocal imaging should eliminate this.  So I can't see how

this could work.

 

                                                                                             Guy

 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
http://www.guycox.net

 

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From: Confocal Microscopy List on behalf of Stephen Cody
Sent: Tue 08/01/29 11:29 AM

To: [hidden email]

Subject: Re: DIC/phase through spinning disk

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

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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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.