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DIC/phase through spinning disk

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Knecht, David

DIC/phase through spinning disk

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

Gary Laevsky-2

Re: DIC/phase through spinning disk

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

Hi Dave,

With the CSUX, there is a disc bypass option, so with the correct optics in the train, you can get true clean DIC/phase with one camera.

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht
Sent: Thursday, January 10, 2008 7:58 AM
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)

Knecht, David

Re: DIC/phase through spinning disk

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Yes, except if I remember correctly, that option costs more than a second camera, so I'm not sure that is the best option. I would rather have a second camera for the same cost. The downside is alignment of the two images if it is separate cameras. Dave

On Jan 10, 2008, at 8:41 AM, Gary Laevsky wrote:

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

With the CSUX, there is a disc bypass option, so with the correct optics in the train, you can get true clean DIC/phase with one camera.

From: Confocal Microscopy List [[hidden email]] On Behalf Of David Knecht
Sent: Thursday, January 10, 2008 7:58 AM
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)

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)

Paul Maddox

Re: DIC/phase through spinning disk

In reply to this post by Knecht, David

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

Hi Dave,

All you need is a shutter for transmitted light and an emission filter
wheel between the CSU head and the camera for DIC. If you put the
analyzer in the filter wheel aligned properly to the polarizer (crossed)
you can make excellent DIC images by rotating the analyzer in and out of
the imaging path. See Maddox, et al. 2003. A spinning disk confocal
microscope system for rapid high resolution, multimode, fluorescence
speckle microscopy and GFP imaging in living cells. In, Biophotonics,
Parts, A and B (G. Marriot and I. Parker, eds.), Methods in Enyzmology,
Vol. 360, Academic Press, SanDiego, CA. for more details.

Paul

Gary Laevsky-2

Re: DIC/phase through spinning disk

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

Hi Paul,

Although you can get excellent DIC images through the disc (using the
very easy mechanism you state), there is some artifact introduced by the
disc.

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
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Paul S. Maddox
Sent: Thursday, January 10, 2008 12:12 PM
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

Hi Dave,

All you need is a shutter for transmitted light and an emission filter
wheel between the CSU head and the camera for DIC. If you put the
analyzer in the filter wheel aligned properly to the polarizer (crossed)

you can make excellent DIC images by rotating the analyzer in and out of

the imaging path. See Maddox, et al. 2003. A spinning disk confocal
microscope system for rapid high resolution, multimode, fluorescence
speckle microscopy and GFP imaging in living cells. In, Biophotonics,
Parts, A and B (G. Marriot and I. Parker, eds.), Methods in Enyzmology,
Vol. 360, Academic Press, SanDiego, CA. for more details.

Paul

Kate Luby-Phelps

Re: DIC/phase through spinning disk

In reply to this post by Knecht, David

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

We bought that option and so far, we are underwhelmed. The DIC is not nearly good enough to
justify the expense.

John Herlihy

Re: DIC/phase through spinning disk -- Commercial Response

In reply to this post by Knecht, David

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

Dear David et al.,

In regards to the question about DIC and spinning disk confocals. I would like to point to the versatile design of the Olympus DSU spinning disk confocal system. With the DSU, the disk is easily moved in and out of the optical path via software, providing an elegant solution to this issue. You can easily configure the system to acquire fluorescent images and brightfield images with or without the disk. Therefore, you utilize the same optical path and the same camera for brightfield, confocal and epifluorescent imaging. This makes acquiring the highest quality fluorescent/DIC overlays effortless. Additionally, there is no requirement for a second camera, which alleviates any issues with aligning/registering two cameras. Additionally, registering a second camera could be either impossible or very expensive given the options for cameras with the same sensor dimensions as the commonly specified EMCCDs. Furthermore, the ease of switching the disk in and out of the optical path makes the DSU increasingly more versatile. For example, the DSU also is a fully functional epifluorescent scope and can be used such with or without deconvolution, or can be used in combination with filter wheels for Ca2+ imaging etc. This allows one scope to be used for multiple purposes, maximizing the expenses invested in high end frames and cameras.

For those requiring the utmost in live cell imaging, by alleviating the need for second cameras, or entire imaging systems, there should be plenty of room in the budget for the Olympus IX81-ZDC; assuring the proper focal plane is maintained during your long-term time lapse acquisitions.

Sincerely,

For those interested in seeing any Olympus systems in the New England area please call or e-mail:

J.D. Herlihy, Ph.D.

Research and Imaging Specialist

Optical Analysis Corporation

Three Bud Way, Suite #25

Nashua, NH 03063-1700

800-588-6054

Cell: 508-965-8894

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)

Stephen Cody

Re: DIC/phase through spinning disk

In reply to this post by Knecht, David

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: 61 3 9341 3155 Fax: 61 3 9341 3104
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

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)

This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error.
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.

Stephen Cody

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

Sorry,

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

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

-----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,

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

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

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

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)

Shalin Mehta

Re: DIC/phase through spinning disk

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: 61 3 9341 3155    Fax: 61 3 9341 3104
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: 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: 61 3 9341 3155    Fax: 61 3 9341 3104
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)

This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error.
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.

--
~~~~~~~~~~~~~~~~~~~~~~~~~
Shalin Mehta
mobile: +65-90694182
blog: shalin.wordpress.com
~~~~~~~~~~~~~~~~~~~~~~~~~~
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
~~~~~~~~~~~~~~~~~~~~~~~~~~~
What is light?
''Light behaves like Voltaire, the French genius, 250 years ago. He was born as a conservative Catholic, he lived as a liberal Protestant, and he returned to Catholicism when he faced the end of his life. Light is born as photons. It travels through space as a wave, and it dies ﬁnally as a photon.'' - Einstein as quoted in Lohmann's paper (JOSA-A, Vol. 17, No.10, pp.1755-1762)

Guy Cox

Re: DIC/phase through spinning disk

In reply to this post by Stephen Cody

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 +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net

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,

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

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

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

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)

lechristophe

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 +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net

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: 61 3 9341 3155    Fax: 61 3 9341 3104
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)

This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error.
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.

Michael Weber-4

Re: DIC/phase through spinning disk

In reply to this post by Guy Cox

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

But the transmitted light beam path usually isn't confocal.

Michael

Guy Cox 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 +61 2 9351 3176 Fax +61 2 9351 7682
> Mobile 0413 281 861
> ______________________________________________
> http://www.guycox.net
> <https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=http://www.guycox.net>
>
> ------------------------------------------------------------------------
> *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 <http://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
> *C*entral* R*esource for* A*dvanced* M*icroscopy
> Ludwig Institute for Cancer Research
> PO Box 2008 Royal Melbourne Hospital
> Victoria, 3050
> Australia
> *Tel:* 61 3 9341 3155 * **Fax:* 61 3 9341 3104
> *email:* [hidden email] <mailto:[hidden email]>
> www.ludwig.edu.au/labs/confocal.html
> <http://www.ludwig.edu.au/labs/confocal.html>
> www.ludwig.edu.au/confocal <http://www.ludwig.edu.au/confocal>
>
> Tip: Learn how to receive reminders about you microscope booking:
> www.ludwig.edu.au/confocal/Local/Booking_Hint.htm
> <http://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)
>
>
>
>
>
> ------------------------------------------------------------------------
> This communication is intended only for the named recipient and may
> contain information that is confidential, legally privileged or subject
> to copyright; the Ludwig Institute for Cancer Research does not waiver
> any rights if you have received this communication in error.
> 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.

Stephen Cody

Re: DIC/phase through spinning disk

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

Yes, Michael and Guy are correct, assuming your brightfield image in non-confocal.

Cheers

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008 Royal Melbourne Hospital
Parkville, Victoria, 3050
Australia
Tel: 61 3 9341 3155 Fax: 61 3 9341 3104
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:
http://www.ludwig.edu.au/confocal/Local/Booking_Hint.htm Type your signature here

________________________________

From: Confocal Microscopy List on behalf of Michael Weber
Sent: Tue 29/01/2008 11:58 PM
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 the transmitted light beam path usually isn't confocal.

Michael

Guy Cox 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 +61 2 9351 3176 Fax +61 2 9351 7682
> Mobile 0413 281 861
> ______________________________________________
> http://www.guycox.net <http://www.guycox.net/>
> <https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=http://www.guycox.net>
>
> ------------------------------------------------------------------------
> *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 <http://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
> *C*entral* R*esource for* A*dvanced* M*icroscopy
> Ludwig Institute for Cancer Research
> PO Box 2008 Royal Melbourne Hospital
> Victoria, 3050
> Australia
> *Tel:* 61 3 9341 3155 * **Fax:* 61 3 9341 3104
> *email:* [hidden email] <mailto:[hidden email]>
> www.ludwig.edu.au/labs/confocal.html
> <http://www.ludwig.edu.au/labs/confocal.html>
> www.ludwig.edu.au/confocal <http://www.ludwig.edu.au/confocal>
>
> Tip: Learn how to receive reminders about you microscope booking:
> www.ludwig.edu.au/confocal/Local/Booking_Hint.htm
> <http://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)
>
>
>
>
>
> ------------------------------------------------------------------------
> This communication is intended only for the named recipient and may
> contain information that is confidential, legally privileged or subject
> to copyright; the Ludwig Institute for Cancer Research does not waiver
> any rights if you have received this communication in error.
> 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.

Urs Utzinger

Re: DIC/phase through spinning disk

In reply to this post by lechristophe

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

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

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

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:

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

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

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)

Stephen Cody

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

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

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 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

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

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,

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

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

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

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)

Guy Cox

Re: DIC/phase through spinning disk

In reply to this post by lechristophe

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

You'r right, of course. I misread the letter as proposing to

process confocal images with QPm.

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 +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
     http://www.guycox.net

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Tuesday, 29 January 2008 11:56 PM
To: [hidden email]
Subject: Re: DIC/phase through spinning disk

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 +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net

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: 61 3 9341 3155    Fax: 61 3 9341 3104
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)

This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error.
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.

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