Nipkow Disc vs. Line-Scanning Confocals (was Deconvolution...)

[O'Malley, Donald]

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Hi Gang,

I wrote a pretty basic review on confocal/2P/deconvolution in 2008, and it is available on the PDFs page of my digital-maze web site:   http://www.digital-maze.com/id3.html

I just wanted to offer an alternative view to the idea that Nipkow discs are faster and better for live preps than line-scanning confocals as I think that just the opposite is true.  These issues are discussed in the 2008 article where I reprint a figure from  Kockskamper et al., 2001 with a side-by-side comparison of the two confocal types, showing their complementary nature.  Very briefly, the problem for both FAST and LIVE imaging is shortage of photons and in many (perhaps most) cases it is useful to widen the aperture to trade away some z-resolution in exchange for more photons, indeed dramatically more photons, which you can easily do with the smoothly variable aperture of the MRC600 (which I still use!) and other line scanners.  This could be done with Nipkow disks if the user can easily switch between disks with different pinhole sizes-- has anyone done this and published improved spatiotemporal resolution or live cell results with it?  [I have not looked into this since 2008].

My bias comes from looking at calcium fluxes thru nuclear pores and seeing others image fluorophores diffusing from dendritic spines into dendritic shafts -- applications that really push the combined spatiotemporal limits of live cell/animal imaging, and this has been done entirely with line scanning instruments, including 2P (which totally trumps confocal if you can afford it).  As an additional wrinkle, I am not sure how much 2P-Nipkow has done in this area, but would be happy to receive PDFs on this at my email: [hidden email]<mailto:[hidden email]>.

As a further aside, please note that I acknowledged you guys in the 2008 review!  [i.e. this listserv was acknowledged-- thanks again to everyone who contributed articles and my apologies that I could not include all of them in the review].

Don O'Malley
Dept. Biology
[hidden email]<mailto:[hidden email]>
617-373-2284

"So much is gone and little is new
and so the sparrow sings dawn chorus for
someone else to hear."  CygnetCom

[Watkins, Simon C]

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In response to Don's comment on the generally fixed nature of the pinholes in spinning disk system limiting efficiency in live cell imaging.  For this very reason we have moved away from this approach to multipinhole systems where the apertures can be readily changed. To my knowledge there are two systems on the market.  The Prarie/Nikon Sweptfield scanner which allows pinholes or slits to be used and which we have been using successfully for many years or the Visitech scanhead(no experience with that system).  Importantly, the ability to change pinhole size at will is important not only to increase throughput when imaging dim specimens but also to match NA with pinhole size.


Simon C. Watkins Ph.D, FRC Path
Professor and Vice Chair Cell Biology
Professor Immunology
Director Center for Biologic Imaging
BSTS 225
University of Pittsburgh
3500 Terrace St
Pittsburgh PA 15261
412-352-2277
www.cbi.pitt.edu


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of O'Malley, Donald
Sent: Wednesday, November 02, 2011 8:36 AM
To: [hidden email]
Subject: Nipkow Disc vs. Line-Scanning Confocals (was Deconvolution...)

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Gang,

I wrote a pretty basic review on confocal/2P/deconvolution in 2008, and it is available on the PDFs page of my digital-maze web site:   http://www.digital-maze.com/id3.html

I just wanted to offer an alternative view to the idea that Nipkow discs are faster and better for live preps than line-scanning confocals as I think that just the opposite is true.  These issues are discussed in the 2008 article where I reprint a figure from  Kockskamper et al., 2001 with a side-by-side comparison of the two confocal types, showing their complementary nature.  Very briefly, the problem for both FAST and LIVE imaging is shortage of photons and in many (perhaps most) cases it is useful to widen the aperture to trade away some z-resolution in exchange for more photons, indeed dramatically more photons, which you can easily do with the smoothly variable aperture of the MRC600 (which I still use!) and other line scanners.  This could be done with Nipkow disks if the user can easily switch between disks with different pinhole sizes-- has anyone done this and published improved spatiotemporal resolution or live cell results with it?  [I have not looked into this since 2008].

My bias comes from looking at calcium fluxes thru nuclear pores and seeing others image fluorophores diffusing from dendritic spines into dendritic shafts -- applications that really push the combined spatiotemporal limits of live cell/animal imaging, and this has been done entirely with line scanning instruments, including 2P (which totally trumps confocal if you can afford it).  As an additional wrinkle, I am not sure how much 2P-Nipkow has done in this area, but would be happy to receive PDFs on this at my email: [hidden email]<mailto:[hidden email]>.

As a further aside, please note that I acknowledged you guys in the 2008 review!  [i.e. this listserv was acknowledged-- thanks again to everyone who contributed articles and my apologies that I could not include all of them in the review].

Don O'Malley
Dept. Biology
[hidden email]<mailto:[hidden email]>
617-373-2284

"So much is gone and little is new
and so the sparrow sings dawn chorus for
someone else to hear."  CygnetCom

[Paul Maddox]

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I agree 100% with Simon here.  I switched 4 years ago from spinning disk to
sweptfield scanners due to the increased flexibility.  I have been very
happy with this decision over the years.  For instance, very dim samples
that can not be imaged well on spinning disk are visible on sweptfield in
slit mode where more photons are delivered/detected.  Additionally, using
the smallest pinhole provides very good out-of-focus rejection for excellent
images.

Paul

Paul S. Maddox, PhD
Assistant Professor
Institute for Research in Immunology and Cancer
Dept of Pathology and Cell Biol, U. de Montreal



On 11/2/11 9:38 AM, "Simon Watkins" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> In response to Don's comment on the generally fixed nature of the pinholes in
> spinning disk system limiting efficiency in live cell imaging.  For this very
> reason we have moved away from this approach to multipinhole systems where the
> apertures can be readily changed. To my knowledge there are two systems on the
> market.  The Prarie/Nikon Sweptfield scanner which allows pinholes or slits to
> be used and which we have been using successfully for many years or the
> Visitech scanhead(no experience with that system).  Importantly, the ability
> to change pinhole size at will is important not only to increase throughput
> when imaging dim specimens but also to match NA with pinhole size.
>
>
> Simon C. Watkins Ph.D, FRC Path
> Professor and Vice Chair Cell Biology
> Professor Immunology
> Director Center for Biologic Imaging
> BSTS 225
> University of Pittsburgh
> 3500 Terrace St
> Pittsburgh PA 15261
> 412-352-2277
> www.cbi.pitt.edu
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of O'Malley, Donald
> Sent: Wednesday, November 02, 2011 8:36 AM
> To: [hidden email]
> Subject: Nipkow Disc vs. Line-Scanning Confocals (was Deconvolution...)
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Gang,
>
> I wrote a pretty basic review on confocal/2P/deconvolution in 2008, and it is
> available on the PDFs page of my digital-maze web site:
> http://www.digital-maze.com/id3.html
>
> I just wanted to offer an alternative view to the idea that Nipkow discs are
> faster and better for live preps than line-scanning confocals as I think that
> just the opposite is true.  These issues are discussed in the 2008 article
> where I reprint a figure from  Kockskamper et al., 2001 with a side-by-side
> comparison of the two confocal types, showing their complementary nature.
> Very briefly, the problem for both FAST and LIVE imaging is shortage of
> photons and in many (perhaps most) cases it is useful to widen the aperture to
> trade away some z-resolution in exchange for more photons, indeed dramatically
> more photons, which you can easily do with the smoothly variable aperture of
> the MRC600 (which I still use!) and other line scanners.  This could be done
> with Nipkow disks if the user can easily switch between disks with different
> pinhole sizes-- has anyone done this and published improved spatiotemporal
> resolution or live cell results with it?  [I have not looked into this since
> 2008].
>
> My bias comes from looking at calcium fluxes thru nuclear pores and seeing
> others image fluorophores diffusing from dendritic spines into dendritic
> shafts -- applications that really push the combined spatiotemporal limits of
> live cell/animal imaging, and this has been done entirely with line scanning
> instruments, including 2P (which totally trumps confocal if you can afford
> it).  As an additional wrinkle, I am not sure how much 2P-Nipkow has done in
> this area, but would be happy to receive PDFs on this at my email:
> [hidden email]<mailto:[hidden email]>.
>
> As a further aside, please note that I acknowledged you guys in the 2008
> review!  [i.e. this listserv was acknowledged-- thanks again to everyone who
> contributed articles and my apologies that I could not include all of them in
> the review].
>
> Don O'Malley
> Dept. Biology
> [hidden email]<mailto:[hidden email]>
> 617-373-2284
>
> "So much is gone and little is new
> and so the sparrow sings dawn chorus for
> someone else to hear."  CygnetCom