Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
You make some interesting points! It does seem that the low threshold
capability of PMT's make them more useful for our photon counting type
experiments. Still, in the case of a brighter sample the speed of an
EMCCD would be useful.
Thanks,
Craig
On 10/6/07, George McNamara <[log in to unmask]> wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> Hi Craig,
>
> The Lightform PARISS has a detection slit in front of the spectral
> dispersion element and the CCD. This makes it half-confocal. If a matching
> slit is placed in the epi-illumination field aperture plane (or a conjugate
> plane) it is a slit confocal (aka line scan confocal). The result is an
> X*Spectral image. If the microscope has a motorized stage, the Y-axis can be
> added = push broom confocal imaging spectrometer. The performance is such
> that the emission filter can be removed from the epi-illumination filter
> cube when used with an Hg lamp (the bright Hg line or lines are one
> nanometer or so side = 1 or a few pixels, so do not bleed into the emission
> areas and can be used to quantify the excitation power). The PARISS can also
> be used for brightfield images.
>
> You can contact Jeremy Lerner at http://www.lightforminc.com/ for
> additional details about the PARISS.
>
>
> With respect to PMT vs CCD or EMCCD, Pawley's rule is that a typical point
> scanning confocal microscope detects a maximum of about 8 photons per pixel
> per scan. This is a nice match to a PMT (or APD for far red and infrared
> photons), not so good for a CCD (see Jim's post about noise). On the other
> hand, a CCD is an area detector (or line * spectrum detector for the
> PARISS), so the CCD is a great way to acquire 2D images fast. A CCD or EMCCD
> system can be used to image a thin optical section, quickly, with judicious
> use of excitation light, as in TIRF, fast multipoint multiphoton scanners
> such as the TriMScope (if it can be made to work reliably) or Scherer's
> stochastic scanner (
> http://www.opticsexpress.org/abstract.cfm?id=89328), or
> hopefully by widefield multiphoton excitation (Brooker 2007 US patent
> 7,170,675 ), or by multiplane acquisition followed by digital deconvolution.
> If you can buy or borrow 3 EMCCD's like Ian Parker, you can acquire multiple
> focal planes simultaneously, see PubMed 17716727, also similar rig by
> Prabhat, PubMed 17384151.
>
> Back to 32-channel PMT confocal microscopes: the Zeiss META detector
> includes blockers for the major laser lines. As far as I am aware, they are
> in the light path all the time (the 488 nm blocker may be responsible for
> zigzag artifacts in CFP emission spectra, though bad PMT channels could also
> be responsible). Nikon may include similar blockers in the si. The Zeiss
> LSM510 light path can be configured to use a microscope filter cube. With
> the right cube and a mirror slide, you can acquire the spectrum of the Hg
> lamp using the META, or you could buy a Lightform MIDL like Bob Zucker's and
> just lay it on the microscope. The META spectrum of the MIDL lamp is not
> even close to the calibration curve that comes with the MIDL. Bob and Jeremy
> have published the close match of the PARISS data for the MIDL.
>
>
> Enjoy,
>
>
> George
>
>
>
> At 07:26 PM 10/5/2007, you wrote:
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Thanks! We are interested in co-localization, for which we sometimes
> need the third dimension. Mainly we want to do spectral unmixing for
> multiple dyes and colocalization, so we really do need optical
> slicing!
>
> Thanks,
>
> Craig
>
>
> On 10/5/07, Robert Zucker <[log in to unmask]> wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Craig
> > you are correct.
> > Currently the PARISS only detects X-Y--not depth.
> > however, most data that I have seen from confocal spectral systems are
> > only a single XY slice. they usually are not used to do a 3D
> > reconstruction in spectral mode.
> > bob
> > .
> >
> >
> > Robert M. Zucker, PhD
> > U.S. Environmental Protection Agency
> > Office of Research and Development
> > National Health and Environmental Effects Research Laboratory.
> > Telephone: 919-541-1585 Fax: 919-541-4017
> > e-mail: [log in to unmask]
> >
> > Mail address: Reproductive Toxicology Division, MD 67
> > 2525 E.NC Highway 54
> > Research Triangle Park, North Carolina, 27711
> >
> > Shipping address: 2525 E.NC Highway 54
> > Durham, NC, 27713
> >
> >
> >
> >
> > Craig Brideau
> > <craig.brideau@G
> > MAIL.COM>
> To
> > Sent by: [log in to unmask]
> > Confocal
> cc
> > Microscopy List
> > <CONFOCAL@LISTSE
> Subject
> > RV.BUFFALO.EDU> Re: Nikon C1si?
> >
> >
> > 10/05/2007 06:44
> > PM
> >
> >
> > Please respond
> > to
> > Confocal
> > Microscopy List
> > <CONFOCAL@LISTSE
> > RV.BUFFALO.EDU>
> >
> >
> >
> >
> >
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > It doesn't seem to deal with depth though? It only detects in X-Y, so
> > can it generate optical slices?
> >
> > Craig
> >
> > On 10/5/07, Robert Zucker <[log in to unmask]> wrote:
> > > Search the CONFOCAL archive at
> > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > The PARISS system uses a Retiga 2000R cooled CCD camera. It is far
> > more
> > > sensitive and accurate than any confocal microscope.
> > > You can detect things in that system that can not be observed with a
> > > confocal spectral system. It is a great system in my opinion.
> > > Bob
> > >
> > > Robert M. Zucker, PhD
> > > U.S. Environmental Protection Agency
> > > Office of Research and Development
> > > National Health and Environmental Effects Research Laboratory.
> > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > e-mail: [log in to unmask]
> > >
> > > Mail address: Reproductive Toxicology Division, MD 67
> > > 2525 E.NC Highway 54
> > > Research Triangle Park, North Carolina, 27711
> > >
> > > Shipping address: 2525 E.NC Highway 54
> > > Durham, NC, 27713
> > >
> > >
> > >
> > >
> > > Craig Brideau
> > > <craig.brideau@G
> > > MAIL.COM>
> > To
> > > Sent by: [log in to unmask]
> > > Confocal
> > cc
> > > Microscopy List
> > > <CONFOCAL@LISTSE
> > Subject
> > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > >
> > >
> > > 10/05/2007 06:19
> > > PM
> > >
> > >
> > > Please respond
> > > to
> > > Confocal
> > > Microscopy List
> > > <CONFOCAL@LISTSE
> > > RV.BUFFALO.EDU>
> > >
> > >
> > >
> > >
> > >
> > >
> > > Search the CONFOCAL archive at
> > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > Will this system have the same sort of sensitivity as a multi-anode
> > > PMT system like the C1si? This PARISS system seems to use a CCD
> > > camera as its detector, which will not be as sensitive, if I
> > > understand correctly?
> > >
> > > Craig
> > >
> > > On 10/5/07, Robert Zucker <[log in to unmask]> wrote:
> > > > Search the CONFOCAL archive at
> > > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > >
> > > > Criag
> > > > If spectral analysis is the endpoint you may want to consider the
> > > PARISS
> > > > system from Lightform. ( http://www.lightforminc.com/) that can be
> > > > placed on a widefield microscope.
> > > > We are using the PARISS system to obtain spectral data --it has 1nm
> > > > resolution and has a spectrum from 400-900nm. I find it to be a
> > great
> > > > asset in the laboratory,
> > > > It is far more sensitive and accurate that any confocal system that
> > I
> > > > have seen.
> > > >
> > > > Contact Jeremy Lerner for details on his system.
> > > > best wishes
> > > > Bob
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > LightForm, Inc.,
> > > > 601 Route 206, Suite 26-479
> > > > Hillsborough NJ 08844
> > > > Tel: (908)281 9098
> > > > Email: [log in to unmask]
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > .
> > > >
> > > > Robert M. Zucker, PhD
> > > > U.S. Environmental Protection Agency
> > > > Office of Research and Development
> > > > National Health and Environmental Effects Research Laboratory.
> > > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > > e-mail: [log in to unmask]
> > > >
> > > > Mail address: Reproductive Toxicology Division, MD 67
> > > > 2525 E.NC Highway 54
> > > > Research Triangle Park, North Carolina, 27711
> > > >
> > > > Shipping address: 2525 E.NC Highway 54
> > > > Durham, NC, 27713
> > > >
> > > >
> > > >
> > > >
> > > > Craig Brideau
> > > > <craig.brideau@G
> > > > MAIL.COM>
> > > To
> > > > Sent by: [log in to unmask]
> > > > Confocal
> > > cc
> > > > Microscopy List
> > > > <CONFOCAL@LISTSE
> > > Subject
> > > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > > >
> > > >
> > > > 10/05/2007 05:48
> > > > PM
> > > >
> > > >
> > > > Please respond
> > > > to
> > > > Confocal
> > > > Microscopy List
> > > > <CONFOCAL@LISTSE
> > > > RV.BUFFALO.EDU>
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > Search the CONFOCAL archive at
> > > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > >
> > > > Thanks for your input! As in everything, there's a trade-off.
> > Still,
> > > > for us the spectral data will be worth it.
> > > >
> > > > Craig
> > > >
> > > > On 10/5/07, Robert Zucker <[log in to unmask]> wrote:
> > > > > Search the CONFOCAL archive at
> > > > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > > >
> > > > > Craig
> > > > > The efficiency of detection is much less in the spectral mode due
> > to
> > > > the
> > > > > design of the multianode detector. In addition as Kurt Thorn said
> > > the
> > > > > light is split into a number of different bandpass channels
> > instead
> > > of
> > > > > being detected by only one channel which also limits the detection
> > > in
> > > > > each channel.
> > > > > Nikon compensates for this decreased light by increasing the pixel
> > > > dwell
> > > > > time. However photons are photons. With less photons you will get
> > a
> > > > > noisier image.
> > > > > The spectral detection of any confocal system using a multianode
> > > > > detector will not be as good as a PMT designed for good
> > sensitivity
> > > > and
> > > > > low light detection. It will produce nosier images but if there is
> > > > > enough light you will be able to determine a valuable spectrum
> > which
> > > > can
> > > > > be used for scientific experiments and to manipulate images. Like
> > > > > everything with confocal microscopy there is trade-offs and no
> > > perfect
> > > > > system.
> > > > > Best wishes.
> > > > > Bob
> > > > >
> > > > > Robert M. Zucker, PhD
> > > > > U.S. Environmental Protection Agency
> > > > > Office of Research and Development
> > > > > National Health and Environmental Effects Research Laboratory.
> > > > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > > > e-mail: [log in to unmask]
> > > > >
> > > > > Mail address: Reproductive Toxicology Division, MD 67
> > > > > 2525 E.NC Highway 54
> > > > > Research Triangle Park, North Carolina, 27711
> > > > >
> > > > > Shipping address: 2525 E.NC Highway 54
> > > > > Durham, NC, 27713
> > > > >
> > > > >
> > > > >
> > > > >
> > > > > Craig Brideau
> > > > > <craig.brideau@G
> > > > > MAIL.COM>
> > > > To
> > > > > Sent by:
> > [log in to unmask]
> > > > > Confocal
> > > > cc
> > > > > Microscopy List
> > > > > <CONFOCAL@LISTSE
> > > > Subject
> > > > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > > > >
> > > > >
> > > > > 10/04/2007 02:49
> > > > > PM
> > > > >
> > > > >
> > > > > Please respond
> > > > > to
> > > > > Confocal
> > > > > Microscopy List
> > > > > <CONFOCAL@LISTSE
> > > > > RV.BUFFALO.EDU>
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > > Search the CONFOCAL archive at
> > > > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > > >
> > > > > I was wondering if you saw this decreased throughput in spectral
> > > mode
> > > > > only, or also in conventional detection mode? The spectral mode
> > > works
> > > > > with a multi-anode PMT, which has a lower quantum efficiency than
> > a
> > > > > conventional PMT. From what I have seen of the design, it also
> > has
> > > > > the option to use a conventional PMT system with the device, and I
> > > am
> > > > > wondering if you have also tried that mode of operation?
> > > > >
> > > > > Thanks,
> > > > >
> > > > > Craig
> > > > >
> > > > > On 10/4/07, Robert Zucker <[log in to unmask]> wrote:
> > > > > > One of the major limitations that we have seen with this unit
> > and
> > > > also
> > > > > > with the Zeiss meta 510 is the decreased light throughput .
> > This
> > > > > > creates images that are noisier than conventional confocal
> > > > > microscopes.
> > > > > > In our hands it appears you will need to have a bright sample to
> > > > make
> > > > > > the spectral system work properly. ,
> > > > >
> > > >
> > >
> >
>
>
>
>
>
>
> George McNamara, Ph.D.
> University of Miami, Miller School of Medicine
> Image Core
> Miami, FL 33010
> [log in to unmask]
> [log in to unmask]
> 305-243-8436 office
> http://home.earthlink.net/~pubspectra/
> http://home.earthlink.net/~geomcnamara/
> http://www.sylvester.org/health_pro/shared_resources/index.asp
> (see Analytical Imaging Core Facility)
>
>
>
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