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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
So this technique is defined by your personal bibliography.
Can anyone who has access to a paper library tell us what is it about ?
I'd be interested to read the 1990 Cytometry paper.
Thanks in advance,
Christophe Leterrier
Carol Heckman wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> Mike and others-
> You are right about complexity. We as a field have never addressed the
> full complexity of the cell with any technique. Judge for yourself how
> the interference approach will slot into the high throughput/high
> content debate. I append the publications list at the end of the
> message. The method is technically "Tolansky interference" and if you
> search that in Google, you come up with the right answers about the
> optics. I just call the method solid substrate interference because it
> seems to make more sense to biologists.
> Carol
>
> Heckman, C.A., J.M. Urban, M.L. Cayer, Y. Li, N. Boudreau, J. Barnes,
> H.K. Plummer, III , J. Barnes, C. Hall, R. Kozma, and L. Lim. Novel
> p21-activated kinase-dependent protrusions characteristically formed at
> the edge of transformed cells. Exp. Cell Res. 295: 432-447, 2004.
> Heckman, C.A., H.K. Plummer, III, and R. Mukherjee. Enhancement of the
> transformed shape phenotype by microtubule inhibitors and reversal by an
> inhibitor combination. Int. J. Oncol. 16:709-723, 2000.
> Heckman, C.A., and R.J. Jamasbi. Describing shape dynamics in
> transformed cells through latent factors. Exp. Cell Res. 246:69-82, 1999.
> Heckman, C.A., H.K. Plummer III, and C.S. Runyeon. Persistent effects
> of phorbol 12-myristate 13-acetate (PMA): Possible implication of
> vesicle traffic. J. Cell. Phys. 166:217-230, 1996.
> Heckman, C.A., K.I. Oravecz, D. Schwab, and J. Pontén. Ruffling and
> locomotion: Role in cell resistance to growth factor-induced
> proliferation. J. Cell. Phys. 154:554-565, 1993.
> Heckman, C.A. Geometrical constraints on the shape of cultured cells.
> Cytometry 11: 771-783, 1990.
> Plummer, H.K., III, and C.A. Heckman. Transient expression of the
> transformed phenotype stimulated by
> 12-O-tetradecanoylphorbol-13-acetate. Exp. Cell Res. 188: 66-74, 1990.
> Heckman, C.A., A.E. Campbell, and B. Wetzel. Characteristic shape and
> surface changes in epithelial transformation. Exp. Cell Res. 169:
> 127-148, 1987.
> Heckman, C.A. "Cell Shape and Growth Control" In_ Advances in Cell
> Culture_, Vol. 4 (K. Maramorosch, ed.), Academic Press, New York, pp.
> 85-156, 1985.
> Olson, A.C., N.M. Larson, and C.A. Heckman. Classification of cultured
> mammalian cells by shape analysis and pattern recognition. PNAS USA 77:
> 1516-1520, 1980.
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>> Carol,
>>
>> I dont' think you'll alienate the community, but I welcome any
>> information on how 'solid substrate interference' can unravel the
>> complex systems biology issues inherent to cell function. Is such an
>> approach amendable to multiplexing, or is it a single point read out
>> you're talking about? Feel free to answer on/off the list.
>>
>> Regarding your estimate of complexity, I think it is much more
>> complicated than the phosphorylation issue since one has to also
>> factor in splicing, ubiquitinylation, sumoylation, neddylation,
>> methylation, and probably other 'ations'......so, the number is much
>> bigger than 100,000. ;)
>>
>> Mike
>>
>>
>>
>> On Jan 7, 2007, at 4:53 PM, Carol Heckman wrote:
>>
>>> Search the CONFOCAL archive at
>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>> <http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>
>>> Re: High throughput
>>> At the risk of alienating the entire confocal community, let me just
>>> point out that the fluorescence techniques are inherently flawed when
>>> it comes to doing work of this type. They don't make maximum
>>> advantage of the one thing cells have to offer optically, i.e.,
>>> refractive index difference, because the indexes of water and cells
>>> do not show a very big difference.
>>>
>>> Meanwhile, the interesting molecules that one might want to locate
>>> number ~25,000 proteins. They exist in possible as many as 5 or 10
>>> phosphorylated variants per protein. Since one can visualize perhaps
>>> 5 channels at once, one is able to answer about maybe 1/100,000 of
>>> the questions. It is an enormous amount of information to be
>>> throwing away! IMHO, solid substrate interference is a much better
>>> solution for high throughput.
>>> Carol
>>>
>>>> Search the CONFOCAL archive at
>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>> This probably one of my favorite topics, so here's my two cents:
>>>>
>>>> We've have had an automated image cytometer/high throughput
>>>> microscope for a couple of years, now, and as part of a larger group
>>>> in the Texas Medical Center, we now have 6 HTMs (which will likely
>>>> double in the next year or two). All the current scopes are
>>>> CCD-based, and likely this will stay the same, unless someone ends
>>>> up demonstrating a need for the confocal scopes.
>>>>
>>>> The various scopes on the market have wildly different specs in
>>>> terms of speed, resolution, focusing abilities/magnification limits,
>>>> and certainly software, and of course different price tags. No one
>>>> makes the perfect machine (as usual). For your stated goal of
>>>> monolayer cultures and 3D high res imaging, no one scope will do
>>>> both great; however, it depends upon how large the 3D structures are
>>>> you want to scope-we don't usually use a confocal unless the
>>>> structures are >~100 microns or more. I agree with previous posts
>>>> that deconvolution capability is important, but the only one i know
>>>> about with built-in Z stacks and decon capabilities (API) is
>>>> severely limited in mag (20x). Indeed, most scopes can't autofocus
>>>> very well at high NA 40x (0.9, 0.95NA), and certainly not well at
>>>> 60-100x; most are spec'ed out for 20x, but can also likely do 40x,
>>>> 0.75NA. A 40x/0.75NA lens isn't too bad, but such a lens certainly
>>>> isn't the choice of users if a 0.95NA will work. The higher NA
>>>> helps tremendously with lower levels of fluorescence. This really
>>>> is important when examining GFP fusions, as the use of low NA lenses
>>>> almost guarantees you'll be limited to looking at only very high,
>>>> non-physiological levels of expressed protein. Even with a much
>>>> brighter 0.9NA lens, from our experience with nuclear receptors,
>>>> only the bottom ~10-20% of transfected cells are close to
>>>> physiological levels; higher levels can be very misleading in terms
>>>> of the biology in question.
>>>>
>>>> The only exceptions i know about are the discontinued Q3DM/Beckman
>>>> IC-100, or the extremely expensive Evotec (700-800K). I don't know
>>>> about others, but if there are some that have validated autofocus at
>>>> high mag/high NA, i'd love to hear from those manufacturers. That
>>>> said, there are plenty of interesting experiments one can do at
>>>> lower res (nuclear translocation, proliferation, cell cycle, etc),
>>>> but the higher res/higher mag facilitates digging out much, much
>>>> more relevant biology....
>>>>
>>>> For an academic lab doing automated microscopy, with or without the
>>>> desire to do high volume screens, getting the best autofocus/mag
>>>> combination is the most important issue as far as i'm concerned.
>>>> Since we can take z stacks with 40, 60, or 100x water/oil lenses,
>>>> monolayers (or some 3D cultures) are now automatically scoped. Now
>>>> that we have a way to automate export to our trusty API decon box,
>>>> we're now adding restorative deconvolution and projection to our
>>>> routines. This is followed by importing the processed data into one
>>>> or two software programs (Cytoshop and more recently, Cell
>>>> Profiler). Determining your software needs and matching them to
>>>> software capabilities is easily the second item on the list to
>>>> consider (if not tied for first). Open access to the software for
>>>> customization (without needing to being a programer) is mandatory in
>>>> my mind, and i'm glad to say some htm software packages have moved
>>>> nicely into that direction (they used to be fully locked down). For
>>>> an academic lab, taking the usual experiments and going from 10-20
>>>> coverslips to a 384 well plate, or many plates, and getting the same
>>>> high res/high sensitivity images at HT rates, is a huge paradigm
>>>> shift in designing and carrying out experiments. These new
>>>> capabilities open the door for very high content, low-to-medium
>>>> scale screening experiments. Doing ~20 384 well plates for focused
>>>> siRNA or compound library screens is the standard experiment, now.
>>>> That isn't a Big Pharma-type screen, of course, but it certainly
>>>> something several postdocs couldn't do in a year.....
>>>>
>>>>
>>>> I'm looking forward to more commercial vendors jumping in and making
>>>> an increasingly versatile high res/high mag HTMs, and more and more
>>>> point and click software to facilitate higher end multiplexing. On
>>>> the fly customization of algorithms/protocols is high on my wish
>>>> list, and it seems that at least one group is moving in that
>>>> direction (PipelinePilot/Accelrys). Hopefully, someone will also
>>>> develop increasingly versatile/powerful database capabilities to
>>>> keep track of the oceans of data that come from these approaches,
>>>> but that's a whole other story....
>>>>
>>>> Mike
>>>>
>>>>
>>>>
>>>>
>>>> Michael A. Mancini, Ph.D.
>>>> Associate Professor
>>>> Director, Integrated Microscopy Core
>>>> Co-Director, Gulf Coast Consortium for Chemical Genomics
>>>> Department of Molecular and Cellular Biology
>>>> Baylor College of Medicine
>>>> Houston, TX 77030
>>>> 713 798 8952 voice
>>>> 713 798 3179 fax
>>>> 713 408 0179 cell
>>>> [log in to unmask] <mailto:[log in to unmask]>
>>>>
>>>>
>>>>
>>>> On Jan 4, 2007, at 12:00 AM, George McNamara wrote:
>>>>
>>>>> Search the CONFOCAL archive at
>>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>> <http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal> Hi Laurent,
>>>>>
>>>>> Try a high content screening system that integrates digital
>>>>> deconvolution. For example, Applied Precision,
>>>>> http://www.api.com/lifescience/arrayworxMF.html
>>>>>
>>>>> Try using more of the dynamic range of the digital CCD camera
>>>>> (binning and/or longer exposure time). this applies to widefield,
>>>>> Yokogawa spinning disk, BD/CARV spinning disk and deconvolution
>>>>> systems. Also, OptiGrid 2.0 (if any HCS systems use it) or the
>>>>> Cellomics system with the Zeiss Apotome (log in to the Cellomics
>>>>> website to see their appnote on this combo).
>>>>>
>>>>> Don't use the META detector (actually, I was impressed with the
>>>>> data in Koushik 2006 Biophys J 91:L99-L101, but that was in
>>>>> cuvettes, not cells).
>>>>>
>>>>> Try the recommendations of Cho and Lockett (2006 J Microsc 223:
>>>>> 15-25) to use the conventional Zeiss 510 PMT's in relatively low
>>>>> gain 12-bit mode.
>>>>>
>>>>> best wishes,
>>>>>
>>>>> George
>>>>>
>>>>>
>>>>> At 09:09 AM 1/3/2007, you wrote:
>>>>>> Search the CONFOCAL archive at
>>>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>>> Dear imagers!
>>>>>> I have a quite broad question regarding instrumentation. I am
>>>>>> currently looking for a confocal platform enabling automatic
>>>>>> scanning for large scale colocalization study on cell lines, as
>>>>>> well as high resolution 3D imaging. By this, I mean a system able
>>>>>> to acquire multi-channel, stacks from a microplate format and
>>>>>> 'designed' to scan for long runs.
>>>>>> I have been evaluating different commercial solutions for high
>>>>>> throughput screening but even though they are definitely faster
>>>>>> than conventional confocal (at least faster than the 510 meta that
>>>>>> am using) I am expecting better image quality. I know that there
>>>>>> is a trade off between throughput and image quality and I don't
>>>>>> think there is any ideal system able to scan fast and image with
>>>>>> amazing quality (and high resolution image is a very subjective
>>>>>> point of view).
>>>>>> But I would be very interesting if some people from the list could
>>>>>> share their opinions and feelings about this kind of equipment.
>>>>>> Any comments are very welcome.
>>>>>>
>>>>>> /Laurent
>>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> George McNamara, Ph.D.
>>>>> UCDMC Stem Cell Program
>>>>> Sacramento, CA
>>>>> [log in to unmask] <mailto:[log in to unmask]>
>>>
>>> --
>>> Carol A. Heckman, Ph.D.
>>> Director, Center for Microscopy & Microanalysis
>>> and Professor of Biological Sciences
>>> Bowling Green State University
>>> Bowling Green, OH 43403
>>> website: http://www.bgsu.edu/departments/biology/facilities/MnM
>
> --
> Carol A. Heckman, Ph.D.
> Director, Center for Microscopy & Microanalysis
> and Professor of Biological Sciences
> Bowling Green State University
> Bowling Green, OH 43403
> website: http://www.bgsu.edu/departments/biology/facilities/MnM
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