What methods are used to image morphology in high content screening systems?
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Shalin Mehta |
What methods are used to image morphology in high content screening systems?
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Dear listers,
DIC and phase-contrast are used widely (albeit, less than fluorescence) for imaging cellular morphology . I am curious about what approaches are used to image morphology in the high content screening applications. I have come across HCS systems on the web that use either DIC or phase-contrast. Do some systems use hoffman modulation contrast? If you could list the system that you have and the type of contrast they employ for imaging the morphology, that will be helpful. Since high-content screening requires automated analysis, and these methods are not really quantitative, do people find it difficult to address some biological questions? My thesis is related to quantitative morphological imaging and I am trying to get the users' perspective on the topic. Thanks very much for your inputs, Shalin 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/people.asp#shalinm |
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George McNamara |
Re: What methods are used to image morphology in high content screening systems?
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Hi Shalin,
BD Pathway 855 HCS ... brightfield Phase contrast (halo), DIC (edges along the shear axis, nothing along the perpendicular axis), Hoffman (similar problem to DIC), are all problematic for clean morphometry. Darkfield is problematic is there is any junk in the lightpath that scatters light (see CytoViva product for interesting NA>1.2 condenser product). A 12-bit CCD camera has plenty of information in it for segmenting brightfield images. I believe GE Healthcare licensed IATIA's quantitative phase map QPm algorithm for (some of?) their InCell HCS instruments ( http://www.iatia.com.au/products/lifeSciences/prodappGe.asp ). Whether anyone is using QPm - ask customers. QPm and similar algorithms (Michael Feld lab, see Holmes chapter in Pawkey, Kam/Agard/Sedat) compute the refractive index map in the image. Graham Dunn published something similar with DRIMAS (PMID: 2592444). These methods are quantitative. By the way, IAITA ( http://www.iatia.com.au/technology/insideQpi.asp ) emphasizes on their web site and literature the conversion of the QPm data to phase contrast, DIC or darkfield like images. This seems bizarre to me because the real value is in the quantitative map of R.I. At least two papers computing QPm maps from the (non-confocal) transmitted light path of confocal microscopes have been published:
Plan B; soluble GFP (if anyone can find a promoter that results in uniform expression in #GFP/um^3 in every cell in a population - good luck ... could be any color FP of course) or much simpler, a fluorescent dye that forms a covalent bond to most cell proteins, see Coates et al 1992, PMID: 1577856, for example (the Texas Red conjugate, not the phalloidin conjugate, in that paper). Best wishes, George p.s. speaking of CytoViva - has anyone used a laser pointer as illuminator with a CytoViva? At 11:40 AM 7/16/2010, you wrote: Dear listers, |
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George McNamara |
Re: What methods are used to image morphology in high content screening systems?
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In reply to this post by Shalin Mehta
Hi Shalin,
Two more items... 1. Interference reflection microscopy (IRM) and reflection interference contrast microscopy (RICM) can enable precise imaging of both cell-substratum adhesive sites (black = focal adhesions) as well as the outlines of cells (including ultrathin lamellipodia and filopodia). Easy to implement on a confocal microscope (no emission filter, though circularly polarized light or antiflex objective lens may help improve image quality). Same optical configuration provides the cell outline as you focus into the cell (provided R.I. or media is different than the plasma membrane and cell!. See PMID: 19816893, 19821476, 3900106, and especially 20013754 (if you do not have access to C.P.C., email the author for the article) for quantitative details. 2. efficient use of cells ... see http://www.nature.com/nmeth/journal/v7/n7/abs/nmeth.1462.html or at least the company's press release at http://www.cytoo.com/cytoo_newsroom.php and be sure to crank up your audio and go to http://www.cellebrate.fr/ Enjoy, George At 11:40 AM 7/16/2010, you wrote: Dear listers, |
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In reply to this post by George McNamara
I was involved as an early evaluation site for the Iatia system
(based on Keith Nugent’s algorithms) so I feel I should comment on this post. The idea behind the system is something that every microscopist is
familiar with, that contrast is lowest at true focus. A bit out of focus and
phase effects enhance the contrast. This is measurable, so by
taking a through-focus series one can recover the phase at the image plane.
The primary output of the software is therefore a phase map of the whole
image. If that is what one wants of course one needs to go no
further. But once one has this information it is easy to synthesize other
imaging modalities such as phase contrast and DIC. The really useful
aspect of this is that one can then control how much of the amplitude image to
feed in to the final output. Optical DIC and phase contrast systems do
not give you that control. So why didn’t this system take the world by storm?
First off, most of us have phase and DIC anyway, and in fact it can be very
hard to buy high-end lenses without phase plates. So unless we need
that full phase map it isn’t going to give us much for the purchase
price. Secondly, and this is the real killer, most people use phase and DIC
to look at living cells and we can’t get them to sit still for a
through-focus series, so it just doesn’t work. The bottom line? A brilliant idea, and one which works, but
still seeking the right niche in the market.
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) Australian Centre for Microscopy & Microanalysis, Madsen Building F09, University of Sydney, NSW 2006 Phone +61 2 9351 3176 Fax +61 2 9351
7682
Mobile 0413 281 861 ______________________________________________ From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of George McNamara Hi Shalin,
You can make arrangements to have test images processed by
IATIA - send them an email. Dear listers,
No virus
found in this incoming message. |
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Pascal Lorentz-2 |
Magnetic fields and microscopy
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Dear list,
this is not exactly a confocal question but anyway related to it. I hope to get some input from people who have some experience with magnetic fields and the influence on microscopy but also on confocal scanners. In our building we are planning to centralize our microscopy facility in the second basement. Right now the architects are doing a feasibility study. Because the new rooms are located between the elevator machine room and the high voltage current room and since the electric cables from the whole house go through these rooms they now ask about magnetic fields. Does somebody know if magnetic fields can influence microscopy (cells for example), the microscopes itself, the lasers or scanners (galvos)? If yes, is there a threshold that is acceptable? I would be very thankful if I could get more information on that topic. Best regards Pascal -- Pascal Lorentz BioOptics Facility Department of Biomedicine University of Basel Mattenstrasse 28 4058 Basel Switzerland |
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Johannes Helm |
Re: Magnetic fields and microscopy
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Good afternoon,
whether the electromagnetic fields will have any non-negligible influence on the biological specimens certainly is a difficult problem. Whether a reviewer refereeing a manuscript for publication would think that the effects of the electromagnetic fields in your lab would cast any doubts on the reliability of the presented observations and results most probably is to a large degree dependent on the reviewer's "personal opinion". There has been and still is a lot of writing about electro smog caused by AC HV lines used to transport electric energy, by AC HV lines for train traffic and, even more important, the effect of the electro magnetic fields to which one is exposed when using cell phones. Concerning the microscopes, lasers, detectors a.s.o.: I cannot see any influence that an electromagnetic field of the strength of one generated by the sources you mention would have on the microscope itself. The magnetic field necessary to alter the plane of polarization of polarized light in a non negligble way, see "Faraday Effect", certainly is much stronger than the fields you are going to experience. There should, hence, not be any noticeable effect even of you plan to do quantitative polarization microscopy. Light detectors using HV, e.g. photomultiplier tubes, are usually mounted in tubes made out of a material, which shields magnetic fields. Best wishes for your work with and in the new lab. Johannes > Dear list, > > this is not exactly a confocal question but anyway related to it. I hope > to get some input from people who have some experience with magnetic > fields and the influence on microscopy but also on confocal scanners. > In our building we are planning to centralize our microscopy facility in > the second basement. Right now the architects are doing a feasibility > study. > Because the new rooms are located between the elevator machine room and > the high voltage current room and since the electric cables from the > whole house go through these rooms they now ask about magnetic fields. > Does somebody know if magnetic fields can influence microscopy (cells > for example), the microscopes itself, the lasers or scanners (galvos)? > If yes, is there a threshold that is acceptable? > I would be very thankful if I could get more information on that topic. > > Best regards > > Pascal > > -- > Pascal Lorentz > BioOptics Facility > Department of Biomedicine > University of Basel > Mattenstrasse 28 > 4058 Basel > Switzerland > -- P. Johannes Helm Voice: (+47) 228 51159 (office) Fax: (+47) 228 51499 (office) |
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Craig Brideau |
Re: Magnetic fields and microscopy
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The electrical noise will probably not be good for your readings.
Unless you are willing to put the whole room in a Faraday cage I would be concerned. You will also probably want line filters as well; this will filter the power coming out of your wall sockets, so any EM they pick up from the nearby high voltage stuff will not make it into your equipment. Overall I'd say your planned location is less than ideal, but with sufficient measures you could still make it work. Good line filters and shielding should keep out the nearby sources of electrical interference. Craig On Tue, Jul 20, 2010 at 8:08 AM, Johannes Helm <[hidden email]> wrote: > Good afternoon, > > whether the electromagnetic fields will have any non-negligible influence > on the biological specimens certainly is a difficult problem. > Whether a reviewer refereeing a manuscript for publication would think > that the effects of the electromagnetic fields in your lab would cast any > doubts on the reliability of the presented observations and results most > probably is to a large degree dependent on the reviewer's "personal > opinion". There has been and still is a lot of writing about electro smog > caused by AC HV lines used to transport electric energy, by AC HV lines > for train traffic and, even more important, the effect of the electro > magnetic fields to which one is exposed when using cell phones. > > Concerning the microscopes, lasers, detectors a.s.o.: > > I cannot see any influence that an electromagnetic field of the strength > of one generated by the sources you mention would have on the microscope > itself. > The magnetic field necessary to alter the plane of polarization of > polarized light in a non negligble way, see "Faraday Effect", certainly is > much stronger than the fields you are going to experience. There should, > hence, not be any noticeable effect even of you plan to do quantitative > polarization microscopy. > Light detectors using HV, e.g. photomultiplier tubes, are usually mounted > in tubes made out of a material, which shields magnetic fields. > > Best wishes for your work with and in the new lab. > > Johannes > > > > >> Dear list, >> >> this is not exactly a confocal question but anyway related to it. I hope >> to get some input from people who have some experience with magnetic >> fields and the influence on microscopy but also on confocal scanners. >> In our building we are planning to centralize our microscopy facility in >> the second basement. Right now the architects are doing a feasibility >> study. >> Because the new rooms are located between the elevator machine room and >> the high voltage current room and since the electric cables from the >> whole house go through these rooms they now ask about magnetic fields. >> Does somebody know if magnetic fields can influence microscopy (cells >> for example), the microscopes itself, the lasers or scanners (galvos)? >> If yes, is there a threshold that is acceptable? >> I would be very thankful if I could get more information on that topic. >> >> Best regards >> >> Pascal >> >> -- >> Pascal Lorentz >> BioOptics Facility >> Department of Biomedicine >> University of Basel >> Mattenstrasse 28 >> 4058 Basel >> Switzerland >> > > > -- > P. Johannes Helm > > Voice: (+47) 228 51159 (office) > Fax: (+47) 228 51499 (office) > |
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Craig Brideau |
Re: Magnetic fields and microscopy
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To clarify my comment; any wires in your system carrying signal will
couple like an antenna to the nearby sources of EM. It's not so much that the EM itself will influence your samples or light, its just that you can expect to see a bunch of 60 Hz or other frequency electrical noise on every wire/cable/connection in the lab if you don't have good shielding. A fun test is to get a long BNC cable, or better yet an unshielded wire, plug it into an oscilloscope, and then see what you pick up in the room as you move the cable around. The frequencies and magnitudes on the oscilloscope will give you some idea of how bad the EM noise signals in the room are. Craig On Tue, Jul 20, 2010 at 4:47 PM, Craig Brideau <[hidden email]> wrote: > The electrical noise will probably not be good for your readings. > Unless you are willing to put the whole room in a Faraday cage I would > be concerned. You will also probably want line filters as well; this > will filter the power coming out of your wall sockets, so any EM they > pick up from the nearby high voltage stuff will not make it into your > equipment. Overall I'd say your planned location is less than ideal, > but with sufficient measures you could still make it work. Good line > filters and shielding should keep out the nearby sources of electrical > interference. > > Craig > > > On Tue, Jul 20, 2010 at 8:08 AM, Johannes Helm <[hidden email]> wrote: >> Good afternoon, >> >> whether the electromagnetic fields will have any non-negligible influence >> on the biological specimens certainly is a difficult problem. >> Whether a reviewer refereeing a manuscript for publication would think >> that the effects of the electromagnetic fields in your lab would cast any >> doubts on the reliability of the presented observations and results most >> probably is to a large degree dependent on the reviewer's "personal >> opinion". There has been and still is a lot of writing about electro smog >> caused by AC HV lines used to transport electric energy, by AC HV lines >> for train traffic and, even more important, the effect of the electro >> magnetic fields to which one is exposed when using cell phones. >> >> Concerning the microscopes, lasers, detectors a.s.o.: >> >> I cannot see any influence that an electromagnetic field of the strength >> of one generated by the sources you mention would have on the microscope >> itself. >> The magnetic field necessary to alter the plane of polarization of >> polarized light in a non negligble way, see "Faraday Effect", certainly is >> much stronger than the fields you are going to experience. There should, >> hence, not be any noticeable effect even of you plan to do quantitative >> polarization microscopy. >> Light detectors using HV, e.g. photomultiplier tubes, are usually mounted >> in tubes made out of a material, which shields magnetic fields. >> >> Best wishes for your work with and in the new lab. >> >> Johannes >> >> >> >> >>> Dear list, >>> >>> this is not exactly a confocal question but anyway related to it. I hope >>> to get some input from people who have some experience with magnetic >>> fields and the influence on microscopy but also on confocal scanners. >>> In our building we are planning to centralize our microscopy facility in >>> the second basement. Right now the architects are doing a feasibility >>> study. >>> Because the new rooms are located between the elevator machine room and >>> the high voltage current room and since the electric cables from the >>> whole house go through these rooms they now ask about magnetic fields. >>> Does somebody know if magnetic fields can influence microscopy (cells >>> for example), the microscopes itself, the lasers or scanners (galvos)? >>> If yes, is there a threshold that is acceptable? >>> I would be very thankful if I could get more information on that topic. >>> >>> Best regards >>> >>> Pascal >>> >>> -- >>> Pascal Lorentz >>> BioOptics Facility >>> Department of Biomedicine >>> University of Basel >>> Mattenstrasse 28 >>> 4058 Basel >>> Switzerland >>> >> >> >> -- >> P. Johannes Helm >> >> Voice: (+47) 228 51159 (office) >> Fax: (+47) 228 51499 (office) >> > |
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Craig Brideau |
Re: Magnetic fields and microscopy
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PMTs need a trans-impedance amplifier to convert the current spikes
caused by the photon impacts into something instrumentation can read. They can be very noise sensitive, especially if your ground lines start picking up surrounding signals. Craig On Tue, Jul 20, 2010 at 5:37 PM, Donnelly, Tom <[hidden email]> wrote: > Don't know how this effects PMT's and galvo's but an CCD or EMCCD are great detectors of EM noise. > > TD > > -----Original Message----- > From: Craig Brideau [mailto:[hidden email]] > Sent: Tuesday, July 20, 2010 3:51 PM > To: [hidden email] > Subject: Re: Magnetic fields and microscopy > > To clarify my comment; any wires in your system carrying signal will couple like an antenna to the nearby sources of EM. It's not so much that the EM itself will influence your samples or light, its just that you can expect to see a bunch of 60 Hz or other frequency electrical noise on every wire/cable/connection in the lab if you don't have good shielding. A fun test is to get a long BNC cable, or better yet an unshielded wire, plug it into an oscilloscope, and then see what you pick up in the room as you move the cable around. The frequencies and magnitudes on the oscilloscope will give you some idea of how bad the EM noise signals in the room are. > > Craig > > > On Tue, Jul 20, 2010 at 4:47 PM, Craig Brideau <[hidden email]> wrote: >> The electrical noise will probably not be good for your readings. >> Unless you are willing to put the whole room in a Faraday cage I would >> be concerned. You will also probably want line filters as well; this >> will filter the power coming out of your wall sockets, so any EM they >> pick up from the nearby high voltage stuff will not make it into your >> equipment. Overall I'd say your planned location is less than ideal, >> but with sufficient measures you could still make it work. Good line >> filters and shielding should keep out the nearby sources of electrical >> interference. >> >> Craig >> >> >> On Tue, Jul 20, 2010 at 8:08 AM, Johannes Helm <[hidden email]> wrote: >>> Good afternoon, >>> >>> whether the electromagnetic fields will have any non-negligible >>> influence on the biological specimens certainly is a difficult problem. >>> Whether a reviewer refereeing a manuscript for publication would >>> think that the effects of the electromagnetic fields in your lab >>> would cast any doubts on the reliability of the presented >>> observations and results most probably is to a large degree dependent >>> on the reviewer's "personal opinion". There has been and still is a >>> lot of writing about electro smog caused by AC HV lines used to >>> transport electric energy, by AC HV lines for train traffic and, even >>> more important, the effect of the electro magnetic fields to which one is exposed when using cell phones. >>> >>> Concerning the microscopes, lasers, detectors a.s.o.: >>> >>> I cannot see any influence that an electromagnetic field of the >>> strength of one generated by the sources you mention would have on >>> the microscope itself. >>> The magnetic field necessary to alter the plane of polarization of >>> polarized light in a non negligble way, see "Faraday Effect", >>> certainly is much stronger than the fields you are going to >>> experience. There should, hence, not be any noticeable effect even of >>> you plan to do quantitative polarization microscopy. >>> Light detectors using HV, e.g. photomultiplier tubes, are usually >>> mounted in tubes made out of a material, which shields magnetic fields. >>> >>> Best wishes for your work with and in the new lab. >>> >>> Johannes >>> >>> >>> >>> >>>> Dear list, >>>> >>>> this is not exactly a confocal question but anyway related to it. I >>>> hope to get some input from people who have some experience with >>>> magnetic fields and the influence on microscopy but also on confocal scanners. >>>> In our building we are planning to centralize our microscopy >>>> facility in the second basement. Right now the architects are doing >>>> a feasibility study. >>>> Because the new rooms are located between the elevator machine room >>>> and the high voltage current room and since the electric cables from >>>> the whole house go through these rooms they now ask about magnetic fields. >>>> Does somebody know if magnetic fields can influence microscopy >>>> (cells for example), the microscopes itself, the lasers or scanners (galvos)? >>>> If yes, is there a threshold that is acceptable? >>>> I would be very thankful if I could get more information on that topic. >>>> >>>> Best regards >>>> >>>> Pascal >>>> >>>> -- >>>> Pascal Lorentz >>>> BioOptics Facility >>>> Department of Biomedicine >>>> University of Basel >>>> Mattenstrasse 28 >>>> 4058 Basel >>>> Switzerland >>>> >>> >>> >>> -- >>> P. Johannes Helm >>> >>> Voice: (+47) 228 51159 (office) >>> Fax: (+47) 228 51499 (office) >>> >> > > > > This email message, together with any attachments, is for the sole use of the intended recipient(s) and is the confidential information of Applied Precision Inc. If you are not the intended recipient, your review, use, disclosure, copying or dissemination of this email message or its attachments, or the information contained therein, is strictly prohibited. If you are not the intended recipient or if you think this email was sent to you in error, please notify the sender by reply email and delete this message and its attachments, as well as all copies, from your system. > |
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George McNamara |
Re: What methods are used to image morphology in high content screening systems?
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In reply to this post by Shalin Mehta
Hi again Shalin,
Check out the HD (high definition imaging pdf at http://essenbioscience.com/products.html I came across this because IncuCyteHD was used by: Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, Russell R, Bayani J, Head R, Lee M, Bernstein M, Squire JA, Smith A, Dirks P. Cell Stem Cell. 2009 Jun 5;4(6):568-80.PMID: 19497285 The authors are to be congratulated for posting so many movies online at http://www.cell.com/cell-stem-cell/supplemental/S1934-5909(09)00149-0 Enjoy, George p.s. listserv - My core facility's web site underwent some changes recently affecting most of the web links. For anyone looking for the Pubspectra dataset, you can go to http://www.sylvester.org/AICF/pubspectra.zip and click on the PubSpectra zip link to download (the dataset is the xlsx file, the zip file has additional files that may be useful). At 11:40 AM 7/16/2010, you wrote: Dear listers, George McNamara, Ph.D. Image Core Manager Analytical Imaging Core Facility University of Miami, Miller School of Medicine Miami, FL 33136 [hidden email] [hidden email] 305-243-8436 office http://www.sylvester.org/AICF (Analytical Imaging Core Facility) http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file) http://home.earthlink.net/~geomcnamara |
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Shalin Mehta |
Re: What methods are used to image morphology in high content screening systems?
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Thank you George and Guy for very informative inputs.
I am *guessing* the reason IATIA incorporated computation of DIC and phase-contrast images from quantitative RI is that our brains tend to pick up sharp features from the images. The quantitative optical path length appears 'smooth', which may not be quite appealing to the eye. DIC and phase contrast highlight edge features, which are easier to interpret by human. best shalin On Sun, Jul 25, 2010 at 3:09 AM, George McNamara <[hidden email]> wrote: > Hi again Shalin, > > Check out the HD (high definition imaging pdf at > http://essenbioscience.com/products.html > I came across this because IncuCyteHD was used by: > > > Glioma stem cell lines expanded in adherent culture have tumor-specific > phenotypes and are suitable for chemical and genetic screens. > > Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, Russell R, Bayani > J, Head R, Lee M, Bernstein M, Squire JA, Smith A, Dirks P. > > Cell Stem Cell. 2009 Jun 5;4(6):568-80.PMID: 19497285 > > The authors are to be congratulated for posting so many movies online at > http://www.cell.com/cell-stem-cell/supplemental/S1934-5909(09)00149-0 > > > Enjoy, > > George > p.s. listserv - My core facility's web site underwent some changes recently > affecting most of the web links. For anyone looking for the Pubspectra > dataset, you can go to http://www.sylvester.org/AICF/pubspectra.zip and > click on the PubSpectra zip link to download (the dataset is the xlsx file, > the zip file has additional files that may be useful). > > > > > At 11:40 AM 7/16/2010, you wrote: > > Dear listers, > DIC and phase-contrast are used widely (albeit, less than fluorescence) for > imaging cellular morphology . I am curious about what approaches are used to > image morphology in the high content screening applications. > I have come across HCS systems on the web that use either DIC or > phase-contrast. > Do some systems use hoffman modulation contrast? > > If you could list the system that you have and the type of contrast they > employ for imaging the morphology, that will be helpful. Since high-content > screening requires automated analysis, and these methods are not really > quantitative, do people find it difficult to address some biological > questions? My thesis is related to quantitative morphological imaging and I > am trying to get the users' perspective on the topic. > > Thanks very much for your inputs, > Shalin > 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/people.asp#shalinm > > > > > > George McNamara, Ph.D. > Image Core Manager > Analytical Imaging Core Facility > University of Miami, Miller School of Medicine > Miami, FL 33136 > [hidden email] > [hidden email] > 305-243-8436 office > http://www.sylvester.org/AICF (Analytical Imaging Core Facility) > http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx > file is in the zip file) > http://home.earthlink.net/~geomcnamara > |
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