Spinning disk requirement
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Dear All
I request all the members to please help me to decide for a spinning disk confocal microscope for our facility which is a multiuser facility for the whole university. We already have a point scanning confocal system from Olympus. We will have another point scanning confocal soon with all the live cell imaging accessories (laser based). For very fast phenomenon like measuring Calcium Flux in the cells, we want to have a multi point scanning system. But i am unable to decide whether i should take a fluorescence based spinning disk or laser based spinning disk confocal. Also, please give me some feedback for CARV II (multi point confocal, fluorescence based). Please help. Thanks in advance. Charu Tanwar Imaging Specialist Advanced Instrumentation Research Facility Jawaharlal Nehru University New Delhi India. |
 
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leoncio vergara |
Re: Spinning disk requirement
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have you tested the Olympus DSU? I am wondering how it would compare with the yokowaga scanhead based systems.
________________________________________ From: Confocal Microscopy List [[hidden email]] On Behalf Of Charu Tanwar [[hidden email]] Sent: Wednesday, December 30, 2009 1:36 AM To: [hidden email] Subject: Spinning disk requirement Dear All I request all the members to please help me to decide for a spinning disk confocal microscope for our facility which is a multiuser facility for the whole university. We already have a point scanning confocal system from Olympus. We will have another point scanning confocal soon with all the live cell imaging accessories (laser based). For very fast phenomenon like measuring Calcium Flux in the cells, we want to have a multi point scanning system. But i am unable to decide whether i should take a fluorescence based spinning disk or laser based spinning disk confocal. Also, please give me some feedback for CARV II (multi point confocal, fluorescence based). Please help. Thanks in advance. Charu Tanwar Imaging Specialist Advanced Instrumentation Research Facility Jawaharlal Nehru University New Delhi India. |
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Dear Charu
Do you really need very fast live cell acquisition, as for as i know the only benefits of DSUs is that you can scan at a very fast rate but you can still achieve at least 250-300 frames per second by any good resonance scanner. I have seen both Ziess and Olympus DSUs i did not find anything great other than speed if one take the huge prices in account. one good thing about these systems is low noise, as they have CCD camera but this reduces sensitivity. On Wed, December 30, 2009 5:19 pm, charu tanwar wrote: > No, i have not yet seen any of the spinning disk systems. Yokogawa is very > famous but i really want to know that how reasonable is to buy a laser based > multipoint scanning system than buying fluorescence based spinning disk. (we > already have single point scanning system, one only confocal and another > confocal with live cell, both laser based.) > > --- On Wed, 30/12/09, Vergara, Leoncio A. <[hidden email]> wrote: > > > From: Vergara, Leoncio A. <[hidden email]> > Subject: Re: Spinning disk requirement > To: [hidden email] > Date: Wednesday, 30 December, 2009, 4:12 PM > > > have you tested the Olympus DSU? I am wondering how it would compare with the > yokowaga scanhead based systems. > ________________________________________ > From: Confocal Microscopy List [[hidden email]] On Behalf Of > Charu Tanwar [[hidden email]] > Sent: Wednesday, December 30, 2009 1:36 AM > To: [hidden email] > Subject: Spinning disk requirement > > Dear All > > I request all the members to please help me to decide for a spinning disk > confocal microscope for our facility which is a multiuser facility for the > whole university. We already have a point scanning confocal system from > Olympus. We will have another point scanning confocal soon with all the live > cell imaging accessories (laser based). > For very fast phenomenon like measuring Calcium Flux in the cells, we want > to have a multi point scanning system. But i am unable to decide whether i > should take a fluorescence based spinning disk or laser based spinning disk > confocal. > Also, please give me some feedback for CARV II (multi point confocal, > fluorescence based). > Please help. > Thanks in advance. > > Charu Tanwar > Imaging Specialist > Advanced Instrumentation Research Facility > Jawaharlal Nehru University > New Delhi > India. > > > > The INTERNET now has a personality. YOURS! See your Yahoo! Homepage. > http://in.yahoo.com/ > -- > This message has been scanned for viruses and > dangerous content by MailScanner, and is > believed to be clean. > > -- ANURAG PANDEY Electro physiology Group M.B.U. I.I.Sc. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. |
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George McNamara |
Re: Spinning disk requirement ... consider alternative array scan confocal
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In reply to this post by Charu Tanwar
Hi Charu,
Check out VT-Infinity and VT-Hawk at Visitech http://www.visitech.co.uk/site/products.php The VT-Hawk adds FRAP/photoactivation capability to the Infinity. Visitech sold Yokogawa's for a long time - Steve Coleman told me in April 2009 that their company was selling off its remaining spinning disk inventory to focus exclusively on their array scanners. A VT with the right EMCCD would be a great combination. My biggest concern with Visitech is how do they support overseas: they are a British company, so I am referring to both here (USA) and you. As for Calcium ion flux, the "standard of care" is Fura-2 for its dynamic range. However, the excitation light path of most fluorescence microscopes are such that UV is usually not parfocal with visible. Until recently, even violet (405 nm) was not parfocal with green and red (and now only a few expensive objective lenses are). Parfocality is not critical in widefield Fura-2, but would be for confocal. The dynamic range of fluorescent protein based biosensors is not anywhere near Fura-2, but are getting better, for example, GCaMP-3, and can be targeted to specific location(s) by appropriate targeting sequences. CARV II - I manage a BD pathway 855 that has a CARV (I?) inside. Nice to be able to see confocal by eye, but the BD (formerly ATTO) software is bad. If you buy a CARV II, be sure to evaluate the software that you will be using with it. Sincerely, George At 02:36 AM 12/30/2009, you wrote: >Dear All > >I request all the members to please help me to decide for a spinning disk >confocal microscope for our facility which is a multiuser facility for the >whole university. We already have a point scanning confocal system from >Olympus. We will have another point scanning confocal soon with all the live >cell imaging accessories (laser based). >For very fast phenomenon like measuring Calcium Flux in the cells, we want >to have a multi point scanning system. But i am unable to decide whether i >should take a fluorescence based spinning disk or laser based spinning disk >confocal. >Also, please give me some feedback for CARV II (multi point confocal, >fluorescence based). >Please help. >Thanks in advance. > >Charu Tanwar >Imaging Specialist >Advanced Instrumentation Research Facility >Jawaharlal Nehru University >New Delhi >India. 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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RICHARD BURRY |
Re: Spinning disk requirement ... consider alternative array scan confocal
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Charu
The spining disk is limited to one pinhole size but the 2D array scanning Infinity3 has sever different pinholes. We have the Infinity3 and find the choice of pinholes is very important. For some samples, thinner optical sections (with smaller pinholes) is important and for some samples more intensity (with larger pinholes) is important. Don't forget the camera, I would recommend the most sensitive you can get. Most samples have too few photons! The Hamamatsu backthinned C9100-13 works well for us. Dick ----- Original Message ----- From: George McNamara <[hidden email]> Date: Wednesday, December 30, 2009 10:47 am Subject: Re: Spinning disk requirement ... consider alternative array scan confocal To: [hidden email] > Hi Charu, > > Check out VT-Infinity and VT-Hawk at Visitech > http://www.visitech.co.uk/site/products.php The VT-Hawk adds > FRAP/photoactivation capability to the Infinity. Visitech sold > Yokogawa's for a long time - Steve Coleman told me in April 2009 > that > their company was selling off its remaining spinning disk > inventory > to focus exclusively on their array scanners. A VT with the > right > EMCCD would be a great combination. My biggest concern with > Visitech > is how do they support overseas: they are a British company, so > I am > referring to both here (USA) and you. > > As for Calcium ion flux, the "standard of care" is Fura-2 for > its > dynamic range. However, the excitation light path of most > fluorescence microscopes are such that UV is usually not > parfocal > with visible. Until recently, even violet (405 nm) was not > parfocal > with green and red (and now only a few expensive objective > lenses > are). Parfocality is not critical in widefield Fura-2, but would > be > for confocal. The dynamic range of fluorescent protein based > biosensors is not anywhere near Fura-2, but are getting better, > for > example, GCaMP-3, and can be targeted to specific location(s) by > appropriate targeting sequences. > > CARV II - I manage a BD pathway 855 that has a CARV (I?) inside. > Nice > to be able to see confocal by eye, but the BD (formerly ATTO) > software is bad. If you buy a CARV II, be sure to evaluate the > software that you will be using with it. > > Sincerely, > > George > > At 02:36 AM 12/30/2009, you wrote: > >Dear All > > > >I request all the members to please help me to decide for a > spinning disk > >confocal microscope for our facility which is a multiuser > facility for the > >whole university. We already have a point scanning confocal > system from > >Olympus. We will have another point scanning confocal soon with > all the live > >cell imaging accessories (laser based). > >For very fast phenomenon like measuring Calcium Flux in the > cells, we want > >to have a multi point scanning system. But i am unable to > decide whether i > >should take a fluorescence based spinning disk or laser based > spinning disk > >confocal. > >Also, please give me some feedback for CARV II (multi point confocal, > >fluorescence based). > >Please help. > >Thanks in advance. > > > >Charu Tanwar > >Imaging Specialist > >Advanced Instrumentation Research Facility > >Jawaharlal Nehru University > >New Delhi > >India. > > > > > > > > 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 > > > -- > BEGIN-ANTISPAM-VOTING-LINKS > ------------------------------------------------------ > > Teach CanIt if this mail (ID 980990647) is spam: > Spam: > https://antispam.osu.edu/b.php?i=980990647&m=9f951fb53215&c=sNot > spam: https://antispam.osu.edu/b.php?i=980990647&m=9f951fb53215&c=n > Forget vote: > https://antispam.osu.edu/b.php?i=980990647&m=9f951fb53215&c=f---- > -------------------------------------------------- > END-ANTISPAM-VOTING-LINKS > Richard W. Burry, Ph.D. Department of Neuroscience, College of Medicine Campus Microscopy and Imaging Facility, Director The Ohio State University Associate Editor, Journal of Histochemistry and Cytochemistry 277 Biomedical Research Tower 460 West Twelfth Avenue Columbus, Ohio 43210 Voice 614.292.2814 Cell 614.638.3345 Fax 614.247.8849 |
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In reply to this post by Charu Tanwar
I just acquired a DSU for live cell imaging. If price is no object
the laser systems are swell but if you are on a budget I think the DSU is a reasonable device. It is my understanding that the CARVII is at end of life. As for sensitivity, a CCD is a good bet. Another nice aspect of a non-laser system is the flexibility of a white light source. You never know if the next great fluor will meet you laser lines. This might be esp the case in a multiuser set up. On Dec 30, 2009, at 1:36 AM, Charu Tanwar wrote: > Dear All > > I request all the members to please help me to decide for a spinning > disk > confocal microscope for our facility which is a multiuser facility > for the > whole university. We already have a point scanning confocal system > from > Olympus. We will have another point scanning confocal soon with all > the live > cell imaging accessories (laser based). > For very fast phenomenon like measuring Calcium Flux in the cells, > we want > to have a multi point scanning system. But i am unable to decide > whether i > should take a fluorescence based spinning disk or laser based > spinning disk > confocal. > Also, please give me some feedback for CARV II (multi point confocal, > fluorescence based). > Please help. > Thanks in advance. > > Charu Tanwar > Imaging Specialist > Advanced Instrumentation Research Facility > Jawaharlal Nehru University > New Delhi > India. Michael J. Herron, U of MN, Dept. of Entomology [hidden email] 612-624-3688 (office) 612-625-5299 (FAX) |
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In reply to this post by Charu Tanwar
Dear Charu,
All types of spinning disk confocals you refer to use fluorescence (i.e. it is not laser versus fluorescence, but rather laser versus arc lamp). To oversimplify, the main differences between systems is (a) the illumination source, and (b) the type of scanner . For the illumination source you can use a laser control unit with a set of individual lasers (each laser providing one or a few wavelengths for fluorescence excitation). Alternatively, you can use a mercury lamp or similar broad band excitation light, and use a filter wheel to select specific bands (range of wavelengths) to excite fluorescence. Some big differences between these options are cost (lasers may cost US $ 10,000-20,000 each), plus the cost of all the electronics controlling them. Old style gas lasers may last a few years and need to be replaced, which may add substantial cost down the road. New solid state lasers will last longer, allegedly, but tend to be still quite expensive to purchase. Typically, with a laser system, you will probably want four lasers (405 and/or 440, 488, 560 or so, 633 or so, maybe 514 for YFP), especially for a multi-user facility. Depending on your anticipated applications, you need to chose your lasers carefully. 440 for instance will be better for CFP, but will be very poor or unusable for nuclear dyes such as DAPI. 405 will work with DAPI and CFP (although less good for CFP than the 440), but will be a bit more toxic if doing CFP, so not as good for live imaging. Lamp based systems will be tens of thousands of dollars cheaper to purchase (but you will have to purchase lamps regularly), and will be more flexible since you can add extra excitation filters at pretty low cost. Most high performance systems tend to use lasers. I don't think it's a matter of power or light intensity, but lasers may have some advantages in terms of speed of switching, beam focusing, synchronization, etc... A second difference is the type of confocal scanner. The gold standard seems to be Yokogawa, which many vendors of spinning disk systems use. In their latest implementation, these are very light efficient (because of the use of microlenses to gather as much light as possible), and may well be the most high performance systems available. They have one single aperture size, which can be a limitation sometimes. Systems such as the CARV use less sophisticated spinning disk technology, and are not quite as high performing, I think. Olympus DSU uses a set of interchangeable apertures, so that users can choose various slit sizes. Combined with an arc lamp illumination, the DSU is a quite affordable system, and does work pretty well for many samples and applications, but I suspect it may not offer the same level of performance as a high end Yokogawa system. The only way to know if this will work for you is probably to run tests on such a system with actual samples and see what happens (or to get feed-back from someone who has both types of systems and has thoroughly compared them). An Olympus DSU with arc lamp illumination may easily cost US $ 100,000-150,000 less than a four laser Yokogawa system, because of the lower cost of the lamp versus lasers, and the lower cost of the DSU scanner versus the Yokogawa (but note that Yokogawa may have several different models). Finally, there are other types of systems that may be somewhere in between these two extremes in terms of cost, performance and functionality, such as the Nikon Live Scan (Prairie technologies swept field), and Zeiss LSM 5 live. The Nikon LiveScan/Prairie swept field allows users to select between pinholes or slits of different sizes and can be tailored to be more sensitive (faster) or more confocal, and is a nice instrument, although possibly requiring more maintenance and calibration than a spinning disk. The Zeiss 5 Live is a slit scanning system. Therefore, these are not strictly speaking spinning disk systems, although they are functionally similar (faster imaging than conventional confocals through simultaneous illumination of multiple points or lines, but with reduced confocality) This being said, I would not necessarily exclude conventional laser point scanning confocals. The way they work is by imaging (reading intensities) one point at a time and building the image by scanning a certain area line by line. Each point is read in the order of milliseconds, and therefore you need much higher illumination intensities. On a spinning disk, you image hundreds of points simultaneously, with illumination intensities that are much lower (and integration times much longer). This is considered better for live cell imaging, but you can do live cell imaging with a point scanning confocal as long as you keep illumination intensities below a critical threshold. Regarding speed, in theory spinning disk can run much faster in terms of frames per second, but you can go pretty fast with a conventional confocal, if you are able to keep the imaged area small (such as 128 x 128 pixels or so), or you can do a line scanning (and obtain an intensity profile over time rather than an image). This may still answer your biological question just fine. If I had one piece of advice to give you, rather than immediately jumping into buying a new instrument, I would try your calcium experiments on the instruments you already have (assuming they have the required lasers, etc...). This will show you whether or not they can be done, will give you some experience about the particular experiment, and you will get a better sense what to look for if you later on can have a demo for a new instrument, because you will know exactly what you are looking for. If you have a demo where people bring samples or try techniques they had never used, and the demo doesn't work, you will never know if it is because of the instrument, or because of the samples... Some vendors may let you have an extended demo, i.e. 1-2 weeks, which will give you a good sense about an instrument, but even so, demos are rarely perfect.... I can not tell you which specific instrument is better for your situation. This will depend on your financial situation (how much money you have for purchase, as well as for maintenance/replacement parts down the road). In addition, you will have to take into account the needs of your customers, such as which dyes/fluorescent proteins they anticipate using, applications, speed requirements, confocality requirements, and such. Maybe you need to consider getting an instrument that satisfies your immediate needs, but than can be upgraded later if needed. If you can have a demo that you can run on actual samples (that is, run an actual experiment and see if it works; ideally, use samples you are familiar with and know they work as I mentioned above), then, that's probably the best. Second best may be to look at your anticipated applications, find publications about similar work, and do a little survey about what these authors used (maybe contact them directly and ask about their experience). This being said, if you have the money (for purchase and long term maintenance), you probably won't go wrong by buying a good laser/yokogawa scanner based spinning disk system. Just make sure you get the right lasers, dichoics, emission filters and objectives for your anticipated applications. Your local vendor should be able to guide you. If funds are limited, you should consider some of the more affordable options, but make sure you test them for your specific applications before you purchase them. They may happen to work just fine... Good luck. Julio. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA On Dec 30, 2009, at 3:49 AM, charu tanwar wrote:
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Charu Tanwar |
Re: Spinning disk requirement ... consider alternative array scan confocal
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In reply to this post by RICHARD BURRY
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In reply to this post by Julio Vazquez
Dear All Wish you a very Happy and a Prosperous New year 2010! Ammasi Ammasi Periasamy, Ph.D. Director, Keck Center for Cellular Imaging (KCCI) Professor of Biology and Biomedical Engineering Biology, Gilmer Hall (064), McCormick Rd University of Virginia Charlottesville, VA 22904 Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:434-982-5210; Email:[hidden email] http://www.kcci.virginia.edu ************************ Workshop on FRET Microscopy, March 9-13, 2010 http://www.kcci.virginia.edu/workshop/workshop2010/index.php ************************* |
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leoncio vergara |
Re: Spinning disk requirement ... consider alternative array scan confocal
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In reply to this post by Charu Tanwar
Array scanners are sold in the US by Nikon and Prairie
Technologies. I am not sure if there are other sources and how they compare to
the Visitech systems. I have no personal experience with eithe of the systems
but I thought the information may help.
There are always trade offs with any design, Yokogawa
scanheads have been around for years and I understand the design has been
improved with the new models. The array scanners are however realtively new
and untried.
I think you should test the systems and compare by
yourself. The best is to get a system to test at your site for an extended
time is the ideal situation but difficult to get. I am not convinced that short
term demos are the best since many times you may get biased by the
skills of the demo crew or you may have problems with a system due to
precarious installations or technical problems... the second best is to travel
to a location were a system has been in operation for some time, there you also
can also talk to experienced users. The problem of traveling to a distant
location is that your live specimen preparations may not travel
well.
Leoncio
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of charu tanwar Sent: Wednesday, December 30, 2009 11:18 PM To: [hidden email] Subject: Re: Spinning disk requirement ... consider alternative array scan confocal
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Boswell, Carl A - (cboswell) |
Re: Spinning disk requirement ... consider alternative array scan confocal
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One system I saw mentioned only casually was the
resonant scanner, particularly Leica's. We have a new SP5 dual-head
system, and we chose the Leica high-speed system after direct comparisons with
DSU and Yokogawa spinning disks. The differences were significant and
consistent. As a test specimen, we tracked GFP and DS-Red-labelled
vessicle transport and exchange. The Leica provided quite useable images
while the spinning disk systems produces unuseable images. The issue was
capturing the low intensity signal at a high rate. From my experience, the
drawback of spinning disk systems is their inability to do a good job gathering
low intensity images. The resonant scanner is noisy, but the 8 kHz speed
allows multi-frame averaging. Also the system uses liquid-cooled
PMT's. One of the users has tested the system with other specimens and
found it to have relatively low photobleaching as compared with the Nikon swept
field.
Slomething to think about, anyway.
c
Carl A. Boswell, Ph.D.
Molecular and Cellular Biology University of Arizona 520-954-7053 FAX 520-621-3709 t |
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Knecht, David |
Re: Spinning disk requirement ... consider alternative array scan confocal
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<base href="x-msg://271/">Were you using an EM camera for the spinning disk? I would have thought the EM camera would have better S/N than the PMT's with low signals. Also, how long does it take to capture each frame on the Leica? I had the impression it was 0.5-1 sec/frame which would be much slower than a spinning disk. Dave
On Jan 4, 2010, at 1:40 PM, Carl Boswell wrote:
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) |
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Boswell, Carl A - (cboswell) |
Re: Spinning disk requirement ... consider alternative array scan confocal
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<BASE
href="x-msg://271/">
It was some time ago so I don't remember the
details. One of the points that this broaches is the lack of decent
support by the manufacturer of the demo teams. We could easily have had a
"standard" camera, thus not presenting the system in the optimal
configuration. I've seen this happen time and again, and wonder at the
short-sighted nature of a company sending out sub-optimal equipement as a
showcase and expecting potential buyers to be impressed with
it.
At any rate, we had each system for a week, then
had to make our decision based on what we had seen, not on what might have
been.
The resonant scanner can do about 25 512x512
frames/sec and more if the y is < 512 lines. Routinely we get 6
z-sections, with 12 frame averaging, in 2-3 seconds for the field of view
we use.
C
Carl A. Boswell, Ph.D.
Molecular and Cellular Biology University of Arizona 520-954-7053 FAX 520-621-3709
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RICHARD BURRY |
Re: Spinning disk requirement ... consider alternative array scan confocal
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In reply to this post by leoncio vergara
Leoncio
The Visitech Infinity system array scanner is not swept field. It uses arrays of seven different sizes of pinholes from 64 to 10 microns and no slits. The technology for the light path is unique. In my experience the through put of the Infinity is better and the optical section thickness is finer. Dick ----- Original Message ----- From: "Vergara, Leoncio A." <[hidden email]> Date: Monday, January 4, 2010 12:19 pm Subject: Re: Spinning disk requirement ... consider alternative array scan confocal To: [hidden email]
Richard W. Burry, Ph.D. Department of Neuroscience, College of Medicine Campus Microscopy and Imaging Facility, Director The Ohio State University Associate Editor, Journal of Histochemistry and Cytochemistry 277 Biomedical Research Tower 460 West Twelfth Avenue Columbus, Ohio 43210 Voice 614.292.2814 Cell 614.638.3345 Fax 614.247.8849 |
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leoncio vergara |
Re: Spinning disk requirement ... consider alternative array scan confocal
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Thanks for the clarification. The source of my information was a
little over a year ago when a group on our campus (UTMB, Galveston) had a demo
of a Nikon swept field, from the talk given by the rep and the literature
distributed I understood the Nikon system is a dual system that uses either a set
of parallel slits or a pinhole array similar to the Visitech system. The
same group also run a demo on a Yokogawa based system. The decision was in
favor of the Nikon system (the purchase later did not materialize, in part due
to Ike I think), nevertheless it is my impression the choice was heavily based
on the circumstantial performance of both the demo systems and the company
teams, one of the faculty even told me they did not like the incubator
included with the Yokogawa system (what about the color of the eyes of the
sales rep?). I am convinced that these kind of demos need to be taken with a
grain of salt as there are many acute factors that can influence the outcome. At
the medical center in Houston I met another group that purchased the same Yokogawa
system that was tested here, they also considered the swept filed system but
their opinion was just the opposite, they purchased the spinning disk system
and are very happy with the performance. The information in this thread has been very useful for me, in
the future if we are again ready to evaluate a fast confocal system for
live cell imaging I will have all these comments in mind. From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of RICHARD
BURRY Leoncio
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