fast confocal imaging -- old vs. new technologies
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O'Malley, Donald |
fast confocal imaging -- old vs. new technologies
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Dear Charu,
Two articles (of mine) address rapid calcium imaging: 2003: illustrates the spatial-temporal limits of calcium imaging achieved with single-point/line scanning confocal. 2008: review discusses some tradeoffs and shows an example where spinning disk was used to get an areal-view and then single-line scanning to obtain highest temporal resolution. [PDFs of both articles can be found on the last page of my ed-tech web site www.digital-maze.com; bottom link on left side navigation bar titled: O'Malley PDFs]. In my 2008 review I have unfortunately not included other newer technologies available only because I had not then been able to find any highest end spatial-temporal resolution images published using any of them. But the bottom line from a neurobiology perspective is that one can often use slower frame-scan imaging to determine a location for single-line scanning to be applied and then the good old fashioned line-scan still seems to provide the most sensitive and durable highest-end combined spatial-temporal resolution images (on the order of 2 msec x microns-squared "dynamic units"). One caveat is that much of the best work has been done by folks using 2-photon single-line scanning instruments (see citations in 2008 review). The issue is not how fast can you acquire, but how fast can you resolve subcellular dynamic events in both space and time. Acquiring fast and binning over time adds nothing to temporal resolution. Figure 3 in my 2003 review illustrates diffusion of calcium across the plasma membrane and across the nuclear envelope, acquired with an MRC600 single-line scanner. My 2008 review covers what I had found thru about late-2007 and I was not then aware of any spatial-temporal dynamic imaging results that exceeded the performance of the venerable MRC600 linescan (if there are any older examples that I have missed -- my apologies!). By trading away some spatial resolution with a partially opened aperture one can enormously increase signal throughput allowing one to reduce excitation intensity-- good old confocal is very good for live cell and live animal imaging. The lack of a continuously-variable aperture on spinning disk confocals is a serious limitation, but one that might be corrected by having a set of disks that trade off spatial resolution for greatly increased signal. If anyone knows of this being implemented and used to obtain highest-end dynamic units (for calcium flux, dextran diffusion, organelle movement, etc.), I am sure that many on the list would like to see published results--ditto for any such results with the resonant scanners. [I have not researched this since 2007, sorry]. In the example in my 2008 review, a Nipkow disk obtained nice 2D images at 17 msec intervals (which with 1 um**2 spatial resolution would yield a dynamic resolution of 17 msec-um**2) after which 2 msec linescans were then used to explore the finest temporal structure of calcium sparks (Fig. 2); this was reprinted work from Lothar Blatter's lab from 2001. My 2008 review discusses some trade offs and issues encountered in fast imaging, and again my apologies for anything that I have missed. Happy 2010, Don Don O'Malley Dept. Biology [hidden email] 617-373-2284 |
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Charu Tanwar |
Re: fast confocal imaging -- old vs. new technologies
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leoncio vergara |
FRAP and fast confocals and wide field systems
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In reply to this post by O'Malley, Donald
I am interested on FRAP and photoactivation in wide field microscopes or fast confocal systems such as spinning disk or array scanning systems used in the study of live cells.
Is anybody familiar with the Mosaic Digital Illumination Systems? http://www.photonic-instruments.com/MicroPoint_Mosaic.aspx Sounds really cool, but is quite expensive. I would appreciate any input regarding experiences with this equipemnt for the above applications. We work with live cells in a multiuser core, so we are planning to setup a fast imaging system and would like to be able to perform FRAP and PA applications. The time course of the events studied will range widely from minutes to seconds and perhaps fractions of a second. Leoncio Vergara MD Assistant Director Center for Biomedical Engineering University of Texas Medical Branch. Galveston, Texas |
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Loralei Dewe-3 |
Re: FRAP and fast confocals and wide field systems
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Hi Dr. Vergara,
This is a commercial response. Perhaps you have heard of TILL Photonics? We specialize in high end microscopy systems just for techniques like FRAP, FRET and TIRF etc. Perhaps you would like to take a look at our website:
We are known for being fast, precise and extremely well made. Our customer service is exceptional!
Please drop me a note if you have any questions...
Loralei Dewe Western US Sales and Applications Engineer TILL Photonics Phone 866-547-8455 x 1446 Cell 585-730-9822 On Wed, Jan 6, 2010 at 3:45 PM, Vergara, Leoncio A. <[hidden email]> wrote: I am interested on FRAP and photoactivation in wide field microscopes or fast confocal systems such as spinning disk or array scanning systems used in the study of live cells. |
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Alison J. North |
Re: FRAP and fast confocals and wide field systems
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In reply to this post by leoncio vergara
Hi Leoncio,
We have two systems that we use for FRAP and photoactivation - a DeltaVision system with the QLM laser module, and a Yokagawa-type spinning disk system fitted with the Photonics Digital Mosaic operated under MetaMorph software. Both systems work really nicely for both purposes. The DeltaVision system, which just targets a single point at a time, gives faster bleaching or activation, but the Mosaic is really cool for the region of interest targeting if speed is less of an issue. So I think your decision may depend on how often you need to be able to both bleach and switch to imaging in under a second, as well as on the optimal type of imaging afterwards (i.e. confocal or widefield). Like you, we are operating a core facility and have a wide range of users and applications on these systems, so rather than going into any more specific details here, please feel free to contact me offline or give me a call to discuss your questions in more detail? I can provide some absolute bleaching times from comparisons of the same experiment performed using the two systems, but am reluctant to post them to the whole world because they were not collected using the most recent hardware and therefore might be misleading as to the current equipment capabilities. All the best - and Happy New Year everybody! Alison Vergara, Leoncio A. wrote: > I am interested on FRAP and photoactivation in wide field microscopes or fast confocal systems such as spinning disk or array scanning systems used in the study of live cells. > > Is anybody familiar with the Mosaic Digital Illumination Systems? > > http://www.photonic-instruments.com/MicroPoint_Mosaic.aspx > > Sounds really cool, but is quite expensive. I would appreciate any input regarding experiences with this equipemnt for the above applications. > > We work with live cells in a multiuser core, so we are planning to setup a fast imaging system and would like to be able to perform FRAP and PA applications. The time course of the events studied will range widely from minutes to seconds and perhaps fractions of a second. > > Leoncio Vergara MD > Assistant Director > Center for Biomedical Engineering > University of Texas Medical Branch. > Galveston, Texas -- Alison J. North, Ph.D., Research Assistant Professor and Director of the Bio-Imaging Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065. Tel: office ++ 212 327 7488 Tel: lab ++ 212 327 7486 Fax: ++ 212 327 7489 |
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Romin, Yevgeniy/Sloan Kettering Institute |
Re: FRAP and fast confocals and wide field systems
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In reply to this post by leoncio vergara
Hello Leoncio
We work with live cells in a multi-user core facility as well, and we use the Carl Zeiss LSM 5 Live Duoscan system for our FRAP and PA experiments. The system gives you a lot of control over the bleaching process and it is very versatile. Speed is also not an issue with this system, it is quite fast. It is a line scanning confocal system, so both bleaching and acquisition can be done in fractions of seconds, depending on the settings of your experiment. If you need any specific information, feel free to contact me directly at [hidden email] best wishes, Yevgeniy ________________________________________ From: Confocal Microscopy List [[hidden email]] On Behalf Of Vergara, Leoncio A. [[hidden email]] Sent: Wednesday, January 06, 2010 6:45 PM To: [hidden email] Subject: FRAP and fast confocals and wide field systems I am interested on FRAP and photoactivation in wide field microscopes or fast confocal systems such as spinning disk or array scanning systems used in the study of live cells. Is anybody familiar with the Mosaic Digital Illumination Systems? http://www.photonic-instruments.com/MicroPoint_Mosaic.aspx Sounds really cool, but is quite expensive. I would appreciate any input regarding experiences with this equipemnt for the above applications. We work with live cells in a multiuser core, so we are planning to setup a fast imaging system and would like to be able to perform FRAP and PA applications. The time course of the events studied will range widely from minutes to seconds and perhaps fractions of a second. Leoncio Vergara MD Assistant Director Center for Biomedical Engineering University of Texas Medical Branch. Galveston, Texas ===================================================================== Please note that this e-mail and any files transmitted with it may be privileged, confidential, and protected from disclosure under applicable law. If the reader of this message is not the intended recipient, or an employee or agent responsible for delivering this message to the intended recipient, you are hereby notified that any reading, dissemination, distribution, copying, or other use of this communication or any of its attachments is strictly prohibited. If you have received this communication in error, please notify the sender immediately by replying to this message and deleting this message, any attachments, and all copies and backups from your computer. |
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James Pawley |
Re: FRAP and fast confocals and wide field systems
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In reply to this post by leoncio vergara
Dear Leoncio,
Thanks for the reference to this instrument. Although I have no experience using it, the system seems to use a computer controlled digital mirror array device (DMD) placed in an image plane of a laser-based, microscope epi-illumination system. Turning a micro-mirror "on" produces a diffraction-limited spot of light in the imaged plane. The advantage of such a system is its flexibility in being able to expose the specimen to an "activation" beam" having an arbitrary 2D shape.* The disadvantage is that laser light that impinges on (the vast majority?) of the micro-mirrors that are turned "off", is lost. Assuming that this wasted light it sent to a well-engineered beam dump, this just means that one needs a (much?) larger laser to do a certain amount of damage. How much larger depends on the fraction of your field-of-view you customarily bleach (and on whether an alternative, single-beam bleach system bleaches in a raster pattern or by "beam steering"). Another consideration for DMD systems is the stray light that diffracts off the corners of the mirrors and their surroundings. Taking the listed contrast ratio of 1,000:1 and assuming that the chip is a 1,024x768 array, then if only one pixel is turned on, this beam will constitute only 1.27 ppm of the light striking the chip while the "stray" light will constitute 1,000 ppm, or about 790x more light. Clearly, you will have to turn on about 790 bright pixels before the photons in the intentional pattern equals those in the stray light striking the rest of the field of view. (These numbers are approximate as I suspect that the 1,000:1 contrast figure given is for the DMD device itself and hidden in this specification are assumptions about the acceptance angle of the following optical system that may be more appropriate to the optics of a digital projector than to that of a microscope illumination system. But these are still matters that should be kept in mind. How much of the "activation" light strikes the specimen when all the mirror are turned off?) More to the point, many of the techniques you mention involve photochemical processes that may involve intermediates that may diffuse some distance from the point where the excitation photon was absorbed. If this is true, one might be interested to know to accurately one needs to specify the pattern of excitation light. For instance, at the power levels "normally" used for bleaching in oxygenated, living cells (bleaching in less than a second, but not in a ms, and probably using continuous rather than pulsed lasers), most bleaching is not caused directly by the absorption of a photon so much as the transfer of the excitation energy to nearby molecules eventually creating one of a number of excited oxygen species. These excited molecules can diffuse on the order of micrometers before reacting. Only some of these reactions are those that cause the fluorescent molecules to cease fluorescing. Others may interfere with the very system you want to analyse. Bleaching at higher power levels (for instance those likely to be provided by a single, diffraction-limited mW beam) may result in singlet-state saturation effects whereby an already-excited molecule absorbs a second photon, perhaps leading to its disintegration without the need for excited oxygen intermediates. Alternatively, some "photodamage systems" utilize pulsed lasers. Pulsed lasers generate peak powers that are 10,000x to 1,000,000x more intense than those produced by continuous lasers of the same average power. The photodamage produced at these power levels can be very different from that produced at lower levels. For instance, the electronic field vector can become strong enough to produce electrical breakdown: (micro lightening?) in which case the resulting damage is not due to (or dependent on) the absorption of the excitation light by a dye molecule. Such systems are often used to make holes in cell membranes, or for other types of microsurgery. I mention this because in other parts of the Photonics www site they do seem to describe pulsed laser ablation systems of this type. Photodamage and microsurgery is discussed at much greater length in Chapters 38 and 39 of The Handbook. Cheers, Jim Pawley * Although this setup can also be used to provide the patterned illumination used in one type of 3D imaging, I have not discussed this here, as it seemed that FRAP and PA were the main interests. ********************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 [hidden email] 3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2010 "If it ain't diffraction, it must be statistics." Anon. >I am interested on FRAP and photoactivation in wide field >microscopes or fast confocal systems such as spinning disk or array >scanning systems used in the study of live cells. > >Is anybody familiar with the Mosaic Digital Illumination Systems? > >http://www.photonic-instruments.com/MicroPoint_Mosaic.aspx > >Sounds really cool, but is quite expensive. I would appreciate any >input regarding experiences with this equipemnt for the above >applications. > >We work with live cells in a multiuser core, so we are planning to >setup a fast imaging system and would like to be able to perform >FRAP and PA applications. The time course of the events studied will >range widely from minutes to seconds and perhaps fractions of a >second. > >Leoncio Vergara MD >Assistant Director >Center for Biomedical Engineering >University of Texas Medical Branch. >Galveston, Texas -- ********************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 [hidden email] 3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2010 "If it ain't diffraction, it must be statistics." Anon. |
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