FW: A1
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal COMMERCIAL INTEREST Dear All In addition to the points already discussed on A1/A1R confocal systems, I would like to throw some light on the lasers and their control through software and hardware. There are three beam introduction ports that allow the connection of two fiber coupled laser sets and one air space coupled laser. Two laser input ports are incorporated in the scan head to use different laser lines. The AOTF modulated 4 laser unit is used as a standard that provides 7 laser lines (choices from 405nm, 448nm, 457nm, 488nm, 514nm, 543nm, 562nm and 638nm) and the AOM modulated 3 laser board can be added as an option that provides additional 3 laser lines, hence 7 lasers with 9 lines are available in maximum. In addition, it can also be coupled to an optional picosecond or faster IR pulsed laser port. Lasers are modulated through power control for each wavelength, return mask and ROI exposure control. Through the software along with AOTF and AOM, the lasers can be controlled in increments of 0.1%. In addition, software variable control with continuous ND is also possible. The input ports are continuously monitored for the laser power that is governed by the control system that ensures quantitative and uniform performance. The scan head has three output ports to allow optical fiber connection to three separate detector units like 4 channel standard fluorescence detector, spectral detector and custom detector for applications like FCS and FLIM. The ability of using this system along with Controlled Light Exposure Microscopy (CLEM) makes this system for long time live cell imaging and confocal analysis. CLEM automatically monitors and varying the laser illumination during time-lapse studies to reduce the risk of laser induced bleaching, biochemical inconsistency and cell death. The complete system along with the inverted microscope can be totally controlled through NIS-Element C that allows diverse image acquisition and analysis methods like Colocalization, 2-D object tracking, rapid volume views, renderings and rotations, automated object counting, multiple binary layer thresholding and processing. Modules such as 3-D Blind Deconvolution, 2-D Real Time Deconvolution and Extended Depth of Focus are also available for the advanced users. Regards K.K.Veeraraghavan Product Specialist Bio- Imaging Division [hidden email] Towa Optics (India) Pvt. Ltd. India. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gene Maverick Sent: 09 February 2008 15:09 To: [hidden email] Subject: Re: A1 Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Heiko Düssmann, Michael Mancini, Simon Watkins, Stephen Cody and other confocal aficionados, In additions to the comments given by Mancini, Simon and Steve, I would like to express some of my inputs related to the newly introduced A1 Confocal system. Though I am working for Nikon, I am expressing my views as a fellow researcher. I presume that Nikon has designed this system to cater the upcoming needs of live cell imaging and molecular interaction analysis. As the molecular biological processes are happening in vivo at nano/micro second levels, we require higher speed without compromising the resolution and generating unwanted artifacts and stray noise interference. Due to high optical efficiency and 16 million pixels resolution, high quality confocal images can be achieved that will bring inter/intra cellular nuance into the limelight. Nikon claims that the most rapid biological events can be seen in ultra high resolution with the new system that is the evolved version of the present real time spectral confocal system C1si that has so many unique features like diffraction efficiency enhancement system based multiple gratings (DEES), weak signal sensitivity through dual integration signal processing (DISP), pre-calibrated synchronized 32 channel multi-anode PMTs, high-efficiency fluorescence transmission technology to achieve high optical transmission and most importantly the faster spectral unmixing algorithm that enables high speed spectral images without any molecular crosstalk in real time. The A1 system has standard paired galvonometers that gives high resolution images up to 4K x 4K pixels, whereas A1R incorporates two independent galvo systems high speed resonant and high resolution non-resonant hybrid scanner system, offering the speed of 30 frames per second at 512 x 512 pixels. The resonant scanner is mounted along with the non-resonant scanner gives industrys fastest 230 fps at 512 x 64 pixels and facilitates ultra-high-speed imaging with out compromising image quality. Scanning can be done by three modes through using the resonant scanner alone for high speed imaging up to 230 fps, using the non-resonant scanner along for high resolution imaging up to 4K x 4K pixels and by combining both resonant scanner and non-resonant scanner for simultaneous photo-stimulation imaging. This mechanism enables simultaneous photo-activation and imaging with improved sensitivity and reduced photo-toxicity that is vital for most of the sensitive functional cell dynamics applications. The hybrid scanning system also enables high speed imaging up to 420fps (2.4ms/frame) at 512 x 32 pixels. This supports advanced live cell imaging works more efficiently. The system comes with the analysis software for FRAP and FRET as standard. The newly fully automated standard fluorescence detector with 4 PMTs enables to acquire 4 color images simultaneously. This detector unit has changeable filters, enabling simple onsite installation of emission filter and mirror sets. In combination with four lasers, simultaneous observation of four different fluorescence labels is possible. When compare to C1si confocal system, the spectral detection performance with A1 is enhanced further along with V-filtering function that expands the range of use of spectral images. Through V-filtering function, up to four preferred spectral ranges can be chosen from 32 channels and the intensity of each range can be adjusted independently and this allows selection of desired spectral range and flexibility to handle new fluorescence probes. Together with the high speed AD conversion circuit, the new signal processing technology allows simultaneous 32 channel spectral image acquisition at 512 x 512 pixels in 0.5 seconds. At 512 x 64 pixels, images can be acquired with the speed of 16 frames per second. The industrys first low incidence 12 degree angle dichroic mirror enhances 30% more fluorescence efficiency and 99% transmission in combination with high performance sputtering as the reflection-transmission characteristics have lower polarization dependence, when compare to conventional incidence angle (45 degree) method, where reflection-transmission characteristics have high polarization dependence. Ideally speaking, the pinhole shape should be circular. With the A1 system, the continuously variable hexagonal pinhole, which replaces the standard four-sided aperture, considerably sharpen the image quality and it allows 30% more light resulting brighter images with the same sectioning performance as the area of hexagon inscribing a circle is bigger than the area of square inscribing to the same circle. With tthis hexagonal pinhole design, maximum confocality is maintained while achieving higher brightness equivalent to that of an ideal circular pinhole. Virtual Adaptable Aperture System (VAAS) pinhole unit provides better images with less flare as the light that a confocal pinhole rejects is collected by another detector. It allows simulation of different sectionings and slice thicknesses after image acquisition and has a better control over the attainment of experimental data. This unique detector system allows virtual adjustment of the confocality and sensitivity by collecting more photons during the initial image acquisition to generate a high resolution image. VAAS detection is an upgrade option that is expected in October 2008. Two laser input ports are incorporated in the scan head to use various laser lines. 4 laser unit is used as a standard and 3 laser board can be added as an option. Hence, 7 lasers with 9 lines are available in maximum. In addition, it also has IR laser port as an option. Along with the newly introduced Ti-E inverted microscope, A1/A1R confocal system set a new standard for advanced time-lapse studies of rapid cellular interactions to bring biological imaging to life. However, to understand this system and the over all performance we need to wait further as the optional spectral detector is yet to be launched in April 2008. In addition, the VAAS system, which is again an optional up grade, will be available only in October 2008. Gene Maverick. |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal When you say "optional" picosecond or faster IR laser - who's option would that be? Bought by the customer, or supplied from yourselves or Nikon? The support for communication with the IR laser is quite interesting too. best wishes Darran Clements |
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In reply to this post by K.K.Veeraraghavan
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The ability of using this system along with Controlled Light Exposure 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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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal You may want to read this article, which idea is implemented by Nikon for their system: Hoebe RA, Van Oven CH, Gadella TW Jr, Dhonukshe PB, Van Noorden CJ, Manders EM. Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging. Nat Biotechnol. 2007 Feb;25(2):249-53. Epub 2007 Jan 21. Christophe Leterrier On Feb 18, 2008 4:50 PM, David Knecht <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > The ability of using this system along with Controlled Light Exposure > Microscopy (CLEM) makes this system for long time live cell imaging and > confocal analysis. CLEM automatically monitors and varying the laser > illumination during time-lapse studies to reduce the risk of laser induced > bleaching, biochemical inconsistency and cell death. > > Can you clarify what this means? I don't see how you can monitor the > imaging and vary the laser in a way that can avoid cell toxicity. > Typically, you don't want the laser intensity to vary. Do you have any > quantitative data on cellular toxicity during imaging in comparison to a > spinning disk system? > > 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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In reply to this post by Knecht, David
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi David, the system measures the amount of fluorescence signal per pixel and controls the AOTF for this pixel based on the measured value. So if there is no light coming to the detector, laser will be restricted from illuminating the sample. Or, if the detector gets close to saturation, laser will be restricted as well. This should reduce photobleaching and phototoxicity also in out-of-focus areas. That's at least how I understand this. "The CLEM control unit is an optional add-on system for the C1. The CLEM unit calculates the integrated detected signal and exposure time for each pixel in real time, performs AOM high speed shutter control and high speed operation processing based on the amount of acquired fluorescence signal, PMT gain and then outputs the resulting fluorescence signal to the C1 controller’s line grabber electronics circuit." source: http://www.nikoninstruments.eu/news.php?n=550 I expect the quantification (measuring intensities) to be difficult afterwards, since one needs to apply a correction. However, it's one of the main things that makes the Nikon system interesting. Michael David Knecht wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> The ability of using this system along with Controlled Light Exposure >> Microscopy (CLEM) makes this system for long time live cell imaging and >> confocal analysis. CLEM automatically monitors and varying the laser >> illumination during time-lapse studies to reduce the risk of laser induced >> bleaching, biochemical inconsistency and cell death. > > Can you clarify what this means? I don't see how you can monitor the > imaging and vary the laser in a way that can avoid cell toxicity. > Typically, you don't want the laser intensity to vary. Do you have any > quantitative data on cellular toxicity during imaging in comparison to a > spinning disk system? > > 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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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear, We were using the CLEM set-up developed by Eric Manders, the inventor of CLEM, and were able to visualize GFP transformed cells for up to 48 hours. We could see confocal mitosis and all the nice biology of GFP labelled cells. From time to time cells escaped our field of view and we had to adjust the focal plane so within 48 hours there is gap, due to some human shortcomings. As compared to conventional confocal it really reduce bleaching and phototoxicity. More over Eric is a very nice person to collaborate with. Bye Patrick Van Oostveldt Quoting Michael Weber <[hidden email]>: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi David, > > the system measures the amount of fluorescence signal per pixel and > controls the AOTF for this pixel based on the measured value. So if > there is no light coming to the detector, laser will be restricted from > illuminating the sample. Or, if the detector gets close to saturation, > laser will be restricted as well. This should reduce photobleaching and > phototoxicity also in out-of-focus areas. That's at least how I > understand this. > > "The CLEM control unit is an optional add-on system for the C1. The > CLEM unit calculates the integrated detected signal and exposure > time for each pixel in real time, performs AOM high speed shutter > control and high speed operation processing based on the amount of > acquired fluorescence signal, PMT gain and then outputs the > resulting fluorescence signal to the C1 controller’s line grabber > electronics circuit." > > source: http://www.nikoninstruments.eu/news.php?n=550 > > I expect the quantification (measuring intensities) to be difficult > afterwards, since one needs to apply a correction. However, it's one of > the main things that makes the Nikon system interesting. > > Michael > > > David Knecht wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> The ability of using this system along with Controlled Light Exposure >>> Microscopy (CLEM) makes this system for long time live cell imaging and >>> confocal analysis. CLEM automatically monitors and varying the laser >>> illumination during time-lapse studies to reduce the risk of laser induced >>> bleaching, biochemical inconsistency and cell death. >> >> Can you clarify what this means? I don't see how you can monitor >> the imaging and vary the laser in a way that can avoid cell >> toxicity. Typically, you don't want the laser intensity to vary. >> Do you have any quantitative data on cellular toxicity during >> imaging in comparison to a spinning disk system? >> >> 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) -- Dep. Moleculaire Biotechnologie Coupure links 653 B 9000 GENT tel 09 264 5969 fax 09 264 6219 |
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In reply to this post by Knecht, David
Search the CONFOCAL archive at
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Search the CONFOCAL archive at
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David Knecht wrote Ø ………Typically, you don't want the laser intensity to vary. …………
My reply is more of a question than a statement, can someone who has used the CLEM device please confirm.
Presumably if you have CLEM fitted to your confocal you can choose to turn it off for quantitative measurements of fluorescence. Turning it on only when required for long time-lapse experiments where reducing bleaching is paramount, and where you are interested primarily in morphology rather than pixel to pixel intensity. Can someone please confirm it is implemented this way on the A1?
Also there has not been in depth discussion of the “VAAS” detector. The conventional confocal detector collects a confocal image after the pinhole, but the light rejected by the pinhole is also collected by a detector. This signal can then be deconvolved with the confocal signal to produce an optical section with more signal compared to conventional confocal. My question: Is this then equivalent to a deconvolved widefield image? Or is the confocal image used in some way to determine what the resulting deconvolved image should look like?
Cheers Steve Stephen H. Cody Tip: Learn how to receive reminders about you microscope
booking: -----Original Message-----
Can you clarify what this means? I don't see how you can monitor the imaging and vary the laser in a way that can avoid cell toxicity. Typically, you don't want the laser intensity to vary. Do you have any quantitative data on cellular toxicity during imaging in comparison to a spinning disk system?
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)
This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error. The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research. |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Stephen Cody wrote: > > Presumably if you have CLEM fitted to your confocal you can choose to > turn it off for quantitative measurements of fluorescence. Turning it on > only when required for long time-lapse experiments where reducing > bleaching is paramount, and where you are interested primarily in > morphology rather than pixel to pixel intensity. Can someone please > confirm it is implemented this way on the A1? > A couple of posts have noted that CLEM might interfere with intensity measurement. I see no reason why this should be so. The CLEM system has all of the information it needs to know how much exposure a given pixel received. That being the case, the resulting intensity levels can be corrected accordingly. Moreover, it would appear that CLEM operation allows one to avoid approaching the edges of the detector's dynamic range, so that the detector can be operated with a more linear response to intensity changes. Therefore, intensity measurements should at least a accurate as a non-CLEM system. Perhaps the developers can comment on whether my understanding is correct. --aryeh -- Aryeh Weiss School of Engineering Bar Ilan University Ramat Gan 52900 Israel Ph: 972-3-5317638 FAX: 972-3-7384050 |
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In reply to this post by Stephen Cody
Search the CONFOCAL archive at
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Somehow I think we've had this discussion about CLEM before
- maybe about a year ago? Not sure of the date but I do recall Jim Pawley
was involved, and Erik Manders was dragged back into the list to explain it
all. this should all be in the archives. I've not used the device in
anger but I have seen it demonstrated and it does work. As it was
explained to me the various parameters can be controlled (eg at what point you
'give up' and don't collect any more from a region which is giving no useful
signal) and yes, of course you can turn it off and you must do so for
quantitation.
The collection of light both in and closely around
the pinhole at the same time was, I
thought, patented by Optiscan. What you can do with it must depend on your
sample but at the least it must help in finding thin
layers.
Guy Optical Imaging Techniques in Cell Biology From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stephen Cody Sent: Tuesday, 19 February 2008 10:25 AM To: [hidden email] Subject: Re: A1 David Knecht wrote Ø ………Typically, you don't want the laser intensity to vary. …………
My reply is more of a question than a statement, can someone who has used the CLEM device please confirm.
Presumably if you have CLEM fitted to your confocal you can choose to turn it off for quantitative measurements of fluorescence. Turning it on only when required for long time-lapse experiments where reducing bleaching is paramount, and where you are interested primarily in morphology rather than pixel to pixel intensity. Can someone please confirm it is implemented this way on the A1?
Also there has not been in depth discussion of the “VAAS” detector. The conventional confocal detector collects a confocal image after the pinhole, but the light rejected by the pinhole is also collected by a detector. This signal can then be deconvolved with the confocal signal to produce an optical section with more signal compared to conventional confocal. My question: Is this then equivalent to a deconvolved widefield image? Or is the confocal image used in some way to determine what the resulting deconvolved image should look like?
Cheers Steve Stephen
H. Cody
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receive reminders about you microscope booking: -----Original
Message-----
Can you clarify what this means? I don't see how you can monitor the imaging and vary the laser in a way that can avoid cell toxicity. Typically, you don't want the laser intensity to vary. Do you have any quantitative data on cellular toxicity during imaging in comparison to a spinning disk system?
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)
This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error. The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research. No virus found in this incoming message. No virus found in this outgoing message. |
 
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Michael Weber-4 |
Re: FW: A1 - IR port and multiphoton compatibility
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In reply to this post by dc-4
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Darran, did you already got a reply off-list? I would be interested in how well the A1 performs in a multiphoton setup (without NDDs?) - and if there are any cooperations a customer could benefit from. cheers, Michael > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > When you say "optional" picosecond or faster IR laser - who's option would > that be? Bought by the customer, or supplied from yourselves or Nikon? The > support for communication with the IR laser is quite interesting too. > > best wishes > > Darran Clements |
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GeneMaverick |
Re: FW: A1 - IR port and multiphoton compatibility
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Michael and Darran, I guess the optional IR Port is given to enable the users to upgrade the system in to multiphoton with IR lasers in future. However, as the patent on multiphoton is not yet expired, Nikon may not make a system as multiphoton ready as a factory assembled unit. The optics of the inverted microscope platforms like TE2000E-PFS and Ti-E are IR optimized. The focal drift control system of the inverted microscope, what Nikon calls perfect focus system (PFS) is also enhanced as they are capable of handling the dyes of far red region. The fast pulsed lasers that are available commercially can be used through the IR port. Gene Maverick. |
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In reply to this post by Michael Weber-4
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Gene, the original question also concerned the ease of implementation of the laser modulation. Is it a basic TTL pulse, the ability to control an external shutter, embedded drivers for the control of AOM/EOM or direct control of the laser? This is probably the more important aspect of the question and will obviously be different in the case of C1 class systems to A1 systems. The physical hooking up of a laser is probably less thorny than the means of controlling the beam when it is hooked up and I just wanted to know the level of preparation and work that would be required to get a fully functioning system and to what specification beam blanking/ROI functionality could be acheived. all the best Darran |
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In reply to this post by K.K.Veeraraghavan
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear All, I would like to add some comments to this discussion. Guy, you are right, we've had a discussion about CLEM about a year ago (and I'm sure, this will not be the last one). Michael, I think there is misunderstanding about data can be obtained with CLEM. For each individual pixel the optimal exposure time is determined. The integrated fluorescence signal is extrapolated at the end of each pixel. So, you are right, this extrapolation is a kind of correction procedure, but this happens internally in the CLEM electronics and you do not notice the difference between CLEM and non-CLEM when you look at the images. So, this means that the extrapolated pixel intensity is unbiased. The only difference is that the noise on the signal is increased for pixels that allow a shorter exposure time. So, CLEM reduces the exposure time in very bright pixels (S/N is too good in non-CLEM) and in dark background pixels (why would you want to have a good S/N if there is no signal!). This reduced S/N in parts of the image where a good s/n is not needed, is the price for a reduction in photobleaching and phototoxicity. The signal does NOT change by CLEM. This brings me to the point of quantitative imaging with CLEM: I do really not agree that CLEM should be switched off when you want to do quantitative imaging as suggested by Stephen. As I explained: CLEM does not change the measured gray values. There is only one reason why people might think that CLEM does not give quantitative data and that is photobleaching. I will here argue that CLEM does give images that are not less quantitative than non-CLEM images. So, now we come to the point of photobleaching. Since CLEM uses different exposure times for different pixels, the sample is non-uniformly exposed to light. This causes non-uniform photobleaching. So, if you scan your sample several times, you cannot use a standard method for photobleaching correction. So, Ron Hoebe and I developed a dedicated correction procedure for CLEM images (which we will make available later). This algorithm calculates how much light each pixels in a 3D volume has received before scanning that pixel and corrects for bleaching. Since we know of every pixel how much the exposure time was, we can correct. This is not difficult, it is only a little bit more complicated. We also measured the difference between this dedicated procedure and a standard correction methods and found out that the difference is not much (only a few percents). Ron did a lot of computer simulations to prove that his program works, by simulating every single photon for every single pixel (lot of computation time...). So, Stephen, you might say: Well when I want to have quantitative images, I just turn of the CLEM. Let's see what would happen. When you do turn it off, your photobleaching will increase again to its non-CLEM value (about 5 to 10 fold) So you will end-up with a strongly bleached image and a dead cell! Great! You do a nice quantitative analysis of an image of a dead cell that you can hardly see anymore! And you do this only because you are afraid that the non-uniform bleaching is not corrected optimally? And what about non-uniform photobleaching in non-CLEM confocal images. Does anyone realize that photobleaching is stronger in the middle of an image than more to the edges? Since we illuminate the sample with a cone of light, the pixels more to the edges of the volume get a lower light dose at the end of a scan. And I have never seen photobleaching correction procedures that take this into account. So, how quantitative is your imaging right now (without CLEM) Finally, we should realize that bleaching is far from linear (especially in confocal and TPE microscopy. All bleaching correction procedures assume linear bleaching (what else can you do??). So, with this in mind: How quantitative are your images now. I prefer a 7-fold reduction of photobleaching so that I do not need any correction procedures!! Aryeh, you are right: CLEM also increases the dynamic range of the system. However, your argument "the detector can be operated with a more linear response to intensity changes" is not fully correct. We do not reduce the intensity of laser light, we only reduce the light exposure time. But since the exposure time is reduced for very bright pixels, clipping of the (integrated) signal is history. This makes that you can see details in the weakly stained parts while not saturating the brightly stained parts. --> larger dynamic range. Stephan asked: "can someone who has used the CLEM device please confirm..." So far, only a hand full of people have used CLEM. Some developers of Nikon rested CLEM and Ron and I as the inventors of CLEM have tested it, but so far only one person seriously applied CLEM for his biological research. Winnok de Vos from the group of Patrick Van Oostveld from Ghent, Belgium visited our lab for months and months to do his live cell imagine. He spent many nights and days behind the CLEM microscope. To his experience, cells can cope with a certain amount of trouble (ROS, light damage). As long as your exposure is under this critical threshold, you are fine. When the light is too much, you are in trouble. By using CLEM he succeeded to stay under this threshold and he just could go on imaging. Without CLEM it was about half an hour; with CLEM for more than 24 hours. So, we should not only focus on the bleaching reduction by CLEM (although, where do you find a anti-bleaching reagent that reduces bleaching by a factor of 5 to 10 ????) but also to the reduction of phototoxicity. At this moment there is only one working CLEM microscope here at the University of Amsterdam and some prototypes of the Nikon-CLEM. But soon there will be much more now Nikon implemented CLEM as a standard option in the new A1. As soon as I have my Nikon-A1, I will give my comments on my findings, especially on my first A1-CLEM experiences. I expect a better CLEM than our own experimental set-up. We'll see... I think this comment satisfies you for the coming few months.... Kind regards, Erik --------------- E.M.M. Manders, PhD Ass. prof. Molecular Cytology Manager Centre for Advanced Microscopy Centre for Advanced Microscopy Swammerdam Institute for Life Sciences Faculty of Science University of Amsterdam E-mail: [hidden email] Tel: +31-(0)20-5256225 Fax: +31-(0)20-5257934 .--. .- ....- . -- |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Erik, Thanks for your reply. It was exactly this kind of informed discussion I was hoping to stimulate. CLEM to me has always seemed very powerful, especially as we have conducted a lot of long term time-lapse experiments with a conventional point scanning confocal. However, CLEM so far does not seemed to have taken off in the market. I did not want to sound critical of the technology, quite the opposite. I think it will be ideal for the types of experiments we have conducted here. I do have another question though if you don't mind. Erik writes > ....and you do not notice the difference between CLEM and non-CLEM when you look at the images. This is great news for qualitative imaging. But from the body of your email you were suggesting it is suitable for more than just qualitative imaging. Have you compared images analysed with Image Correlation Spectroscopy techniques such as RICS? Are the CLEM and non-CLEM images considered equivalent when analysed with such techniques? It is given that photo-bleaching will presumably be worse in the non-CLEM images. Cheers Steve Stephen H. Cody Microscopy Manager Central Resource for Advanced Microscopy Ludwig Institute for Cancer Research PO Box 2008 Royal Melbourne Hospital Victoria, 3050 Australia Tel: 61 3 9341 3155 Fax: 61 3 9341 3104 email: [hidden email] www.ludwig.edu.au/labs/confocal.html www.ludwig.edu.au/confocal Tip: Learn how to receive reminders about you microscope booking: www.ludwig.edu.au/confocal/Local/Booking_Hint.htm -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Erik Manders Sent: Thursday, 21 February 2008 5:54 AM To: [hidden email] Subject: Re: A1 Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear All, I would like to add some comments to this discussion. Guy, you are right, we've had a discussion about CLEM about a year ago (and I'm sure, this will not be the last one). Michael, I think there is misunderstanding about data can be obtained with CLEM. For each individual pixel the optimal exposure time is determined. The integrated fluorescence signal is extrapolated at the end of each pixel. So, you are right, this extrapolation is a kind of correction procedure, but this happens internally in the CLEM electronics and you do not notice the difference between CLEM and non-CLEM when you look at the images. So, this means that the extrapolated pixel intensity is unbiased. The only difference is that the noise on the signal is increased for pixels that allow a shorter exposure time. So, CLEM reduces the exposure time in very bright pixels (S/N is too good in non-CLEM) and in dark background pixels (why would you want to have a good S/N if there is no signal!). This reduced S/N in parts of the image where a good s/n is not needed, is the price for a reduction in photobleaching and phototoxicity. The signal does NOT change by CLEM. This brings me to the point of quantitative imaging with CLEM: I do really not agree that CLEM should be switched off when you want to do quantitative imaging as suggested by Stephen. As I explained: CLEM does not change the measured gray values. There is only one reason why people might think that CLEM does not give quantitative data and that is photobleaching. I will here argue that CLEM does give images that are not less quantitative than non-CLEM images. So, now we come to the point of photobleaching. Since CLEM uses different exposure times for different pixels, the sample is non-uniformly exposed to light. This causes non-uniform photobleaching. So, if you scan your sample several times, you cannot use a standard method for photobleaching correction. So, Ron Hoebe and I developed a dedicated correction procedure for CLEM images (which we will make available later). This algorithm calculates how much light each pixels in a 3D volume has received before scanning that pixel and corrects for bleaching. Since we know of every pixel how much the exposure time was, we can correct. This is not difficult, it is only a little bit more complicated. We also measured the difference between this dedicated procedure and a standard correction methods and found out that the difference is not much (only a few percents). Ron did a lot of computer simulations to prove that his program works, by simulating every single photon for every single pixel (lot of computation time...). So, Stephen, you might say: Well when I want to have quantitative images, I just turn of the CLEM. Let's see what would happen. When you do turn it off, your photobleaching will increase again to its non-CLEM value (about 5 to 10 fold) So you will end-up with a strongly bleached image and a dead cell! Great! You do a nice quantitative analysis of an image of a dead cell that you can hardly see anymore! And you do this only because you are afraid that the non-uniform bleaching is not corrected optimally? And what about non-uniform photobleaching in non-CLEM confocal images. Does anyone realize that photobleaching is stronger in the middle of an image than more to the edges? Since we illuminate the sample with a cone of light, the pixels more to the edges of the volume get a lower light dose at the end of a scan. And I have never seen photobleaching correction procedures that take this into account. So, how quantitative is your imaging right now (without CLEM) Finally, we should realize that bleaching is far from linear (especially in confocal and TPE microscopy. All bleaching correction procedures assume linear bleaching (what else can you do??). So, with this in mind: How quantitative are your images now. I prefer a 7-fold reduction of photobleaching so that I do not need any correction procedures!! Aryeh, you are right: CLEM also increases the dynamic range of the system. However, your argument "the detector can be operated with a more linear response to intensity changes" is not fully correct. We do not reduce the intensity of laser light, we only reduce the light exposure time. But since the exposure time is reduced for very bright pixels, clipping of the (integrated) signal is history. This makes that you can see details in the weakly stained parts while not saturating the brightly stained parts. --> larger dynamic range. Stephan asked: "can someone who has used the CLEM device please confirm..." So far, only a hand full of people have used CLEM. Some developers of Nikon rested CLEM and Ron and I as the inventors of CLEM have tested it, but so far only one person seriously applied CLEM for his biological research. Winnok de Vos from the group of Patrick Van Oostveld from Ghent, Belgium visited our lab for months and months to do his live cell imagine. He spent many nights and days behind the CLEM microscope. To his experience, cells can cope with a certain amount of trouble (ROS, light damage). As long as your exposure is under this critical threshold, you are fine. When the light is too much, you are in trouble. By using CLEM he succeeded to stay under this threshold and he just could go on imaging. Without CLEM it was about half an hour; with CLEM for more than 24 hours. So, we should not only focus on the bleaching reduction by CLEM (although, where do you find a anti-bleaching reagent that reduces bleaching by a factor of 5 to 10 ????) but also to the reduction of phototoxicity. At this moment there is only one working CLEM microscope here at the University of Amsterdam and some prototypes of the Nikon-CLEM. But soon there will be much more now Nikon implemented CLEM as a standard option in the new A1. As soon as I have my Nikon-A1, I will give my comments on my findings, especially on my first A1-CLEM experiences. I expect a better CLEM than our own experimental set-up. We'll see... I think this comment satisfies you for the coming few months.... Kind regards, Erik --------------- E.M.M. Manders, PhD Ass. prof. Molecular Cytology Manager Centre for Advanced Microscopy Centre for Advanced Microscopy Swammerdam Institute for Life Sciences Faculty of Science University of Amsterdam E-mail: [hidden email] Tel: +31-(0)20-5256225 Fax: +31-(0)20-5257934 .--. .- ....- . -- This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error. The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research. |
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In reply to this post by Michael Weber-4
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal On 19/02/2008, at 3:41 AM, Michael Weber wrote: > Hi David, > > the system measures the amount of fluorescence signal per pixel and > controls the AOTF for this pixel based on the measured value. So if > there is no light coming to the detector, laser will be restricted > from illuminating the sample. Or, if the detector gets close to > saturation, laser will be restricted as well. This should reduce > photobleaching and phototoxicity also in out-of-focus areas. That's > at least how I understand this. Does it work with the AOTF on the A1? When I enquired about it on the C1 I was told it was only available with the AOM on the 3-laser board, not the AOTF on the 4 laser board? Regards, Adrian Smith Centenary Institute, Sydney, Australia |
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