Help with EMCCD evaluation
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, I am in the market for an EMCCD for widefield fluorescence time-lapse imaging of genetically-labeled cultured cells. I am acquiring images frequently (every 10 minutes) for several days. The cells seem to be very sensitive to phototoxicity and photobleaching (GFP is the fluorophore), so I am hoping that replacing my Roper CoolSnapHQ CCD with an EMCCD will allow me to use much shorter exposure times. I realize that this is a confocal list and my application is widefield but since EMCCDs are also used with spinning disk confocal, I thought I'd ask. Apologies if this question has been hashed out before... I searched the archives without finding definitive recommendations. (Maybe that's asking for too much!) Any help on the following would be much appreciated: 1) What EMCCDs have people found to be the most *sensitive* (nice picture in low-light) and the most *reliable* (driver doesn't crash, etc.)? I'd especially be interested in any experiences or opinions with Roper/Photometrics's CascadeII or QuantEM and the Andor iXon cameras. 2) How have people been testing cameras? I've set up demos of several cameras so I'd like to do some standard tests. I've seen some references on the web for calculating dark noise, read noise, and gain, such as: http://www.qsimaging.com/ccd_noise_measure.html http://www.mirametrics.com/tech_note_ccdgain.htm http://www.photomet.com/library/library_encyclopedia/library_enc_gain.php but I'd love to hear what other tests people think are relevant for these cameras. Obviously, I'll try imaging my samples (using fiber-coupled LED as a constant illumination source)... what else should I do? Fluorescent beads instead of my cells perhaps? 3) For coupling the camera to my Olympus IX71, is there something like the Zeiss Optovar (which, as I understand it, has multiple selectable magnifications of the intermediate lens) made by a third party? This would be helpful for changing between achieving the full NA of the objective and getting less resolution but a bigger field of view for different experiments. Thanks for your help. Best, - Neville --- Neville Sanjana Dept. of Brain and Cognitive Science Massachusetts Institute of Technology |
 
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Nowell, Cameron |
Re: Help with EMCCD evaluation
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Neville, While an EMCCD camera will decrease your exposure time, you may find that it will not really help solve your phototoxicity problem. The time your cells are exposed to fluorecent light will not neccesarily be dependant on the exposure time of your camera, but on how fast your entire sytem (microscope, shutters, filters, etc.) shuffles everything around. What software etc are you using to capture your images? We have a wide filed live cell rig here that uses an Orca ER camera on an IX81 microscope and it is all driven by metamorph. Even when the camera is capturing at 100ms exposures, the samples are exposed to flourecent light for about 2 seconds. To get rid of these sort of delays you need to move to using filter wheels, or for even shorter exposuers something like olympus' CellR system Cheers Cam Cameron Nowell B. Sc (Hons) Microscopy Imaging and Research Core Facility Peter MacCallum Cancer Centre 7 St Andrews Place East Melbourne, Victoria 3002 AUSTRALIA Phone: +61396561242 Mobile: +61422882700 Fax: +61396561411 ________________________________ From: Confocal Microscopy List on behalf of Neville Sanjana Sent: Wed 28/11/2007 7:57 AM To: [hidden email] Subject: Help with EMCCD evaluation Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, I am in the market for an EMCCD for widefield fluorescence time-lapse imaging of genetically-labeled cultured cells. I am acquiring images frequently (every 10 minutes) for several days. The cells seem to be very sensitive to phototoxicity and photobleaching (GFP is the fluorophore), so I am hoping that replacing my Roper CoolSnapHQ CCD with an EMCCD will allow me to use much shorter exposure times. I realize that this is a confocal list and my application is widefield but since EMCCDs are also used with spinning disk confocal, I thought I'd ask. Apologies if this question has been hashed out before... I searched the archives without finding definitive recommendations. (Maybe that's asking for too much!) Any help on the following would be much appreciated: 1) What EMCCDs have people found to be the most *sensitive* (nice picture in low-light) and the most *reliable* (driver doesn't crash, etc.)? I'd especially be interested in any experiences or opinions with Roper/Photometrics's CascadeII or QuantEM and the Andor iXon cameras. 2) How have people been testing cameras? I've set up demos of several cameras so I'd like to do some standard tests. I've seen some references on the web for calculating dark noise, read noise, and gain, such as: http://www.qsimaging.com/ccd_noise_measure.html http://www.mirametrics.com/tech_note_ccdgain.htm http://www.photomet.com/library/library_encyclopedia/library_enc_gain.php but I'd love to hear what other tests people think are relevant for these cameras. Obviously, I'll try imaging my samples (using fiber-coupled LED as a constant illumination source)... what else should I do? Fluorescent beads instead of my cells perhaps? 3) For coupling the camera to my Olympus IX71, is there something like the Zeiss Optovar (which, as I understand it, has multiple selectable magnifications of the intermediate lens) made by a third party? This would be helpful for changing between achieving the full NA of the objective and getting less resolution but a bigger field of view for different experiments. Thanks for your help. Best, - Neville --- Neville Sanjana Dept. of Brain and Cognitive Science Massachusetts Institute of Technology This email (including any attachments) may contain confidential and/or legally privileged information and is intended only to be read or used by the addressee. If you are not the intended addressee, any use, distribution, disclosure or copying of this email is strictly prohibited. Confidentiality and legal privilege attached to this email (including any attachments) are not waived or lost by reason of its mistaken delivery to you. If you have received this email in error, please delete it and notify us immediately by telephone or email. Peter MacCallum Cancer Centre provides no guarantee that this transmission is free of virus or that it has not been intercepted or altered and will not be liable for any delay in its receipt. |
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Gary Laevsky-2 |
Re: Help with EMCCD evaluation**Commercial Response**
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal The Andor iQ system employs what we call "active blanking." The EMCCD and our AOTF communicate directly with each other so that the AOTF only passes excitation light while the camera is exposing. This great reduces effects of phototoxicity due to exposure of cells to excitation light between captures. Best, Gary Laevsky, Ph.D. Imaging Application Specialist Andor Technology discover new ways of seeing [hidden email] Cell (774) 291 - 9992 Office (860) 290 - 9211 x219 Fax (860) 290 - 9566 Web: www.andor.com -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Nowell, Cameron Sent: Wednesday, November 28, 2007 2:51 AM To: [hidden email] Subject: Re: Help with EMCCD evaluation Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Neville, While an EMCCD camera will decrease your exposure time, you may find that it will not really help solve your phototoxicity problem. The time your cells are exposed to fluorecent light will not neccesarily be dependant on the exposure time of your camera, but on how fast your entire sytem (microscope, shutters, filters, etc.) shuffles everything around. What software etc are you using to capture your images? We have a wide filed live cell rig here that uses an Orca ER camera on an IX81 microscope and it is all driven by metamorph. Even when the camera is capturing at 100ms exposures, the samples are exposed to flourecent light for about 2 seconds. To get rid of these sort of delays you need to move to using filter wheels, or for even shorter exposuers something like olympus' CellR system Cheers Cam Cameron Nowell B. Sc (Hons) Microscopy Imaging and Research Core Facility Peter MacCallum Cancer Centre 7 St Andrews Place East Melbourne, Victoria 3002 AUSTRALIA Phone: +61396561242 Mobile: +61422882700 Fax: +61396561411 ________________________________ From: Confocal Microscopy List on behalf of Neville Sanjana Sent: Wed 28/11/2007 7:57 AM To: [hidden email] Subject: Help with EMCCD evaluation Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, I am in the market for an EMCCD for widefield fluorescence time-lapse imaging of genetically-labeled cultured cells. I am acquiring images frequently (every 10 minutes) for several days. The cells seem to be very sensitive to phototoxicity and photobleaching (GFP is the fluorophore), so I am hoping that replacing my Roper CoolSnapHQ CCD with an EMCCD will allow me to use much shorter exposure times. I realize that this is a confocal list and my application is widefield but since EMCCDs are also used with spinning disk confocal, I thought I'd ask. Apologies if this question has been hashed out before... I searched the archives without finding definitive recommendations. (Maybe that's asking for too much!) Any help on the following would be much appreciated: 1) What EMCCDs have people found to be the most *sensitive* (nice picture in low-light) and the most *reliable* (driver doesn't crash, etc.)? I'd especially be interested in any experiences or opinions with Roper/Photometrics's CascadeII or QuantEM and the Andor iXon cameras. 2) How have people been testing cameras? I've set up demos of several cameras so I'd like to do some standard tests. I've seen some references on the web for calculating dark noise, read noise, and gain, such as: http://www.qsimaging.com/ccd_noise_measure.html http://www.mirametrics.com/tech_note_ccdgain.htm http://www.photomet.com/library/library_encyclopedia/library_enc_gain.ph p but I'd love to hear what other tests people think are relevant for these cameras. Obviously, I'll try imaging my samples (using fiber-coupled LED as a constant illumination source)... what else should I do? Fluorescent beads instead of my cells perhaps? 3) For coupling the camera to my Olympus IX71, is there something like the Zeiss Optovar (which, as I understand it, has multiple selectable magnifications of the intermediate lens) made by a third party? This would be helpful for changing between achieving the full NA of the objective and getting less resolution but a bigger field of view for different experiments. Thanks for your help. Best, - Neville --- Neville Sanjana Dept. of Brain and Cognitive Science Massachusetts Institute of Technology This email (including any attachments) may contain confidential and/or legally privileged information and is intended only to be read or used by the addressee. If you are not the intended addressee, any use, distribution, disclosure or copying of this email is strictly prohibited. Confidentiality and legal privilege attached to this email (including any attachments) are not waived or lost by reason of its mistaken delivery to you. If you have received this email in error, please delete it and notify us immediately by telephone or email. Peter MacCallum Cancer Centre provides no guarantee that this transmission is free of virus or that it has not been intercepted or altered and will not be liable for any delay in its receipt. |
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In reply to this post by Nowell, Cameron
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi! If it is for cell imaging you might consider a BT camera. Forcing the previous point, which I thing is very important: I'm having the same problem with an ORCA AG with ImagePro Plus. What I have now is a small program called by macros that controls an external shutter via TTL, which makes a delay till it opens the shutter. I found out that the driver takes almost 500ms to get the camera taking a picture which is unacceptable! However this solution is far from being elegant and sometimes the pseudo synchronization fails! Is this a Hamamatsu problem? With a Hamamatsu BT1024 which has a QE>90% it happends the same thing with metamorph. Regarding to the previous commercial response, does anyone have a cheaper and more flexible solution? Regards, NM Nowell, Cameron wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi Neville, > While an EMCCD camera will decrease your exposure time, you may find that it will not really help solve your phototoxicity problem. The time your cells are exposed to fluorecent light will not neccesarily be dependant on the exposure time of your camera, but on how fast your entire sytem (microscope, shutters, filters, etc.) shuffles everything around. > > What software etc are you using to capture your images? > > We have a wide filed live cell rig here that uses an Orca ER camera on an IX81 microscope and it is all driven by metamorph. Even when the camera is capturing at 100ms exposures, the samples are exposed to flourecent light for about 2 seconds. To get rid of these sort of delays you need to move to using filter wheels, or for even shorter exposuers something like olympus' CellR system > > > Cheers > > > Cam > > > Cameron Nowell B. Sc (Hons) > > > > Microscopy Imaging and Research Core Facility > > Peter MacCallum Cancer Centre > > 7 St Andrews Place > > East Melbourne, Victoria 3002 > > AUSTRALIA > > > > Phone: +61396561242 > > Mobile: +61422882700 > > Fax: +61396561411 > > > > ________________________________ > > From: Confocal Microscopy List on behalf of Neville Sanjana > Sent: Wed 28/11/2007 7:57 AM > To: [hidden email] > Subject: Help with EMCCD evaluation > > > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi, > > I am in the market for an EMCCD for widefield fluorescence > time-lapse imaging of genetically-labeled cultured cells. I > am acquiring images frequently (every 10 minutes) for > several days. The cells seem to be very sensitive to > phototoxicity and photobleaching (GFP is the fluorophore), > so I am hoping that replacing my Roper CoolSnapHQ CCD with > an EMCCD will allow me to use much shorter exposure times. > > I realize that this is a confocal list and my application is > widefield but since EMCCDs are also used with spinning disk > confocal, I thought I'd ask. Apologies if this question has > been hashed out before... I searched the archives without > finding definitive recommendations. (Maybe that's asking for > too much!) Any help on the following would be much appreciated: > > 1) What EMCCDs have people found to be the most *sensitive* > (nice picture in low-light) and the most *reliable* (driver > doesn't crash, etc.)? I'd especially be interested in any > experiences or opinions with Roper/Photometrics's CascadeII > or QuantEM and the Andor iXon cameras. > > 2) How have people been testing cameras? I've set up demos > of several cameras so I'd like to do some standard tests. > I've seen some references on the web for calculating dark > noise, read noise, and gain, such as: > http://www.qsimaging.com/ccd_noise_measure.html > http://www.mirametrics.com/tech_note_ccdgain.htm > http://www.photomet.com/library/library_encyclopedia/library_enc_gain.php > but I'd love to hear what other tests people think are > relevant for these cameras. Obviously, I'll try imaging my > samples (using fiber-coupled LED as a constant illumination > source)... what else should I do? Fluorescent beads instead > of my cells perhaps? > > 3) For coupling the camera to my Olympus IX71, is there > something like the Zeiss Optovar (which, as I understand it, > has multiple selectable magnifications of the intermediate > lens) made by a third party? This would be helpful for > changing between achieving the full NA of the objective and > getting less resolution but a bigger field of view for > different experiments. > > Thanks for your help. Best, > > - Neville > > --- > Neville Sanjana > Dept. of Brain and Cognitive Science > Massachusetts Institute of Technology > > > > > This email (including any attachments) may contain > confidential and/or legally privileged information and is > intended only to be read or used by the addressee. If you > are not the intended addressee, any use, distribution, > disclosure or copying of this email is strictly > prohibited. > Confidentiality and legal privilege attached to this email > (including any attachments) are not waived or lost by > reason of its mistaken delivery to you. > If you have received this email in error, please delete it > and notify us immediately by telephone or email. Peter > MacCallum Cancer Centre provides no guarantee that this > transmission is free of virus or that it has not been > intercepted or altered and will not be liable for any delay > in its receipt. > -- Nuno Moreno Cell Imaging Unit Instituto Gulbenkian de Ciência http://uic.igc.gulbekian.pt http://www.igc.gulbekian.pt phone +351 214464606 fax +351 214407970 |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Nuno Moreno wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi! > > If it is for cell imaging you might consider a BT camera. > > Forcing the previous point, which I thing is very important: > > I'm having the same problem with an ORCA AG with ImagePro Plus. What I > have now is a small program called by macros that controls an external > shutter via TTL, which makes a delay till it opens the shutter. I found > out that the driver takes almost 500ms to get the camera taking a > picture which is unacceptable! However this solution is far from being > elegant and sometimes the pseudo synchronization fails! Is this a > Hamamatsu problem? With a Hamamatsu BT1024 which has a QE>90% it > happends the same thing with metamorph. > > Regarding to the previous commercial response, does anyone have a > cheaper and more flexible solution? > The way to do this is by directly controlling the shutter from the chip-exposure line of the camera. This bypasses the software completely. Most high end cameras have such a control line, although it goes by various names. I know that the PCO sensicam series, Roper Coolsnap series, Orca AG (probably the other Orcas also), QImaging, all have such a line. I use it with a Uniblitz shutter. You still have to worry about the the 5-10ms shutter opening and closing time. In a camera like the PCO (again, probably in others) the camera can be set up to start integrating after a delay time set by the user. This increases your sample exposure by 10-20ms, but that is really not bad compared to the 1-2 seconds that some software controlled systems waste before closing the shutter. Of course, if you connect the control line directly to a system like CoolLed or an AOTF, you can get very fast switching. You may need to build a little bit of digital logic. For example, when doing a live preview for focusing, we want the shutter to remain open continuously. But when using the chip expose line, the shutter will cycle rapidly as the camera switches between integration and image transfer. Therefore, we set it up so that in live preview mode the Uniblitz system was commanded to stay open independent of the hardware control line. I also added an external switch that could force the input to the Uniblitz control to be on (ie, I OR'ed the camera control line with a manually switched voltage). This was a matter of a few NAND gates on two 74LSxx series integrated circuits (your electronics shop will certainly have these). I know that this solution is used in other labs, so maybe I am missing something in this thread about the difficulty that is being discussed. --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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Neville Sanjana |
Re: Help with EMCCD evaluation
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi confocal list, First, thanks to everyone for their wonderful suggestions. Both commercial entities and scientists had lots of nice advice. As for shuttering, I'm currently doing exactly what Aryeh mentions: Using the direct chip-exposure hardware trigger from the camera (CoolSnapHQ) to control a Uniblitz shutter so that no software is invovled. (We use some OR and AND gates on a breadboard as Aryeh described to control when the shutter is responsive to the exposure signal of the CoolSnap.) The CoolSnap can also be told to use a pre-set delay between shutter opening and exposure, which we set at 5msec (the Uniblitz is rated for 3msec). Since we use a blue LED as our excitation source, I could just turn that on and off directly too (we were using a Hg lamp before). I think the take-home message is that it is generally better to have the camera directly control the shutter/LED rather than worry about software synchronization, which will never be as precise. Since Cameron asked, I'm using custom Matlab software to control the stage, camera, shutters, filters, etc. The PVCAM C interface makes it easy to directly control the camera. I am confident that the software is taking exposures accurately. In particular, I'm taking about 200msec exposures with 3x3 binning (blue LED, 60x/0.7NA objective, CoolSnapHQ) every 10 minutes and the cells stay viable but the signal is weak making it difficult for me to trace fine structures in my cultured neurons. I'm working on changing to a better objective and, as you know, a more sensitive CCD. Still, I would welcome any further ideas on experiences with different EMCCDs (not from commercial vendors) and ideas on how to test EMCCDs. Right now, for demoing, I'm hoping to take some dark frames at various exposure times (dark noise) and some pictures of fluorescent beads with a set exposure time (overall QE test). I could also test the read noise with some bias images, but I'm hoping that these EMCCDs will have no issue there. Thanks again for all the help... I've certainly learned a lot from your replies! Best, - Neville Aryeh Weiss wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Nuno Moreno wrote: > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hi! >> >> If it is for cell imaging you might consider a BT camera. >> >> Forcing the previous point, which I thing is very important: >> >> I'm having the same problem with an ORCA AG with ImagePro Plus. What >> I have now is a small program called by macros that controls an >> external shutter via TTL, which makes a delay till it opens the >> shutter. I found out that the driver takes almost 500ms to get the >> camera taking a picture which is unacceptable! However this solution >> is far from being elegant and sometimes the pseudo synchronization >> fails! Is this a Hamamatsu problem? With a Hamamatsu BT1024 which has >> a QE>90% it happends the same thing with metamorph. >> >> Regarding to the previous commercial response, does anyone have a >> cheaper and more flexible solution? >> > > The way to do this is by directly controlling the shutter from the > chip-exposure line of the camera. This bypasses the software completely. > Most high end cameras have such a control line, although it goes by > various names. I know that the PCO sensicam series, Roper Coolsnap > series, Orca AG (probably the other Orcas also), QImaging, all have > such a line. I use it with a Uniblitz shutter. You still have to worry > about the the 5-10ms shutter opening and closing time. In a camera like > the PCO (again, probably in others) the camera can be set up to start > integrating after a delay time set by the user. This increases your > sample exposure by 10-20ms, but that is really not bad compared to the > 1-2 seconds that some software controlled systems waste before closing > the shutter. > > Of course, if you connect the control line directly to a system like > CoolLed or an AOTF, you can get very fast switching. > > You may need to build a little bit of digital logic. For example, when > doing a live preview for focusing, we want the shutter to remain open > continuously. But when using the chip expose line, the shutter will > cycle rapidly as the camera switches between integration and image > transfer. Therefore, we set it up so that in live preview mode the > Uniblitz system was commanded to stay open independent of the hardware > control line. I also added an external switch that could force the input > to the Uniblitz control to be on (ie, I OR'ed the camera control line > with a manually switched voltage). This was a matter of a few NAND gates > on two 74LSxx series integrated circuits (your electronics shop will > certainly have these). > > I know that this solution is used in other labs, so maybe I am missing > something in this thread about the difficulty that is being discussed. > > --aryeh |
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In reply to this post by Nuno Moreno
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Nuno,
The drivers for all Hamamatsu cameras are written by Hamamatsu. I would suggest that you contact them for help with your issue. In my experience as a user of Image-Pro Plus, and as an employee of Media Cybernetics and as an employee of several of their dealers, Hamamatsu is very good about supporting their cameras on any platform and will gladly help you get it running as smoothly as possible. Chris Tully On Nov 28, 2007 8:59 AM, Nuno Moreno <[hidden email]> wrote: Hi! |
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In reply to this post by Neville Sanjana
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Neville Sanjana <[hidden email]> writes: > is taking exposures accurately. In particular, I'm taking about > 200msec exposures with 3x3 binning (blue LED, 60x/0.7NA objective, Totally unrelated to your EMCCD question but a cheaper probably more effective solution may be a 60x 1.4NA oil immersion lens. This will collect 4 times as much light and hence much brighter pictures (as well as the improved resolution). Of course your particular application may make this impossible. Ian |
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Neville Sanjana |
Re: Help with EMCCD evaluation
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal That's a great idea but there are a few particulars of my application (automated long-term time lapse) that make that difficult. First, my autofocus algorithms (second derivative/Laplacian filter) seem to break at higher NAs due to the smaller depth of field (and, hence, lower tolerance for small errors in Z). Also, for long-term timelapse with stage tiling, after a while the oil can get bubbles in it, which ruins the image. I am however thinking along the lines you suggested and trying a 40x/NA0.9 air objective to see if that could work. - Neville Ian Dobbie wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Neville Sanjana <[hidden email]> writes: > > >>is taking exposures accurately. In particular, I'm taking about >>200msec exposures with 3x3 binning (blue LED, 60x/0.7NA objective, > > > Totally unrelated to your EMCCD question but a cheaper probably more > effective solution may be a 60x 1.4NA oil immersion lens. This will > collect 4 times as much light and hence much brighter pictures (as > well as the improved resolution). Of course your particular > application may make this impossible. > > Ian |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Neville,
First let me say that I work for Vashaw Scientific (a Leica Microsystems dealer) and have worked for other Microscope dealers and Media Cybernetics (makers of Image-Pro Plus). I have also been a user of microscopes and imaging systems prior to my tenure on the commercial side. With that said I would like to suggest trying less illumination (lower voltage to the LED) and longer exposures, since phototoxicity seems to be an issue in your experiments. I do have experience with two customers who had similar problems. One used a Photometics Sensys 1400 (the best camera at the time) with a 16 bit digitizer in maximum gain mode and still only acquired for long enough to get ~200 gray levels from the camera in the brightest pixels. The second put three neutral density filters (totaling 1/512 of the Hg bulb's illumination reaching the sample). With the neutral density filters in place, his cells which bleached within seconds at full 100W Hg intensity survived 24 hours of illumination in good health. We did have to extend his exposures to 1 minute to get reasonable signal levels and perform a dark frame subtraction. But with those two steps he got beautiful, detailed images. Although I cannot guarantee that my suggestion will work for you I think that it may be worth playing with different illumination intensities and exposure times. Chris Tully Vashaw Scientific Inc. [hidden email] On Dec 12, 2007 5:17 PM, Neville Sanjana <[hidden email]> wrote: That's a great idea but there are a few particulars of my |
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