Two unrelated questions
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Dear List,
I have two completely unrelated questions, one of which is confocal related, the other not particularly. Firstly, can anyone enlighten me as to why on a resonant scanner-based system you can only do an optical zoom retaining a fixed number of pixels in the x dimension (usually 512)? Secondly, does anyone have any experience making automated measurements of distances between FISH spots in 3 dimensions? What software is out there that can reliably perform such analysis? Does anyone have any experience of using high-throughput FISH systems which capture images and do the analysis as well (e.g. Metafer MetaCyte)? Any feedback positive or negative would be much appreciated. Thanks, Simon |
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I'm not sure what you mean by your first question. It's basic that a resonant scanner (being resonant) can only scan over a fixed amplitude, and at a fixed speed. A non-resonant scanner, on the other hand, can zoom in and out by varying its scan amplitude, and can also, by varying its scan rate, accommodate varying pixel numbers in a given scan field. Since the aim of a resonant scanner is to capture images at high speed, one assumes that the microscope will acquire pixels just as fast as the system can handle them, so there is no possibility of acquiring 1024 pixels in place of 512 - though one should be able to go down to 256 by binning. The sample area scanned by the mirror can be varied by an optical zoom system (though this will mean extra lenses and extra losses), but you are still stuck with the rate at which the computer can acquire pixels.
This all seems very simplistic, so maybe your question was deeper and I've missed the point. Just put that down to it being late at night here in Oz. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Walker Sent: Friday, 19 December 2008 10:04 PM To: [hidden email] Subject: Two unrelated questions Dear List, I have two completely unrelated questions, one of which is confocal related, the other not particularly. Firstly, can anyone enlighten me as to why on a resonant scanner-based system you can only do an optical zoom retaining a fixed number of pixels in the x dimension (usually 512)? Secondly, does anyone have any experience making automated measurements of distances between FISH spots in 3 dimensions? What software is out there that can reliably perform such analysis? Does anyone have any experience of using high-throughput FISH systems which capture images and do the analysis as well (e.g. Metafer MetaCyte)? Any feedback positive or negative would be much appreciated. Thanks, Simon No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM |
 
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simon walker (BI) |
Re: Two unrelated questions
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It's probably an ignorant question based on insufficient information, so
apologies. A recent demo of a Nikon A1R system implied that when using the resonant scanner the number of pixels in x was fixed (at 512), but the number in y was variable. Ok, based on the differences between the x and y scanners I understand that, but what I couldn't understand was why that still applied on optically zoomed regions (basically because I didn't understand how the zooming was working). From what you say, zooming using the resonant scanner is done using additional lenses and therefore the number of zoom factors will be fixed. Again this makes sense and could explain the slight loss in signal we saw when we zoomed using the resonant scanner. Cheers, Simon -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox Sent: 19 December 2008 11:46 To: [hidden email] Subject: Re: Two unrelated questions I'm not sure what you mean by your first question. It's basic that a resonant scanner (being resonant) can only scan over a fixed amplitude, and at a fixed speed. A non-resonant scanner, on the other hand, can zoom in and out by varying its scan amplitude, and can also, by varying its scan rate, accommodate varying pixel numbers in a given scan field. Since the aim of a resonant scanner is to capture images at high speed, one assumes that the microscope will acquire pixels just as fast as the system can handle them, so there is no possibility of acquiring 1024 pixels in place of 512 - though one should be able to go down to 256 by binning. The sample area scanned by the mirror can be varied by an optical zoom system (though this will mean extra lenses and extra losses), but you are still stuck with the rate at which the computer can acquire pixels. This all seems very simplistic, so maybe your question was deeper and I've missed the point. Just put that down to it being late at night here in Oz. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Walker Sent: Friday, 19 December 2008 10:04 PM To: [hidden email] Subject: Two unrelated questions Dear List, I have two completely unrelated questions, one of which is confocal related, the other not particularly. Firstly, can anyone enlighten me as to why on a resonant scanner-based system you can only do an optical zoom retaining a fixed number of pixels in the x dimension (usually 512)? Secondly, does anyone have any experience making automated measurements of distances between FISH spots in 3 dimensions? What software is out there that can reliably perform such analysis? Does anyone have any experience of using high-throughput FISH systems which capture images and do the analysis as well (e.g. Metafer MetaCyte)? Any feedback positive or negative would be much appreciated. Thanks, Simon No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM |
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Simon,
You're quite right. The zooming on a resonant scanner (if it has that facility) is, and can only be, created by optical elements. On an analogy with other optical systems, that is true zooming. On a non-resonant confocal 'zooming' is just reducing the scan amplitude, which is not really equivalent to zooming with a wide-field system (or a camera). But this terminology is a minefield given that many digital cameras offer 'electronic zooming' which is nothing more than blowing up pixels. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of simon walker (BI) Sent: Friday, 19 December 2008 11:17 PM To: [hidden email] Subject: Re: Two unrelated questions It's probably an ignorant question based on insufficient information, so apologies. A recent demo of a Nikon A1R system implied that when using the resonant scanner the number of pixels in x was fixed (at 512), but the number in y was variable. Ok, based on the differences between the x and y scanners I understand that, but what I couldn't understand was why that still applied on optically zoomed regions (basically because I didn't understand how the zooming was working). From what you say, zooming using the resonant scanner is done using additional lenses and therefore the number of zoom factors will be fixed. Again this makes sense and could explain the slight loss in signal we saw when we zoomed using the resonant scanner. Cheers, Simon -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox Sent: 19 December 2008 11:46 To: [hidden email] Subject: Re: Two unrelated questions I'm not sure what you mean by your first question. It's basic that a resonant scanner (being resonant) can only scan over a fixed amplitude, and at a fixed speed. A non-resonant scanner, on the other hand, can zoom in and out by varying its scan amplitude, and can also, by varying its scan rate, accommodate varying pixel numbers in a given scan field. Since the aim of a resonant scanner is to capture images at high speed, one assumes that the microscope will acquire pixels just as fast as the system can handle them, so there is no possibility of acquiring 1024 pixels in place of 512 - though one should be able to go down to 256 by binning. The sample area scanned by the mirror can be varied by an optical zoom system (though this will mean extra lenses and extra losses), but you are still stuck with the rate at which the computer can acquire pixels. This all seems very simplistic, so maybe your question was deeper and I've missed the point. Just put that down to it being late at night here in Oz. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Walker Sent: Friday, 19 December 2008 10:04 PM To: [hidden email] Subject: Two unrelated questions Dear List, I have two completely unrelated questions, one of which is confocal related, the other not particularly. Firstly, can anyone enlighten me as to why on a resonant scanner-based system you can only do an optical zoom retaining a fixed number of pixels in the x dimension (usually 512)? Secondly, does anyone have any experience making automated measurements of distances between FISH spots in 3 dimensions? What software is out there that can reliably perform such analysis? Does anyone have any experience of using high-throughput FISH systems which capture images and do the analysis as well (e.g. Metafer MetaCyte)? Any feedback positive or negative would be much appreciated. Thanks, Simon No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM |
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Dear Guy, Simon,
I'm not so sure about zooming needing extra optical elements in a resonant scanning system. i) The Y deflector (I was always told this is the one responsible for the top to bottom motion on the scanner) is generally (possibly always?) a galvanometer. Therefore it can be controlled in the same manner as for normal scanning ii) The resonating X deflecting mirror is more challenging though. Pixels output as different X locations are not really discrete elements as they are on a camera, but only derived from the time signatures, so some time t gives the beginning of one pixel, t+1 the beginning of the next etc. It is not necessary to actually zoom the resonating scanner in the same fashion as you would a galvanometrically driven one. You can just map the pixels into a different set of times, so the pixels are harvesting light from a shorter time frame - also in this scenario you would spend (relatively) more time with the signal blanked than would be the case in an unzoomed format. The weakness here is that you have to be able to be accurate with timing, but also either compensate in some way for the lower light yield per pixel (as t decreases so do the number of photons) or be straight about exactly what is happening. The strength is the lack of a requirement for additional optical elements, and more control, at a faster rate, for imaging modes. I haven't seen the A1R, so I'm not suggesting this is how Nikon approach the control of the zoom function, I'm merely suggesting it as a possibility, which could also explain the reduction in the light levels seen in the sample. all the best Nadolig Llawen Darran -------------------------------------------------- From: "Guy Cox" <[hidden email]> Sent: Friday, December 19, 2008 12:29 PM To: <[hidden email]> Subject: Re: Two unrelated questions > Simon, > > You're quite right. The zooming on a resonant scanner (if it has > that facility) is, and can only be, created by optical elements. On an > analogy with other optical systems, that is true zooming. On a > non-resonant > confocal 'zooming' is just reducing the scan amplitude, which is not > really > equivalent to zooming with a wide-field system (or a camera). But this > terminology is a minefield given that many digital cameras offer > 'electronic > zooming' which is nothing more than blowing up pixels. > > Guy > > > > Optical Imaging Techniques in Cell Biology > by Guy Cox CRC Press / Taylor & Francis > http://www.guycox.com/optical.htm > ______________________________________________ > Associate Professor Guy Cox, MA, DPhil(Oxon) > Electron Microscope Unit, Madsen Building F09, > University of Sydney, NSW 2006 > ______________________________________________ > Phone +61 2 9351 3176 Fax +61 2 9351 7682 > Mobile 0413 281 861 > ______________________________________________ > http://www.guycox.net > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of simon walker (BI) > Sent: Friday, 19 December 2008 11:17 PM > To: [hidden email] > Subject: Re: Two unrelated questions > > It's probably an ignorant question based on insufficient information, so > apologies. A recent demo of a Nikon A1R system implied that when using > the resonant scanner the number of pixels in x was fixed (at 512), but the > number in y was variable. Ok, based on the differences between the x and > y scanners I understand that, but what I couldn't understand was why that > still applied on optically zoomed regions (basically because I didn't > understand how the zooming was working). From what you say, zooming using > the resonant scanner is done using additional lenses and therefore the > number of zoom factors will be fixed. Again this makes sense and could > explain the slight loss in signal we saw when we zoomed using the resonant > scanner. > Cheers, > Simon > > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Guy Cox > Sent: 19 December 2008 11:46 > To: [hidden email] > Subject: Re: Two unrelated questions > > I'm not sure what you mean by your first question. It's basic that a > resonant scanner (being resonant) can only scan over a fixed amplitude, > and at a fixed speed. A non-resonant scanner, on the other hand, can zoom > in and out by varying its scan amplitude, and can also, by varying its > scan rate, accommodate varying pixel numbers in a given scan field. > Since the aim of a resonant scanner is to capture images at high speed, > one assumes that the microscope will acquire pixels just as fast as the > system can handle them, so there is no possibility of acquiring 1024 > pixels in place of 512 - though one should be able to go down to 256 by > binning. The sample area scanned by the mirror can be varied by an > optical zoom system (though this will mean extra lenses and extra losses), > but you are still stuck with the rate at which the computer can acquire > pixels. > > This all seems very simplistic, so maybe your question was deeper and I've > missed the point. Just put that down to it being late at night here in > Oz. > > Guy > > > > Optical Imaging Techniques in Cell Biology > by Guy Cox CRC Press / Taylor & Francis > http://www.guycox.com/optical.htm > ______________________________________________ > Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, > Madsen Building F09, University of Sydney, NSW 2006 > ______________________________________________ > Phone +61 2 9351 3176 Fax +61 2 9351 7682 > Mobile 0413 281 861 > ______________________________________________ > http://www.guycox.net > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Simon Walker > Sent: Friday, 19 December 2008 10:04 PM > To: [hidden email] > Subject: Two unrelated questions > > Dear List, > > I have two completely unrelated questions, one of which is confocal > related, the other not particularly. > > Firstly, can anyone enlighten me as to why on a resonant scanner-based > system you can only do an optical zoom retaining a fixed number of pixels > in the x dimension (usually 512)? > > Secondly, does anyone have any experience making automated measurements of > distances between FISH spots in 3 dimensions? What software is out there > that can reliably perform such analysis? Does anyone have any experience > of using high-throughput FISH systems which capture images and do the > analysis as well (e.g. Metafer MetaCyte)? Any feedback positive or > negative would be much appreciated. > Thanks, > Simon > > No virus found in this incoming message. > Checked by AVG. > Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: > 16/12/2008 6:11 PM > > > No virus found in this outgoing message. > Checked by AVG. > Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: > 16/12/2008 6:11 PM > > > No virus found in this incoming message. > Checked by AVG. > Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: > 16/12/2008 6:11 PM > > > No virus found in this outgoing message. > Checked by AVG. > Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: > 16/12/2008 6:11 PM > > > |
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In reply to this post by simon walker (BI)
Simon Walker asked:
Can anyone enlighten me as to why on a resonant scanner-based system you can only do an optical zoom retaining a fixed number of pixels in the x dimension (usually 512)? How bout this answer: If you change the zoom of a resonant scanner you are basically changing the amplitude of a tuning fork. The time it takes to scan a line remains constant no matter if it is scanning 100 microns of a sample or 200 microns. If you divide that line scan into a constant number of pixels (512 in your example) the dwell time for each pixel remains the same (ignore for now that the tuning fork scan mirror speeds up near the center and slows down at the edges of its travel). The electronics of the PMT detector have a time constant that sort of makes a running average of light intensity signal which fits the pixel dwell time. I mean that it is sort of, kind of, integrating the signal that hits the PMT during the pixel dwell then erasing it for the next pixel dwell. That time constant is in the electronics and we hope the manufacture has designed it to agree with the resonant scanner speed. If you changed the number of pixels you would have to change the electronics. Too dificult. Otherwise you would be just be throwing away free information (in the case of fewer pixels) or smearing the same information across more pixels. Remember that part about the tuning fork scan mirror speeding up and slowing down? To keep the pixel width constant the microscope does change the pixel dwell time to shorter in the middle than the edges. And fudges the electronics. Paul Herzmark Specialist [hidden email] Department of Molecular and Cell Biology 479 Life Science Addition University of California, Berkeley Berkeley, CA 94720-3200 (510) 643-9603 (510) 643-9500 fax On Fri, Dec 19, 2008 at 3:03 AM, Simon Walker <[hidden email]> wrote: Dear List, |
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Jerry Sedgewick-2 |
Re: Two unrelated questions
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Yes, the resonant scanner uses a "tuning fork" scanner for the x or y
along with a galvo. And it's true that the amplitude of the resonant scanner can be changed to zoom in and out (with a change, as well, in the rate of the galvo mirror). And the scan speed can be clocked to the video board to add more pixels (so that the video rate slows down from 30fps to 15fps to 8fps to increase sampling and the number of pixels collected as the line is scanned). But, additionally, the distance between the eyepiece and the resonant mirror can be increased or decreased without any change in the scan frequencies, and this can also be used to zoom in and out. However, the size of the projected spot on the back aperture of the objective will enlarge or contract, affecting the z-resolution and penetration, so I wouldn't think that this method would be used on a commercially made confocal. I don't know if I'm very good at explaining "why," but having built a resonant mirror confocal through blood, sweat and tears I've tested every component of the system, mostly by mistake, and have seen the results on the screen. Don't really know, however, how Nikon solved the zoom issue. Jerry Paul Herzmark wrote: > Simon Walker asked: > Can anyone enlighten me as to why on a resonant scanner-based > system you can only do an optical zoom retaining a fixed number of > pixels in > the x dimension (usually 512)? > > How bout this answer: > If you change the zoom of a resonant scanner you are basically > changing the amplitude of a tuning fork. The time it takes to scan a > line remains constant no matter if it is scanning 100 microns of a > sample or 200 microns. If you divide that line scan into a constant > number of pixels (512 in your example) the dwell time for each pixel > remains the same (ignore for now that the tuning fork scan mirror > speeds up near the center and slows down at the edges of its travel). > The electronics of the PMT detector have a time constant that sort of > makes a running average of light intensity signal which fits the pixel > dwell time. I mean that it is sort of, kind of, integrating the signal > that hits the PMT during the pixel dwell then erasing it for the next > pixel dwell. > > That time constant is in the electronics and we hope the manufacture > has designed it to agree with the resonant scanner speed. If you > changed the number of pixels you would have to change the electronics. > Too dificult. Otherwise you would be just be throwing away free > information (in the case of fewer pixels) or smearing the same > information across more pixels. > > Remember that part about the tuning fork scan mirror speeding up and > slowing down? To keep the pixel width constant the microscope does > change the pixel dwell time to shorter in the middle than the edges. > And fudges the electronics. > > > > > Paul Herzmark > Specialist > [hidden email] <mailto:[hidden email]> > > Department of Molecular and Cell Biology > 479 Life Science Addition > University of California, Berkeley > Berkeley, CA 94720-3200 > (510) 643-9603 > (510) 643-9500 fax > > > On Fri, Dec 19, 2008 at 3:03 AM, Simon Walker > <[hidden email] <mailto:[hidden email]>> wrote: > > Dear List, > > I have two completely unrelated questions, one of which is > confocal related, > the other not particularly. > > Firstly, can anyone enlighten me as to why on a resonant scanner-based > system you can only do an optical zoom retaining a fixed number of > pixels in > the x dimension (usually 512)? > > Secondly, does anyone have any experience making automated > measurements > of distances between FISH spots in 3 dimensions? What software is > out there > that can reliably perform such analysis? Does anyone have any > experience of > using high-throughput FISH systems which capture images and do the > analysis > as well (e.g. Metafer MetaCyte)? Any feedback positive or > negative would be > much appreciated. > Thanks, > Simon > > -- Jerry (Gerald) Sedgewick Program Director, Biomedical Image Processing Lab (BIPL) Department of Neuroscience, University of Minnesota 312 Church St. SE, 1-205 Hasselmo Hall Minneapolis, MN 55455 (612) 624-6607 [hidden email] http://www.bipl.umn.edu Author: "Scientific Imaging with Photoshop: Methods, Measurement and Output." Rawlight.com (dba Sedgewick Initiatives) 965 Cromwell Avenue Saint Paul, MN 55114 [hidden email] (651) 308-1466 http://www.quickphotoshop.com http://www.heartFROMstone.com http://www.rawlight.com --- Get FREE High Speed Internet from USFamily.Net! -- http://www.usfamily.net/mkt-freepromo.html --- |
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In reply to this post by Guy Cox
>Simon,
> > You're quite right. The zooming on a resonant scanner (if it has >that facility) is, and can only be, created by optical elements. On an >analogy with other optical systems, that is true zooming. On a non-resonant >confocal 'zooming' is just reducing the scan amplitude, which is not really >equivalent to zooming with a wide-field system (or a camera). But this >terminology is a minefield given that many digital cameras offer 'electronic >zooming' which is nothing more than blowing up pixels. > > Guy > Hi all, I don't think this is right. Resonant means that it only "rings" at one frequency. Not that it only rings at a specific loudness (amplitude). If you put more power at the resonant frequency into a resonant mechanical system, it will swing over a larger range of positions and when the system in question is a scan mirror, the spot will move over a larger field on the specimen (lower zoom magnification). Now there are limits on this: if you put in too much power, the galvo may melt or resonant spring may fracture. Absent that, there is no reason inherent in the operation of resonant galvos that prevents them from providing scan-based zoom. However, the fact that the frequency is fixed (and that one usually only uses the "fairly linear 30% of the scan amplitude) means that, no matter what range it scans over, you have a fixed amount of time in which to sample and store useful signal. For instance, an 8khz scanner will collect data in bursts about 20 µs long. Even collecting 512 samples in this time requires a pretty fast digitizer (about 40 ns/sample), but more to the point, you don't capture much signal in 40 ns. (4µs is a more usual for a 512x512, 1 second frame time) If you want 1,024 or 2,048 pixels/line, scan slower. You can't get big pictures fast when you only scan one beam because the data rate is limited by singlet-state saturation. Hope that this helps, Jim Pawley -- ********************************************** 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 13-25, 2009, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2009 "If it ain't diffraction, it must be statistics." Anon. |
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Actually you are not limited to the 'fairly linear' part of
the scan - Nikon and I guess other manufacturers linearise their output so that they can use more of the scan (and also because even the 'fairly linear' part of the scan isn't really sufficiently linear.) Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley Sent: Saturday, 20 December 2008 10:36 AM To: [hidden email] Subject: Re: Two unrelated questions >Simon, > > You're quite right. The zooming on a resonant scanner (if it >has that facility) is, and can only be, created by optical elements. >On an analogy with other optical systems, that is true zooming. On a >non-resonant confocal 'zooming' is just reducing the scan amplitude, >which is not really equivalent to zooming with a wide-field system (or >a camera). But this terminology is a minefield given that many digital >cameras offer 'electronic zooming' which is nothing more than blowing up pixels. > > Guy > Hi all, I don't think this is right. Resonant means that it only "rings" at one frequency. Not that it only rings at a specific loudness (amplitude). If you put more power at the resonant frequency into a resonant mechanical system, it will swing over a larger range of positions and when the system in question is a scan mirror, the spot will move over a larger field on the specimen (lower zoom magnification). Now there are limits on this: if you put in too much power, the galvo may melt or resonant spring may fracture. Absent that, there is no reason inherent in the operation of resonant galvos that prevents them from providing scan-based zoom. However, the fact that the frequency is fixed (and that one usually only uses the "fairly linear 30% of the scan amplitude) means that, no matter what range it scans over, you have a fixed amount of time in which to sample and store useful signal. For instance, an 8khz scanner will collect data in bursts about 20 µs long. Even collecting 512 samples in this time requires a pretty fast digitizer (about 40 ns/sample), but more to the point, you don't capture much signal in 40 ns. (4µs is a more usual for a 512x512, 1 second frame time) If you want 1,024 or 2,048 pixels/line, scan slower. You can't get big pictures fast when you only scan one beam because the data rate is limited by singlet-state saturation. Hope that this helps, Jim Pawley -- ********************************************** 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 13-25, 2009, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2009 "If it ain't diffraction, it must be statistics." Anon. No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.19/1857 - Release Date: 19/12/2008 10:09 AM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.19/1857 - Release Date: 19/12/2008 10:09 AM |
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>Actually you are not limited to the 'fairly linear' part of
>the scan - Nikon and I guess other manufacturers linearise >their output so that they can use more of the scan (and also >because even the 'fairly linear' part of the scan isn't >really sufficiently linear.) > > Guy > Hi again, I agree. No part of a sine wave is really linear. Print one out and then try to run a ruler along it... You may decide that accepting 30% of the horizontal space (time) as being relatively linear is really quite generous. One can linearize more of it to some extent, but again there are limits. One usually linearizes to preserve the linear geometry of the image by varying the pixel clock so that the pixels near the center of the scan (where the mirror is moving fastest but accelerating least) are shorter in time compared to those at the edges of the scan, with the idea that each pixel can then represent the same area of the specimen. However, as both the dose to the specimen (bleaching) and the number of photons emitted (and collected) during a pixel depend on the pixel time, one begins to have rather non-linear (doughnut-shaped, in 2D) bleaching if you use too much of the sinusoidal scan pattern. In addition, the S/N of the image on axis becomes noticeably worse than that around the periphery (where more photons are collected, making the Poisson noise less). In any case, you must also adjust the gain of the signal collection system (or the range of the digitizer) so that the longer pixels don't also look brighter. This can be done but quantification becomes tricky as so many other factors cause the signal to vary between on- and off-axis (apodization, curvature of field, chromatic magnification error etc.). Cheers, Jim P. -- ********************************************** 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 13-25, 2009, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2009 "If it ain't diffraction, it must be statistics." Anon. |
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In reply to this post by Guy Cox
Guy,
I'm Okugawa from Nikon. Let me have a comment about resonant scanning system, please. I'm afraid people misunderstand the resonant scanning system. If you mean the optical system is some optical magnifying elements, A1R doesn't use it. Zooming is done by changing the amplitude like non-resonant scanner. The difference between resonant and non-resonant of A1R is the way to create the pixel clock. A1R uses unique optical clock generation method which can create even clocks spatially. It adopts plural gratings which create 512 clocks in a line to get stable images. Hisashi Okugawa ----- Original Message ----- From: "Guy Cox" <[hidden email]> To: <[hidden email]> Sent: Friday, December 19, 2008 9:29 PM Subject: Re: Two unrelated questions Simon, You're quite right. The zooming on a resonant scanner (if it has that facility) is, and can only be, created by optical elements. On an analogy with other optical systems, that is true zooming. On a non-resonant confocal 'zooming' is just reducing the scan amplitude, which is not really equivalent to zooming with a wide-field system (or a camera). But this terminology is a minefield given that many digital cameras offer 'electronic zooming' which is nothing more than blowing up pixels. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of simon walker (BI) Sent: Friday, 19 December 2008 11:17 PM To: [hidden email] Subject: Re: Two unrelated questions It's probably an ignorant question based on insufficient information, so apologies. A recent demo of a Nikon A1R system implied that when using the resonant scanner the number of pixels in x was fixed (at 512), but the number in y was variable. Ok, based on the differences between the x and y scanners I understand that, but what I couldn't understand was why that still applied on optically zoomed regions (basically because I didn't understand how the zooming was working). From what you say, zooming using the resonant scanner is done using additional lenses and therefore the number of zoom factors will be fixed. Again this makes sense and could explain the slight loss in signal we saw when we zoomed using the resonant scanner. Cheers, Simon -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox Sent: 19 December 2008 11:46 To: [hidden email] Subject: Re: Two unrelated questions I'm not sure what you mean by your first question. It's basic that a resonant scanner (being resonant) can only scan over a fixed amplitude, and at a fixed speed. A non-resonant scanner, on the other hand, can zoom in and out by varying its scan amplitude, and can also, by varying its scan rate, accommodate varying pixel numbers in a given scan field. Since the aim of a resonant scanner is to capture images at high speed, one assumes that the microscope will acquire pixels just as fast as the system can handle them, so there is no possibility of acquiring 1024 pixels in place of 512 - though one should be able to go down to 256 by binning. The sample area scanned by the mirror can be varied by an optical zoom system (though this will mean extra lenses and extra losses), but you are still stuck with the rate at which the computer can acquire pixels. This all seems very simplistic, so maybe your question was deeper and I've missed the point. Just put that down to it being late at night here in Oz. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Walker Sent: Friday, 19 December 2008 10:04 PM To: [hidden email] Subject: Two unrelated questions Dear List, I have two completely unrelated questions, one of which is confocal related, the other not particularly. Firstly, can anyone enlighten me as to why on a resonant scanner-based system you can only do an optical zoom retaining a fixed number of pixels in the x dimension (usually 512)? Secondly, does anyone have any experience making automated measurements of distances between FISH spots in 3 dimensions? What software is out there that can reliably perform such analysis? Does anyone have any experience of using high-throughput FISH systems which capture images and do the analysis as well (e.g. Metafer MetaCyte)? Any feedback positive or negative would be much appreciated. Thanks, Simon No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM |
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simon walker (BI) |
Re: Two unrelated questions
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Well, maybe it wasn't such a simple question..
Thanks as ever to the list for the informed responses. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Hisashi Okugawa Sent: 20 December 2008 01:37 To: [hidden email] Subject: Re: Two unrelated questions Guy, I'm Okugawa from Nikon. Let me have a comment about resonant scanning system, please. I'm afraid people misunderstand the resonant scanning system. If you mean the optical system is some optical magnifying elements, A1R doesn't use it. Zooming is done by changing the amplitude like non-resonant scanner. The difference between resonant and non-resonant of A1R is the way to create the pixel clock. A1R uses unique optical clock generation method which can create even clocks spatially. It adopts plural gratings which create 512 clocks in a line to get stable images. Hisashi Okugawa ----- Original Message ----- From: "Guy Cox" <[hidden email]> To: <[hidden email]> Sent: Friday, December 19, 2008 9:29 PM Subject: Re: Two unrelated questions Simon, You're quite right. The zooming on a resonant scanner (if it has that facility) is, and can only be, created by optical elements. On an analogy with other optical systems, that is true zooming. On a non-resonant confocal 'zooming' is just reducing the scan amplitude, which is not really equivalent to zooming with a wide-field system (or a camera). But this terminology is a minefield given that many digital cameras offer 'electronic zooming' which is nothing more than blowing up pixels. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of simon walker (BI) Sent: Friday, 19 December 2008 11:17 PM To: [hidden email] Subject: Re: Two unrelated questions It's probably an ignorant question based on insufficient information, so apologies. A recent demo of a Nikon A1R system implied that when using the resonant scanner the number of pixels in x was fixed (at 512), but the number in y was variable. Ok, based on the differences between the x and y scanners I understand that, but what I couldn't understand was why that still applied on optically zoomed regions (basically because I didn't understand how the zooming was working). From what you say, zooming using the resonant scanner is done using additional lenses and therefore the number of zoom factors will be fixed. Again this makes sense and could explain the slight loss in signal we saw when we zoomed using the resonant scanner. Cheers, Simon -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox Sent: 19 December 2008 11:46 To: [hidden email] Subject: Re: Two unrelated questions I'm not sure what you mean by your first question. It's basic that a resonant scanner (being resonant) can only scan over a fixed amplitude, and at a fixed speed. A non-resonant scanner, on the other hand, can zoom in and out by varying its scan amplitude, and can also, by varying its scan rate, accommodate varying pixel numbers in a given scan field. Since the aim of a resonant scanner is to capture images at high speed, one assumes that the microscope will acquire pixels just as fast as the system can handle them, so there is no possibility of acquiring 1024 pixels in place of 512 - though one should be able to go down to 256 by binning. The sample area scanned by the mirror can be varied by an optical zoom system (though this will mean extra lenses and extra losses), but you are still stuck with the rate at which the computer can acquire pixels. This all seems very simplistic, so maybe your question was deeper and I've missed the point. Just put that down to it being late at night here in Oz. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Walker Sent: Friday, 19 December 2008 10:04 PM To: [hidden email] Subject: Two unrelated questions Dear List, I have two completely unrelated questions, one of which is confocal related, the other not particularly. Firstly, can anyone enlighten me as to why on a resonant scanner-based system you can only do an optical zoom retaining a fixed number of pixels in the x dimension (usually 512)? Secondly, does anyone have any experience making automated measurements of distances between FISH spots in 3 dimensions? What software is out there that can reliably perform such analysis? Does anyone have any experience of using high-throughput FISH systems which capture images and do the analysis as well (e.g. Metafer MetaCyte)? Any feedback positive or negative would be much appreciated. Thanks, Simon No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.9.18/1852 - Release Date: 16/12/2008 6:11 PM |
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