Apotome tile scan
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear list Not exactly a confocal question but I am sure I can get some feedback to my problem here: We observed a strange behavior when using our Zeiss Apotome system for tile scanning. We acquired a 3x3 tile and can clearly see that some of the images are brighter than others (I used a chroma plastic slide here). A single image really pops out. If I draw a line profile over the three images in a row I can see a jump in intensity from one image to the next. If I acquire the same tile again with the exact settings the pattern changes. So this time another image pops out or if we are lucky they all have the same intensity. Do you have any idea why this happens? If we run a tile scan in wide field with the Apotome removed and the correct shading correction in place the line profile is perfectly flat. So I assume there is an issue with the Apotome but I am not sure if this is hardware or software related. I am also not exactly sure how to properly correct for shading. As far as I understood we should use a shading correction acquired in wide field mode and use this during Apotome acquisition. I would be very glad to hear from other people if they observed similar things with their Apotome and if there is a solution to this problem. Thanks a lot and best regards Pascal |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Pascal, This is a known limitation of all structured illumination methods, including Apotome. Structured illumination uses a grid illumination to encode the depth of the structures in the specimen relative to the focal plane: if a grid projection on a structure is sharp, the object is known to be in the focal plane. If it's blurred, it lies outside the focal plane and is removed from the final image. In case of a homogeneous "sea of fluorescence" (such as Chroma slides), the algorithm cannot distinguish well between the blurred out-of-focus signal and the "real" blur resulting from the homogeneous fluorophore distribution, and therefore generates images with arbitrary levels of background. This results in a tile-scans with varying level of signal in different tiles that you observed. As a guideline for Apotome processing, only samples should be used that demonstrate a visible Apotome illumination grid (either in Live-Mode on the screen, or through the Oculars). Shading-correction should be done in wide-field mode. If you want to make sure that the Apotome processing works fine with a tile-scan, you could test it e.g. on a sample with a layer of fluorescent beads (that have some space between them) instead of your homogeneous fluorescent slide. I hope this helps! With best regards, Uros Krzic from ZEISS -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pascal Lorentz Sent: 06 November 2017 11:27 To: [hidden email] Subject: Apotome tile scan ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear list Not exactly a confocal question but I am sure I can get some feedback to my problem here: We observed a strange behavior when using our Zeiss Apotome system for tile scanning. We acquired a 3x3 tile and can clearly see that some of the images are brighter than others (I used a chroma plastic slide here). A single image really pops out. If I draw a line profile over the three images in a row I can see a jump in intensity from one image to the next. If I acquire the same tile again with the exact settings the pattern changes. So this time another image pops out or if we are lucky they all have the same intensity. Do you have any idea why this happens? If we run a tile scan in wide field with the Apotome removed and the correct shading correction in place the line profile is perfectly flat. So I assume there is an issue with the Apotome but I am not sure if this is hardware or software related. I am also not exactly sure how to properly correct for shading. As far as I understood we should use a shading correction acquired in wide field mode and use this during Apotome acquisition. I would be very glad to hear from other people if they observed similar things with their Apotome and if there is a solution to this problem. Thanks a lot and best regards Pascal |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Uros Thanks a lot for your feedback. I was not aware that this technique is so much sample depended. The user is actually working with tissue sections that have quite a homogeneous staining. Could it be that I observe the same problem there as with a chroma slide? However I will have a closer look with a bead slide then. Best regards Pascal Am 06.11.2017 um 17:19 schrieb Krzic, Uros: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Dear Pascal, > > This is a known limitation of all structured illumination methods, including Apotome. Structured illumination uses a grid illumination to encode the depth of the structures in the specimen relative to the focal plane: if a grid projection on a structure is sharp, the object is known to be in the focal plane. If it's blurred, it lies outside the focal plane and is removed from the final image. In case of a homogeneous "sea of fluorescence" (such as Chroma slides), the algorithm cannot distinguish well between the blurred out-of-focus signal and the "real" blur resulting from the homogeneous fluorophore distribution, and therefore generates images with arbitrary levels of background. This results in a tile-scans with varying level of signal in different tiles that you observed. > > As a guideline for Apotome processing, only samples should be used that demonstrate a visible Apotome illumination grid (either in Live-Mode on the screen, or through the Oculars). Shading-correction should be done in wide-field mode. > > If you want to make sure that the Apotome processing works fine with a tile-scan, you could test it e.g. on a sample with a layer of fluorescent beads (that have some space between them) instead of your homogeneous fluorescent slide. > > I hope this helps! > > With best regards, > Uros Krzic from ZEISS > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pascal Lorentz > Sent: 06 November 2017 11:27 > To: [hidden email] > Subject: Apotome tile scan > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Dear list > > Not exactly a confocal question but I am sure I can get some feedback to my problem here: > > We observed a strange behavior when using our Zeiss Apotome system for tile scanning. We acquired a 3x3 tile and can clearly see that some of the images are brighter than others (I used a chroma plastic slide here). A single image really pops out. If I draw a line profile over the three images in a row I can see a jump in intensity from one image to the next. > If I acquire the same tile again with the exact settings the pattern changes. So this time another image pops out or if we are lucky they all have the same intensity. > Do you have any idea why this happens? If we run a tile scan in wide field with the Apotome removed and the correct shading correction in place the line profile is perfectly flat. > So I assume there is an issue with the Apotome but I am not sure if this is hardware or software related. I am also not exactly sure how to properly correct for shading. As far as I understood we should use a shading correction acquired in wide field mode and use this during Apotome acquisition. > I would be very glad to hear from other people if they observed similar things with their Apotome and if there is a solution to this problem. > > Thanks a lot and best regards > > Pascal |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Pascal, Apart from homogeneously distributed fluorescence (including a case of NO fluorescence), Apotome is rather robust against the choice of specimen. As long as you have a minimal amount of structure in your images that is not completely submerged in background, you should be fine. I can't say exactly how much structured signal is enough, but it's not much! :) Best regards, Uros -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pascal Lorentz Sent: 07 November 2017 11:22 To: [hidden email] Subject: Re: Apotome tile scan ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Uros Thanks a lot for your feedback. I was not aware that this technique is so much sample depended. The user is actually working with tissue sections that have quite a homogeneous staining. Could it be that I observe the same problem there as with a chroma slide? However I will have a closer look with a bead slide then. Best regards Pascal Am 06.11.2017 um 17:19 schrieb Krzic, Uros: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Dear Pascal, > > This is a known limitation of all structured illumination methods, including Apotome. Structured illumination uses a grid illumination to encode the depth of the structures in the specimen relative to the focal plane: if a grid projection on a structure is sharp, the object is known to be in the focal plane. If it's blurred, it lies outside the focal plane and is removed from the final image. In case of a homogeneous "sea of fluorescence" (such as Chroma slides), the algorithm cannot distinguish well between the blurred out-of-focus signal and the "real" blur resulting from the homogeneous fluorophore distribution, and therefore generates images with arbitrary levels of background. This results in a tile-scans with varying level of signal in different tiles that you observed. > > As a guideline for Apotome processing, only samples should be used that demonstrate a visible Apotome illumination grid (either in Live-Mode on the screen, or through the Oculars). Shading-correction should be done in wide-field mode. > > If you want to make sure that the Apotome processing works fine with a tile-scan, you could test it e.g. on a sample with a layer of fluorescent beads (that have some space between them) instead of your homogeneous fluorescent slide. > > I hope this helps! > > With best regards, > Uros Krzic from ZEISS > > > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]] On Behalf Of Pascal Lorentz > Sent: 06 November 2017 11:27 > To: [hidden email] > Subject: Apotome tile scan > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Dear list > > Not exactly a confocal question but I am sure I can get some feedback to my problem here: > > We observed a strange behavior when using our Zeiss Apotome system for tile scanning. We acquired a 3x3 tile and can clearly see that some of the images are brighter than others (I used a chroma plastic slide here). A single image really pops out. If I draw a line profile over the three images in a row I can see a jump in intensity from one image to the next. > If I acquire the same tile again with the exact settings the pattern changes. So this time another image pops out or if we are lucky they all have the same intensity. > Do you have any idea why this happens? If we run a tile scan in wide field with the Apotome removed and the correct shading correction in place the line profile is perfectly flat. > So I assume there is an issue with the Apotome but I am not sure if this is hardware or software related. I am also not exactly sure how to properly correct for shading. As far as I understood we should use a shading correction acquired in wide field mode and use this during Apotome acquisition. > I would be very glad to hear from other people if they observed similar things with their Apotome and if there is a solution to this problem. > > Thanks a lot and best regards > > Pascal |
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In reply to this post by Krzic, Uros
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Colleagues: *This is a known limitation of all structured illumination methods....* I am afraid I can not agree with this statement. Pascal says that in WF mode, all the images have the same intensity, indicating that the light source is stable and the camera is behaving normally. What this means is that there is something in the Apotome data processing that is somehow re-scaling the images, and it is doing so wrongly. Recall that there are many flavors of structured illumination microscopy, and many different ways to reconstruct the data. Other SIM reconstruction methods (for example scaled subtraction) would not be expected to exhibit the described problem. Cheers, Guy Hagen On Mon, Nov 6, 2017 at 9:19 AM, Krzic, Uros <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Dear Pascal, > > This is a known limitation of all structured illumination methods, > including Apotome. Structured illumination uses a grid illumination to > encode the depth of the structures in the specimen relative to the focal > plane: if a grid projection on a structure is sharp, the object is known to > be in the focal plane. If it's blurred, it lies outside the focal plane and > is removed from the final image. In case of a homogeneous "sea of > fluorescence" (such as Chroma slides), the algorithm cannot distinguish > well between the blurred out-of-focus signal and the "real" blur resulting > from the homogeneous fluorophore distribution, and therefore generates > images with arbitrary levels of background. This results in a tile-scans > with varying level of signal in different tiles that you observed. > > As a guideline for Apotome processing, only samples should be used that > demonstrate a visible Apotome illumination grid (either in Live-Mode on the > screen, or through the Oculars). Shading-correction should be done in > wide-field mode. > > If you want to make sure that the Apotome processing works fine with a > tile-scan, you could test it e.g. on a sample with a layer of fluorescent > beads (that have some space between them) instead of your homogeneous > fluorescent slide. > > I hope this helps! > > With best regards, > Uros Krzic from ZEISS > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Pascal Lorentz > Sent: 06 November 2017 11:27 > To: [hidden email] > Subject: Apotome tile scan > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Dear list > > Not exactly a confocal question but I am sure I can get some feedback to > my problem here: > > We observed a strange behavior when using our Zeiss Apotome system for > tile scanning. We acquired a 3x3 tile and can clearly see that some of the > images are brighter than others (I used a chroma plastic slide here). A > single image really pops out. If I draw a line profile over the three > images in a row I can see a jump in intensity from one image to the next. > If I acquire the same tile again with the exact settings the pattern > changes. So this time another image pops out or if we are lucky they all > have the same intensity. > Do you have any idea why this happens? If we run a tile scan in wide field > with the Apotome removed and the correct shading correction in place the > line profile is perfectly flat. > So I assume there is an issue with the Apotome but I am not sure if this > is hardware or software related. I am also not exactly sure how to properly > correct for shading. As far as I understood we should use a shading > correction acquired in wide field mode and use this during Apotome > acquisition. > I would be very glad to hear from other people if they observed similar > things with their Apotome and if there is a solution to this problem. > > Thanks a lot and best regards > > Pascal > |
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