Topographical pits volume
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear All, We have one user working on dentine slice with osteoclasts and interested in the resorption pits volume on the surface. We used reflection mode to generate 3D images for the dentine slice on our LSM 510 Meta confocal system and showed the topographical profiles and depth coding map in Fiji by using 3D shape and SurfCharJ plugin. Could you kindly suggest Fiji plugin to calculate the pits volume? Or any other software is able to do this job? Thank you for your advice and help in advance. Kind regards, TONG Yan (Ms) Senior Scientific Officer:: NUS Centre for BioImaging Sciences :: Department of Biological Sciences :: National University of Singapore :: 14 Science Drive 4, Singapore 117543 :: (65) 6516 7202 (DID) :: (65) 6779 2486 (Fax) ::) :: [hidden email]<mailto:[hidden email]> (E) :: www.nus.edu.sg<http://www.nus.edu.sg/> (W)Company Registration No: 200604346E |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all, I would be interested in this reply too. I have a client working with cuticle elevation and volume, as well as trichome volume and length from 3D stacks. THank you all! Shanna Banman |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** The volume of holes can be obtained from a stack thresholded to show the voids. then the Z projected sum (ImageJ) gives the thickness in Z. The sum of the thickness for each void is its volume, which you can get from analyse particles in ImageJ You also need to know where the voids starts in Z - perhaps the trickiest part, and remove areas outside the void from the analysis. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Shanna Banman Sent: den 7 mars 2014 18:33 To: [hidden email] Subject: Re: Topographical pits volume ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all, I would be interested in this reply too. I have a client working with cuticle elevation and volume, as well as trichome volume and length from 3D stacks. THank you all! Shanna Banman |
 
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Stanislav Vitha-2 |
Re: Topographical pits volume
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In reply to this post by dbstongy
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I created a PDF that shows the steps with Fiji/ImageJ: http://microscopy.tamu.edu/lab-protocols/Measuringpitvolume.pdf In summary (without pictures): 1. generate TOPO projection with TopoJ plugin. Use the Quadratic Refinement option for more precise result. The result is a 32-bit file where pixel value corresponds to surface height (in the calibrated units, e.g, micrometers) 2. Crop the image as needed to remove processing artifacts on the edges of the image 3. display the interactive 3D surface plot to check if there are any outliers (hot or black pixels). If necessary, remove the outliers (process-Noise- Remove Outliers) 4. Level the TOPO image (Plugins-TopoJ-Level) 5. Threshold the image to highlight the flat surface. Measure the mean pixel value of the surface (make sure the Analyze- Set Measurements-Limit to Threshold is set and that you have sufficient number of decimal places for the result) , subtract this value from the image (Process-Math- Subtract). The flat surface now have pixel values close to zero. 6. Threshold the resulting image to highlight the pits. 7. Analyze-Set Measurements – make sure to check the Integrated Density. Then Process-Analyze Objects (EXCLUDE ON EDGES, INCLUDE HOLES, SHOW OUTLINE). The IntDen corresponds to the well volume. In this case, measuring an AFM test target with ~1.4 micron square wells ~1 micron deep, the volume is about two cubic micrometers each well. Results:(in micrometers) # label Area mean IntDen RawIntDen ----------------------------------------------------------------------------- 1 QR_AFT- 2.0430 -0.9844 -2.0110 -461.6724 2 QR_AFT- 2.0691 -1.0024 -2.0740 -476.1324 3 QR_AFT- 2.0604 -0.9930 -2.0460 -469.7013 4 QR_AFT- 2.0647 -0.9901 -2.0443 -469.3108 5 QR_AFT- 2.0430 -0.9811 -2.0043 -460.1183 6 QR_AFT- 2.0647 -0.9914 -2.0469 -469.9020 7 QR_AFT- 2.0560 -0.9771 -2.0090 -461.1930 8 QR_AFT- 2.0430 -0.9807 -2.0036 -459.9545 9 QR_AFT- 2.0299 -0.9745 -1.9781 -454.1175 I hope this helps. Stan Vitha Microscopy and Imaging Center Texas A&M University On Wed, 5 Mar 2014 11:28:11 +0800, Tong Yan <[hidden email]> wrote: >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >Dear All, > >We have one user working on dentine slice with osteoclasts and interested in the resorption pits volume on the surface. > >We used reflection mode to generate 3D images for the dentine slice on our LSM 510 Meta confocal system and showed the topographical profiles and depth coding map in Fiji by using 3D shape and SurfCharJ plugin. > >Could you kindly suggest Fiji plugin to calculate the pits volume? Or any other software is able to do this job? > >Thank you for your advice and help in advance. >Kind regards, > >TONG Yan (Ms) >Senior Scientific Officer:: NUS Centre for BioImaging Sciences :: Department of Biological Sciences :: National University of Singapore :: 14 Science Drive 4, Singapore 117543 :: (65) 6516 7202 (DID) :: (65) 6779 2486 (Fax) ::) :: [hidden email]<mailto:[hidden email]> (E) :: www.nus.edu.sg<http://www.nus.edu.sg/> (W)Company Registration No: 200604346E |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear All, Thanks all for your response and suggestions, James, Michael and Jeremy. Especially the protocol from Stan, following which we get the successful calculation of the pits volume for our preliminary samples. Best regards, Iris -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stanislav Vitha Sent: Tuesday, March 11, 2014 11:42 PM To: [hidden email] Subject: Re: Topographical pits volume ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I created a PDF that shows the steps with Fiji/ImageJ: http://microscopy.tamu.edu/lab-protocols/Measuringpitvolume.pdf In summary (without pictures): 1. generate TOPO projection with TopoJ plugin. Use the Quadratic Refinement option for more precise result. The result is a 32-bit file where pixel value corresponds to surface height (in the calibrated units, e.g, micrometers) 2. Crop the image as needed to remove processing artifacts on the edges of the image 3. display the interactive 3D surface plot to check if there are any outliers (hot or black pixels). If necessary, remove the outliers (process-Noise- Remove Outliers) 4. Level the TOPO image (Plugins-TopoJ-Level) 5. Threshold the image to highlight the flat surface. Measure the mean pixel value of the surface (make sure the Analyze- Set Measurements-Limit to Threshold is set and that you have sufficient number of decimal places for the result) , subtract this value from the image (Process-Math- Subtract). The flat surface now have pixel values close to zero. 6. Threshold the resulting image to highlight the pits. 7. Analyze-Set Measurements - make sure to check the Integrated Density. Then Process-Analyze Objects (EXCLUDE ON EDGES, INCLUDE HOLES, SHOW OUTLINE). The IntDen corresponds to the well volume. In this case, measuring an AFM test target with ~1.4 micron square wells ~1 micron deep, the volume is about two cubic micrometers each well. Results:(in micrometers) # label Area mean IntDen RawIntDen ----------------------------------------------------------------------------- 1 QR_AFT- 2.0430 -0.9844 -2.0110 -461.6724 2 QR_AFT- 2.0691 -1.0024 -2.0740 -476.1324 3 QR_AFT- 2.0604 -0.9930 -2.0460 -469.7013 4 QR_AFT- 2.0647 -0.9901 -2.0443 -469.3108 5 QR_AFT- 2.0430 -0.9811 -2.0043 -460.1183 6 QR_AFT- 2.0647 -0.9914 -2.0469 -469.9020 7 QR_AFT- 2.0560 -0.9771 -2.0090 -461.1930 8 QR_AFT- 2.0430 -0.9807 -2.0036 -459.9545 9 QR_AFT- 2.0299 -0.9745 -1.9781 -454.1175 I hope this helps. Stan Vitha Microscopy and Imaging Center Texas A&M University On Wed, 5 Mar 2014 11:28:11 +0800, Tong Yan <[hidden email]> wrote: >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >Dear All, > >We have one user working on dentine slice with osteoclasts and interested in the resorption pits volume on the surface. > >We used reflection mode to generate 3D images for the dentine slice on our LSM 510 Meta confocal system and showed the topographical profiles and depth coding map in Fiji by using 3D shape and SurfCharJ plugin. > >Could you kindly suggest Fiji plugin to calculate the pits volume? Or >any other software is able to do this job? > >Thank you for your advice and help in advance. >Kind regards, > >TONG Yan (Ms) >Senior Scientific Officer:: NUS Centre for BioImaging Sciences :: Department of Biological Sciences :: National University of Singapore :: 14 Science Drive 4, Singapore 117543 :: (65) 6516 7202 (DID) :: (65) 6779 2486 (Fax) ::) :: [hidden email]<mailto:[hidden email]> (E) :: www.nus.edu.sg<http://www.nus.edu.sg/> (W)Company Registration No: 200604346E |
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