TY - JOUR
T1 - Large-scale, three-dimensional tissue cytometry of the human kidney
T2 - a complete and accessible pipeline
AU - for the Kidney Precision Medicine Project
AU - Ferkowicz, Michael J.
AU - Winfree, Seth
AU - Sabo, Angela R.
AU - Kamocka, Malgorzata M.
AU - Khochare, Suraj
AU - Barwinska, Daria
AU - Eadon, Michael T.
AU - Cheng, Ying Hua
AU - Phillips, Carrie L.
AU - Sutton, Timothy A.
AU - Kelly, Katherine J.
AU - Dagher, Pierre C.
AU - El-Achkar, Tarek M.
AU - Dunn, Kenneth W.
AU - Knight, Richard
AU - Lecker, Stewart
AU - Stillman, Isaac
AU - Mcmahon, Gearoid
AU - Waikar, Sus
AU - Weins, Astrid
AU - Hacohen, Nir
AU - Hoover, Paul
AU - Aulisio, Mark
AU - Cooperman, Leslie
AU - Herlitz, Leal
AU - O’toole, John
AU - Poggio, Emilio
AU - Sedor, John
AU - Appelbaum, Paul
AU - Barasch, Jonathan
AU - Bomback, Andrew
AU - D’agati, Vivette
AU - Kiryluk, Krzysztof
AU - Mehl, Karla
AU - Shang, Ning
AU - Weng, Chenhua
AU - Barisoni, Laura
AU - Alexandrov, Theodore
AU - Ashkar, Tarek
AU - Barwinska, Daria
AU - Dagher, Pierre
AU - Dunn, Kenneth
AU - Eadon, Michael
AU - Ferkowicz, Michael
AU - Kelly, Katherine
AU - Sutton, Timothy
AU - Winfree, Seth
AU - Gaut, Joe
AU - Jain, Sanjay
AU - Vijayan, Anitha
N1 - Publisher Copyright:
© 2021, The Author(s), under exclusive licence to United States and Canadian Academy of Pathology.
PY - 2021/5
Y1 - 2021/5
N2 - The advent of personalized medicine has driven the development of novel approaches for obtaining detailed cellular and molecular information from clinical tissue samples. Tissue cytometry is a promising new technique that can be used to enumerate and characterize each cell in a tissue and, unlike flow cytometry and other single-cell techniques, does so in the context of the intact tissue, preserving spatial information that is frequently crucial to understanding a cell’s physiology, function, and behavior. However, the wide-scale adoption of tissue cytometry as a research tool has been limited by the fact that published examples utilize specialized techniques that are beyond the capabilities of most laboratories. Here we describe a complete and accessible pipeline, including methods of sample preparation, microscopy, image analysis, and data analysis for large-scale three-dimensional tissue cytometry of human kidney tissues. In this workflow, multiphoton microscopy of unlabeled tissue is first conducted to collect autofluorescence and second-harmonic images. The tissue is then labeled with eight fluorescent probes, and imaged using spectral confocal microscopy. The raw 16-channel images are spectrally deconvolved into 8-channel images, and analyzed using the Volumetric Tissue Exploration and Analysis (VTEA) software developed by our group. We applied this workflow to analyze millimeter-scale tissue samples obtained from human nephrectomies and from renal biopsies from individuals diagnosed with diabetic nephropathy, generating a quantitative census of tens of thousands of cells in each. Such analyses can provide useful insights that can be linked to the biology or pathology of kidney disease. The approach utilizes common laboratory techniques, is compatible with most commercially-available confocal microscope systems and all image and data analysis is conducted using the VTEA image analysis software, which is available as a plug-in for ImageJ.
AB - The advent of personalized medicine has driven the development of novel approaches for obtaining detailed cellular and molecular information from clinical tissue samples. Tissue cytometry is a promising new technique that can be used to enumerate and characterize each cell in a tissue and, unlike flow cytometry and other single-cell techniques, does so in the context of the intact tissue, preserving spatial information that is frequently crucial to understanding a cell’s physiology, function, and behavior. However, the wide-scale adoption of tissue cytometry as a research tool has been limited by the fact that published examples utilize specialized techniques that are beyond the capabilities of most laboratories. Here we describe a complete and accessible pipeline, including methods of sample preparation, microscopy, image analysis, and data analysis for large-scale three-dimensional tissue cytometry of human kidney tissues. In this workflow, multiphoton microscopy of unlabeled tissue is first conducted to collect autofluorescence and second-harmonic images. The tissue is then labeled with eight fluorescent probes, and imaged using spectral confocal microscopy. The raw 16-channel images are spectrally deconvolved into 8-channel images, and analyzed using the Volumetric Tissue Exploration and Analysis (VTEA) software developed by our group. We applied this workflow to analyze millimeter-scale tissue samples obtained from human nephrectomies and from renal biopsies from individuals diagnosed with diabetic nephropathy, generating a quantitative census of tens of thousands of cells in each. Such analyses can provide useful insights that can be linked to the biology or pathology of kidney disease. The approach utilizes common laboratory techniques, is compatible with most commercially-available confocal microscope systems and all image and data analysis is conducted using the VTEA image analysis software, which is available as a plug-in for ImageJ.
UR - http://www.scopus.com/inward/record.url?scp=85098755345&partnerID=8YFLogxK
U2 - 10.1038/s41374-020-00518-w
DO - 10.1038/s41374-020-00518-w
M3 - Article
C2 - 33408350
AN - SCOPUS:85098755345
SN - 0023-6837
VL - 101
SP - 661
EP - 676
JO - Laboratory Investigation
JF - Laboratory Investigation
IS - 5
ER -