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Cryostat Slice Irregularities May Introduce Bias in Tissue Thickness Estimation: Relevance for Cell Counting Methods

Published online by Cambridge University Press:  15 July 2015

Anna Puigdellívol-Sánchez ,
Albert Giralt ,
Anna Casanovas ,
Jordi Alberch  and
Alberto Prats-Galino
Anna Puigdellívol-Sánchez*
Affiliation:
Human Anatomy and Embryology Unit, Facultat de Medicina, Universitat de Barcelona, c/Casanova 143, 08036 Barcelona, Spain CAP Anton de Borja, Consorci Sanitari de Terrassa, c/Edison-cantonada Marconi, 08091 Rubí, Barcelona, Spain
Albert Giralt
Affiliation:
Departament de Biologia Cellular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, c/Casanova 143, 08036 Barcelona, Spain. Barcelona, Spain Institut d’ Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, c/Casanova 143, 08036 Barcelona, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, c/Casanova 143, 08036 Barcelona, Spain
Anna Casanovas
Affiliation:
Unit of Cellular Neurobioloy, Departament de Medicina Experimental, Facultat de Medicina, Universitat de Lleida, c/Montserrat Roig 2, 25008 Lleida, Spain
Jordi Alberch
Affiliation:
Departament de Biologia Cellular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, c/Casanova 143, 08036 Barcelona, Spain. Barcelona, Spain Institut d’ Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, c/Casanova 143, 08036 Barcelona, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, c/Casanova 143, 08036 Barcelona, Spain
Alberto Prats-Galino
Affiliation:
Human Anatomy and Embryology Unit, Facultat de Medicina, Universitat de Barcelona, c/Casanova 143, 08036 Barcelona, Spain
*
*Corresponding author.[email protected]
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Abstract

Stereological techniques using the optical disectors require estimation of final section thickness, but frozen tissue irregularities may interfere with this estimation. Cryostat slices from rodent nerve tissues (dorsal root ganglia, spinal cord, and brain), cut at 16, 40, and 50 μm, were digitized with a confocal microscope and visualized through 3D software. Geometric section thickness of tissue (Tgeom) was defined as tissue volume/area. Maximal section thicknesses (Tmax), from the top to the bottom of the section, were measured in a random sample of vertical ZX planes. Irregularities were mostly related to blood vessels traversing the tissue and neuronal somas protruding over the cut surfaces, with other neuron profiles showing a fragmented appearance. Irregularities contributed to increasing the distance between the tops and bottoms of slices sectioned in different laboratories. Significant differences were found between Tmax and Tgeom for all thickness studies and counting frames (p<0.01). The Tgeom/Tmax average rate was 68.4–85.7% in volumes around cell profiles (∼600–1,200 μm2) and 83.3–91.8% in subcellular samples (∼25–160 μm2). Confocal microscopy may help to assess tissue irregularities, which might lead to an overestimation of tissue volume if section thickness is estimated by focusing on the top and bottom of the sections.

Keywords

quantitative microscopyfrozen sectionsconfocal microscopy3D analysis
Type
Biological Applications and Techniques
Information
Microscopy and Microanalysis , Volume 21 , Issue 4 , August 2015 , pp. 893 - 901
Copyright
© Microscopy Society of America 2015 

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