Multi-User Equipment approved in grant 15/26818-2: computerized stereology system with motorized microscope

• Grant number: 16/08976-5
• Support Opportunities: Multi-user Equipment Program
• Start date: February 01, 2017
• End date: January 31, 2024
• Field of knowledge: Humanities - Psychology - Physiological Psychology
• Principal Investigator: Dora Selma Fix Ventura
• Grantee: Dora Selma Fix Ventura
• Host Institution: Instituto de Psicologia (IP). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 14/26818-2 - Development and implementation of visual evaluation methods: clinical applications and animal models, AP.TEM
• EMU web page: http://www.ip.usp.br/site/laboratorio-de-psicofisiologia-sensorial-3/
• Type of equipment: Caracterização de Materiais - Microscopia ótica - Estéreo (metalúrgico)
• Manufacturer: Leica
• Model: Microscópio trinocular Leica DM5500B

Abstract

Morphological studies of the vertebrate retina allow assessing a number of aspects of the visual capabilities in normal and abnormal physiological states. By being physically isolated from the rest of the central nervous system, the retina allows analyzing how different species perceive their environment, and how the visual information is filtered and processed by the brain. The technique of cell sampling in whole mounted retinas is an important tool to evaluate the structure and organization of the retina. In recent years, a stereological approach based on the optical fractionation method was developed for use in flat mount retinas. The stereology method enables a precise, efficient and reliable quantification of the number of retinal cells, and allows estimating the quality of the quantitative values obtained by the error coefficient calculation (EC). This approach is already currently the most widely accepted standard in this field. The retinal mapping is done using a compound microscope equipped with a motorized stage X, Y and Z attached to a computer running a stereology software such as the Stereo Investigator (MicroBrightField, Colchester, VT). On the computer, the limits of retinal edges are drawn with an objective of low magnification, as well as all internal structures, such as the optical nerve. Using the stereology software, random and systematic count boxes are positioned on the retina drawn, in a standard grid, covering its entire length. To estimate the total number of neurons using stereological method, a tridimensional probe, the "optical dissector", is used to count nuclei of neurons, combined with a systematic, uniform sampling scheme. The optical fractionation procedure takes into account the following factors: 1) the relationship between the number of cuts of the sample and the total number of sections (section sampling fraction, SSF); 2) the relationship between the area of the sampling box and the area of the grid (area sampling fraction, asf); and 3) the relationship between the height of the dissector and the average measured thickness (thickness sampling factor, tsf). In each counting point, the thickness of the cell layer is assessed using the fine focus of the microscope. (AU)