A Pipeline for Dynamic Analysis of Mitochondrial Content in Developing T Cells: Bridging the Gap Between High-Throughput Flow Cytometry and Single-Cell Microscopy Analysis

Vaibhav Dhyani; Anchi S Chann; Lopamudra Giri; Sarah M Russell; Mirren Charnley

doi:10.1007/978-1-0716-3834-7_12

A Pipeline for Dynamic Analysis of Mitochondrial Content in Developing T Cells: Bridging the Gap Between High-Throughput Flow Cytometry and Single-Cell Microscopy Analysis

Methods Mol Biol. 2024:2800:167-187. doi: 10.1007/978-1-0716-3834-7_12.

Authors

Vaibhav Dhyani^{1

2}, Anchi S Chann^{2

3

4}, Lopamudra Giri¹, Sarah M Russell^{5

6

7}, Mirren Charnley^{8

9}

Affiliations

¹ Bioimaging and Data Analysis Lab, Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Sangareddy, Telangana, India.
² Optical Science Centre, Faculty of Science, Engineering & Technology, Swinburne University of Technology, Hawthorn, Australia.
³ Immune Signalling Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.
⁴ Department of Immunology, Mayo Clinic, Scottsdale, AZ, USA.
⁵ Optical Science Centre, Faculty of Science, Engineering & Technology, Swinburne University of Technology, Hawthorn, Australia. [email protected].
⁶ Immune Signalling Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. [email protected].
⁷ Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, VIC, Australia. [email protected].
⁸ Optical Science Centre, Faculty of Science, Engineering & Technology, Swinburne University of Technology, Hawthorn, Australia. [email protected].
⁹ Immune Signalling Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. [email protected].

PMID: 38709484
DOI: 10.1007/978-1-0716-3834-7_12

Abstract

Analyzing the dynamics of mitochondrial content in developing T cells is crucial for understanding the metabolic state during T cell development. However, monitoring mitochondrial content in real-time needs a balance of cell viability and image resolution. In this chapter, we present experimental protocols for measuring mitochondrial content in developing T cells using three modalities: bulk analysis via flow cytometry, volumetric imaging in laser scanning confocal microscopy, and dynamic live-cell monitoring in spinning disc confocal microscopy. Next, we provide an image segmentation and centroid tracking-based analysis pipeline for automated quantification of a large number of microscopy images. These protocols together offer comprehensive approaches to investigate mitochondrial dynamics in developing T cells, enabling a deeper understanding of their metabolic processes.

Keywords: 3D imaging; DN3; Density-based spatial clustering of applications with noise (DBSCAN); Image segmentation; MitoView Green; Mitochondria.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Flow Cytometry* / methods
Humans
Image Processing, Computer-Assisted / methods
Mice
Microscopy, Confocal* / methods
Mitochondria* / metabolism
Mitochondrial Dynamics
Single-Cell Analysis* / methods
T-Lymphocytes* / cytology
T-Lymphocytes* / metabolism