Researchers from Sechenov University (Project 5-100 participant) and University College Cork (Ireland) developed a new method for studying mitochondrial polarisation in live cells and tissues. This approach enables studies of mitochondria in live cells, tissues and organoids and complements existing microscopy methods. The research was published in Cytometry Part A.
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Mitochondria play important roles in the live cell, providing energy, taking part in the regulation of the cell cycle and programmed cell death. However, capacities to study mitochondria in a quantitative manner and within the live cell are still rather limited. Fluorescence Lifetime Imaging Microscopy (FLIM) enables deeper understanding of mitochondrial function. The method is based on the environment-sensitive (polarity, temperature and other conditions) emission of fluorescent dyes, proteins and nanoparticles. Modern FLIM microscopes allow to measure fluorescence lifetime in sub-nanosecond time range and help reconstructing two- and three-dimensional images of live cells, tissues or organoids. This method is already actively used in studies of tissue oxygenation and hypoxia, pH and cellular redox status.
The authors of the paper found out that several well-known dyes, such as tetramethylrhodamine methyl ester (TMRM) and SYTO family of dyes, significantly improve traditional microscopy methods by examining mitochondria in a FLIM mode.
‘The work started with the attempts to perform FLIM of green fluorescent SYTO dyes to analyse the DNA and chromatin compaction in live cells. To our surprise, we found that most dyes did not show exclusively nuclear staining but also resided in mitochondria, in a membrane potential-dependent manner. Thus, the project was suddenly driven to the new and exciting direction – understanding of the mitochondrial membrane potential’, recalls Ruslan Dmitriev, group leader at the Institute for Regenerative Medicine, Sechenov University.
The study reports that fluorescence lifetime-dependent detection of mitochondrial polarisation (changes in mitochondrial membrane potential) allows distinguishing cell types more accurately, continuously monitoring ‘mitochondria at work’, and facilitates assessing cell oxygenation (hypoxia). Using FLIM, researchers confirmed the increase of mitochondrial polarisation at the border between G1 and S phases, – the critical moment for cell cycle progression. Alterations in the normal cell cycle progression are directly related to reduced ability of the tissue to regenerate (i.e. aging) and proliferation of cancer cells.
FLIM becomes an even more powerful approach when it is applied to three-dimensional tissue models, such as stem cell-derived intestinal organoids. Such ‘mini-gut’ tissue is a multicellular structure with the diversity of intestinal epithelium cell types including stem cells, enterocytes, Paneth, enteroendocrine and goblet cells. Organoid cultures enable the study of development and function of the gastrointestinal tract, its interactions with microbiota and pharmaceutical drugs, and is highly useful for studies of diabetes, colitis and cancer. The study demonstrated that FLIM is well-suited for analysis of live intestinal organoid culture and enables discrimination of cells within stem cell niche and monitoring of their proliferation. Multiplexed monitoring of mitochondrial polarisation and cell cycle S phase helped to find different subpopulations of stem cells. At the same time, studies of live three-dimensional objects using FLIM require improved data analysis algorithms and the use of state-of-the-art microscopes.
The reported approach opens bright prospects for tissue engineering with stem cells including monitoring of their quality. It should promote the development of new tissue engineering methods and microscopy techniques and stimulate basic research in the stem cell field.