Multiphoton Microscopy and Mass Spectrometry for Revealing Metabolic Heterogeneity of Hepatocytes .

S A Rodimova, D S Kuznetsova, N V Bobrov, A A Gulin, A A Vasin, M V Gubina, V I Scheslavsky, V V Elagin, M M Karabut, V E Zagainov, E V Zagaynova

Journal: Sovremennye tekhnologii v meditsine 2021;13(2):18-29

PMID: 34513073

Free Full Text on PubMed Central View on PubMed

Abstract

UNLABELLED

was to study the possibility of revealing the heterogeneity of normal liver hepatocytes in terms of metabolic status using the modern methods of multiphoton microscopy and mass spectrometry.

MATERIALS AND METHODS

Heterogeneity of hepatocytes in terms of total metabolic activity was assessed using multiphoton microscopy based on the autofluorescence intensity of intracellular cofactors NAD(P)H and FAD. Hepatocyte heterogeneity in terms of intensity of intracellular metabolic processes was determined using the fluorescence lifetime imaging (FLIM) method based on the data about fluorescence lifetime contributions of various forms of NAD(P)H. The method of time-of-flight secondary ion mass spectrometry (TоF-SIMS) was used to study the lipid and amino acid composition of hepatocytes.

RESULTS

It has been revealed using multiphoton microscopy that hepatocytes are heterogeneous in terms of general metabolic activity. Using FLIM, it was established that the heterogeneity degree was high in terms of intensity of oxidative phosphorylation, glycolysis, and synthetic processes (lipogenesis, nucleic acid synthesis, and the pentose phosphate pathway). The TоF-SIMS method revealed the presence of hepatocyte heterogeneity in terms of amino acid and lipid composition, which points to various intensities of synthetic processes in individual hepatocytes. Moreover, differences in the content of PO ions were revealed. The results of ToF-SIMS study correlate with the data obtained by multiphoton microscopy and FLIM, confirming the revealed heterogeneity of hepatocytes in terms of general metabolic activity and intensity of intercellular metabolic processes.

CONCLUSION

The latest methods of fluorescence bioimaging and mass spectrometry proved to be effective in revealing hepatocyte heterogeneity in terms of metabolic status. The presence of heterogeneity should be taken into account in studying the liver tissue under various conditions with the application of fluorescence bioimaging methods.

Address: Junior Researcher, Laboratory of Regenerative Medicine, Research Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia; PhD Student, Institute of Biology and Biomedicine, National Research Lobachevsky State University of Nizhni Novgorod, 23 Prospekt Gagarina, Nizhny Novgorod, 603950, Russia.; Researcher, Laboratory of Regenerative Medicine, Research Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia.; Assistant, Department of Theoretical Surgery and Transplantology, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia; Surgeon, Oncology Department, Volga District Medical Centre of Federal Medical Biological Agency of Russia, 14 Ilyinskaya St., Nizhny Novgorod, 603109, Russia.; Senior Researcher, Acting Head of the Laboratory of Biophotonics, N.N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, 4 Kosygina St., Moscow, 119991, Russia; Researcher, Faculty of Chemistry, Lomonosov Moscow State University, 1 Leninskiye Gory, Moscow, 119991, Russia.; Research Engineer, Laboratory of Nanophotonics, N.N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, 4 Kosygina St., Moscow, 119991, Russia; Student, Faculty of Chemistry, Lomonosov Moscow State University, 1 Leninskiye Gory, Moscow, 119991, Russia.; Research Engineer, Laboratory of Nanophotonics, N.N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, 4 Kosygina St., Moscow, 119991, Russia; Student, Phystech School of Electronics, Photonics and Molecular Physics, Moscow Institute of Physics and Technology (National Research University), 9 Institutskiy per., Dolgoprudny, Moscow Region, 141701, Russia.; Senior Researcher, Becker & Hickl, GmbH, Nunsdorfer Ring 7-9, Berlin, 12277, Germany; Head of the Laboratory of High-Resolution Microscopy, Research Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia.; Researcher, Laboratory of High-Resolution Microscopy, Research Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia.; Researcher, Laboratory of Genomics and Adaptive Antitumor Immunity, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia.; Head of the Department of Theoretical Surgery and Transplantology, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia; Chief Specialist in Surgery, Volga District Medical Centre of Federal Medical Biological Agency of Russia, 14 Ilyinskaya St., Nizhny Novgorod, 603109, Russia.; Rector, National Research Lobachevsky State University of Nizhni Novgorod, 23 Prospekt Gagarina, Nizhny Novgorod, 603950, Russia; Senior Researcher, Research Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia.

DOI: 10.17691/stm2021.13.2.02

Link outs

Free resources

Full Text Sources:

Miscellaneous:

https://assays.cancer.gov/non-CPTAC-1157

MeSH Terms: Hepatocytes, Microscopy, Fluorescence, Multiphoton, NAD, Oxidative Phosphorylation, Spectrometry, Mass, Secondary Ion