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.
Sovrem Tekhnologii Med. 2021;13(2):18-29. doi: 10.17691/stm2021.13.2.02. Epub 2021 Apr 30.
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.
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.
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.
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.
使用多光子显微镜和质谱等现代方法，研究正常肝细胞在代谢状态方面表现出异质性的可能性。
使用基于细胞内辅因子 NAD(P)H 和 FAD 自发荧光强度的多光子显微镜评估肝细胞在总代谢活性方面的异质性。使用基于各种形式 NAD(P)H 荧光寿命贡献数据的荧光寿命成像 (FLIM) 方法确定肝细胞在细胞内代谢过程强度方面的异质性。采用飞行时间二次离子质谱法 (TоF-SIMS) 研究肝细胞的脂质和氨基酸组成。
多光子显微镜显示，肝细胞在一般代谢活性方面存在异质性。FLIM 研究表明，氧化磷酸化、糖酵解和合成过程（脂肪生成、核酸合成和戊糖磷酸途径）的强度具有高度的异质性程度。TоF-SIMS 方法揭示了氨基酸和脂质组成方面的肝细胞异质性，表明个别肝细胞中合成过程的强度存在差异。此外，还发现了 PO 离子含量的差异。TоF-SIMS 研究的结果与多光子显微镜和 FLIM 获得的数据相关，证实了肝细胞在一般代谢活性和细胞间代谢过程强度方面存在异质性。
荧光生物成像和质谱的最新方法在揭示代谢状态下肝细胞的异质性方面是有效的。在应用荧光生物成像方法研究各种条件下的肝组织时，应考虑到异质性的存在。
