Organelle Proximity Analysis for Enhanced Quantification of Mitochondria–Endoplasmic Reticulum Interactions in Single Cells via Super-Resolution Microscopy
Yucheng Sun, Shiyu Chen, Ying Hou, Seong Ho Kang* and Jin-Ming Lin*,
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引用次数: 0
Abstract
Mitochondria (MT) and the endoplasmic reticulum (ER) maintain lipid and calcium homeostasis through membrane contacts, particularly MT–ER contacts (MERCs), spanning distances from 10 to 50 nm. However, the variation of different distance ranges and the metabolic factors influencing this variation remain poorly understood. This study employed microfluidic chip-based super-resolution microscopy in conjunction with a Moore-Neighbor tracing-incorporated organelle proximity analysis algorithm. This approach enabled precise three-dimensional localization of single-fluorescence protein molecules within narrow and irregular membrane proximities. It achieved lateral localization precision of less than 20 nm, resulting in a minimum MERC distance of approximately 8 nm in spatial and mean distances across multiple threshold ranges. Additionally, we demonstrated that the MERC distance variation was correlated with MT size rather than ER width. The proportion of each distance range varied significantly after the stimuli. Free cholesterol showed a negative correlation with various distances, while distances of 10–30 nm were associated with glucose, glutamine, and pyruvic acid. Furthermore, the 30–40 nm range was influenced by citric acid. These results underscore the role of advanced subcellular organelle analysis in elucidating the single-molecule behavior and organelle morphology in single-cell studies.
期刊介绍:
Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.