Timelapse and volumetric imaging of mitochondrial networking using NAD(P)H autofluorescence via 2-photon microscopy

2025 · doi:10.1101/2025.01.15.633075
preprint OA: closed
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The paper describes and validates a microscopy protocol to image mitochondrial dynamics using NAD(P)H autofluorescence with 2-photon microscopy, including timelapse (2D) and volumetric (3D) imaging. Using MDA-MB-231 cells, the authors optimized imaging parameters (laser power, image size, dwell time, interval time, and total duration) to minimize photodamage and maintain signal, and used the mitochondrial dye MitoSpy Orange to validate that the NAD(P)H signal reflects mitochondria. They report NAD(P)H-based timelapse acquisition at 0.4 FPS for observing mitochondrial movement in 2D and 3D imaging at about 0.5 FPS through ~15 µm for visualizing mitochondrial network distribution, with the main limitation being that only a single cell type was tested in this protocol description. This paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.

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Abstract

Significance Mitochondria are dynamic organelles that play a key role in energy production and maintaining cellular homeostasis. The regulation of mitochondrial dynamics, involving both fission and fusion, is vital for maintaining a healthy population of mitochondria within the cell. Alterations in mitochondrial dynamics have been associated with various disease states, such as metabolic and neurodegenerative diseases and cancer. Aim We describe a protocol for imaging and analyzing NAD(P)H intensity to visualize the movement of mitochondria over time and in 3D to visualize the distribution of the mitochondrial network within the cell. Approach A multiphoton (MP) laser scanning microscope was used to image NAD(P)H autofluorescence signal of MDA-MB-231 cells at 750 nm excitation. A mitochondrial fluorescent dye, MitoSpy Orange, was used to validate the signal. Laser power, image size, dwell time, interval time, and imaging duration were optimized for timelapse and 3D imaging to minimize photodamage and maximize autofluorescence signal.

Results

The NAD(P)H signal in 2D was imaged with a frame rate of 0.4 frames per second (FPS) allowing for visualization of mitochondria movement. 3D imaging was performed with a frame rate of 0.5 FPS for a single cell with a thickness of approximately 15 microns that allowed for visualization of the mitochondria network within the cell.

Conclusions

This protocol motivates using label-free imaging techniques to study mitochondrial dynamics in a non- destructive manner, suitable for drug screening and understanding the effects of mitochondrial dynamic alterations in disease. Competing Interest Statement The authors have declared no competing interest.

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last seen: 2026-05-20T01:45:00.602351+00:00