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

ter Hofstede, B.; Walsh, A. J.

doi:10.1101/2025.01.15.633075

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bioRxiv · 10.1101/2025.01.15.633075

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

ter Hofstede, B.·Walsh, A. J.

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Abstract

SignificanceMitochondria 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. AimWe 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. ApproachA 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. ResultsThe 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. ConclusionsThis 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.

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ter Hofstede, B., Walsh, A. J.. 2025-01-19. Timelapse and volumetric imaging of mitochondrial networking using NAD(P)H autofluorescence via 2-photon microscopy. https://doi.org/10.1101/2025.01.15.633075

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