Assessing mitochondrial morphology and dynamics using fluorescence wide-field microscopy and 3D image processing

Abstract

Mitochondrial morphology and length change during fission and fusion and mitochondrial movement varies dependent upon the cell type and the physiological conditions. Here, we describe fundamental wide-field fluorescence microscopy and 3D imaging techniques to assess mitochondrial shape, number and length in various cell types including cancer cell lines, motor neurons and astrocytes. Furthermore, we illustrate how to assess mitochondrial fission and fusion events by 3D time-lapse imaging and to calculate mitochondrial length and numbers as a function of time. These imaging methods provide useful tools to investigate mitochondrial dynamics in health, aging and disease.

Figure 1. Mitochondrial morphology in different cell types

Mitochondrial morphology of HeLa cells (left panels), astrocytes (middle panels), and motor neurons (right panels). HeLa cells express Mito-mPlum to visualize mitochondria. Astrocytes and motor neurons express DsRed2-Mito. Images were acquired by collecting 10 z-planes of 1 μm spacing. (A) Maximum projection of unfiltered, raw images. (B) Maximum projections of Fourier transformed images. (C) Maximum projections of median filter subtracted images. Scale bar: 25 μm.

Figure 2. Quantitation of mitochondrial fission by OPA1 siRNA transfection in Mito-mPlum transfected HeLa cells

Representative images of Mito-mPlum expressing HeLa cells transfected with (A) scrambled siRNA or (B) OPA1 siRNA after median filter subtraction, following an acquisition of 15 z-planes of 0.2 μm spacing. Mitochondria in (A) exhibit elongated and tubular morphology, while mitochondria in (B) exhibit fragmented morphology. Dashed lines indicate selected regions of interest (ROI). Scale bar: 10 μm. (C) Comparison of mitochondrial length distribution in OPA1 siRNA and scrambled siRNA transfected HeLa cells (n=10). * indicates significant differences between the two cell populations in ROI (p<0.01; Student's t-test, two-sided). (D) Average number of mitochondria in OPA1 and scrambled siRNA transfected HeLa cells (n=10) in ROI, excluding perinuclear and nuclear area. (E) Average mitochondrial length (μm) in ROI (excluding perinuclear and nuclear area) of OPA1 and scrambled siRNA transfected HeLa cells (n=10). ** indicates significant difference between the two cell populations (P<0.01; Student's t-test, two-sided).

Figure 3. Mitochondrial fission in a motor neuron addressed by 3D time-lapse imaging

(A) Representative images of a 190-minute time-lapse series of a motor neuron transfected with DsRed2-Mito undergoing mitochondrial fission due to neurotrophic factor withdrawal. Images were maximum projected and medium filtered. The inset is a 3× zoom of the indicated ROI showing a single mitochondrion undergoing fission over time. Scale bar: 25 μm. The arrows indicate fission of selected mitochondrial filaments. The arrowheads indicates condensation. (B) The mean mitochondrial length of the motor neuron shown in (A) is plotted over time. (C) Mitochondrial numbers within the ROI of the same motor neuron is plotted over time. An increase mitochondrial numbers occurs at ~ 100 minutes concomitant with a decrease in size (B).