The Matrix of Mitochondrial Imaging: Exploring Spatial Dimensions

Abstract

Mitochondria play a crucial role in human biology, affecting cellular processes at the smallest spatial scale as well as those involved in the functionality of the whole system. Imaging is the most important research tool for studying the fundamental role of mitochondria across these diverse spatial scales. A wide array of available imaging techniques have enabled us to visualize mitochondrial structure and behavior, as well as their effect on cells and tissues in a range from micrometers to centimeters. Each of the various imaging techniques that are available offers unique advantages tailored to specific research needs. Selecting an appropriate technique suitable for the scale and application of interest is therefore crucial, but can be challenging due to the large range of possibilities. The aim of this review is two-fold. First, we provide an overview of the available imaging techniques and discuss their strengths and limitations for applications across the sub-mitochondrial, cellular, tissue and organ levels for the imaging of mitochondria. Second, we identify opportunities for novel applications and advancement in the field. We emphasize the importance of integration across scales in mitochondrial imaging studies, particularly to bridge the gap between microscopic and non-invasive techniques. While integrating these diverse scales is challenging, primarily because such multi-scale approaches require expertise that spans different imaging modalities, we argue that integration has the potential to provide groundbreaking insights into mitochondrial biology. By providing a comprehensive overview of imaging techniques, this review paves the way for multi-scale imaging initiatives in mitochondrial research.

Keywords: MRI; imaging; microscopy; mitochondria.

Figure 1

Sub-mitochondrial structure visualization. This timeline provides a historical overview of the various sub-mitochondrial imaging techniques. First FIB/SEM paper on mitochondria [17]. EM tomography image from Mannella, C.A. [18], super-resolution fixed mitochondria from Schmidt, R. [19] and super-resolution live mitochondria from Shao, L. [20], all obtained with permission. TEM = transmission electron microscopy; FIB/SEM = focused ion beam/scanning electron microscopy. Scale bars from left to right equal 1 µm, 250 nm, 100 nm, 250 nm and 1 µm.

Figure 2

Imaging applications at a cellular level. (A) Changes in mitochondrial network morphology as the result of an altered balance between fission and fusion can be determined using imaging and quantification of the mitochondrial morphology. (B) Tracking of fission and fusion events using photoactivatable fluorescent proteins. The presence of a signal in non-activated mitochondria is indicative of fusion, while a break in the activated signal indicates fission. (C) Tracking of fission and fusion events using photoconvertable proteins. The presence of both activated and non-activated signals indicates fusion events, while the breaking of an activated signal indicates fission. (D) Tracking of mitophagy through the loss of green fluorescence upon mitophagosome and lysosome fusion. (E) Co-localization of proteins with mitochondria can determined through staining the mitochondria and protein of interest. The overlap between the two stains is indicative of the mitochondrial localization of a protein. (F) Mitochondrial membrane potential changes can be visualized through the use of membrane potential-dependent dyes, like TMRM. Both a global change and local decrease in membrane potential can be visualized using imaging. (G) Increases in ROS formation are observable through the binding of ROS to fluorescent dyes, which increases the emitted fluorescence. (H) Increases in mitochondrial calcium are observable through the binding of calcium with fluorescent dyes, which increases the emitted fluorescence.

Figure 3

Overview of the three commonly used microscopy techniques and their suitability to various mitochondrial imaging applications. LSCM: laser scanning confocal microscopy; SDCM: spinning disk confocal microscopy.

Figure 4

Impact of magnetic field strength and acquisition contrast on MRI images. Comparison of the effects of a 1.5, 3 and 7 Tesla magnetic field strength on image resolution for a T₂-FLAIR-weighted image. The arrow indicates a white matter lesion (images from Zwanenburg, J.J.M. et al. [177], obtained with permission).

Figure 5

Mitochondrial imaging techniques across scales. All imaging techniques can span various spatial scales; however, a complete picture of all levels can only be obtained by combining various techniques. Tissue image from Kuznetsov, A.V. et al. [179]. Scale bars from left to right indicate 15 µm and 1 µm.