Proc. Natl. Acad. Sci. U.S.A.: Development of new probe for mitochondrial multicolor STED imaging

Mitochondria are the power source of cells, affecting key signaling pathways
for cell homeostasis, proliferation, and death.
Due to the dynamic behavior of mitochondria and the rich interaction with other organelles, the development of fluorescence microscopy has particularly advanced mitochondrial research
.
The inner membrane (IM) inwardly concaves to form many lamellar or tubular inner crests, often less than 100 nm apart, making its internal fine structure
impossible to observe with conventional fluorescence microscopy.
Therefore, fixed-sample-based electron microscopy has been a mainstream tool
for capturing mitochondrial membrane structure.
In recent years, mitochondrial nanoimaging of living cells has evolved from proof-of-principle to a feasible method
for structural and functional studies.
Among them, stimulated emission loss nanomicroscopy (STED) and structured light illumination microscopy (SIM) have been reported for intramitochondrial crest imaging
of living cells.
However, at present, the visualization of mitochondrial nanostructures is mostly limited to two-dimensional (2D) monochromatic imaging of cancer cells, orthogonal strategies have not yet been established, and STED image acquisition will be affected by photodamage or rapid photobleaching of organelles, and it is difficult to observe mitochondrial morphology
in the native state.

The research group of Professor Chen Zhixing of the School of Future Technology of Peking University and the Peking University-Tsinghua Joint Center for Life Sciences was established in the research group of Prof.
Natl.
Acad.
Sci.
U.
S.
A.
published a research paper entitled "Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain" and was selected as the cover article
。 The study reported a mitochondrial crest dye PK Mito Orange (PKMO) suitable for STED nanomicroscopy with excellent photostability and significantly reduced phototoxicity, enabling long-term, super-resolution mitochondrial dynamic imaging in immortalized mammalian cell lines, primary cells, and tissues, 3D-STED imaging of individual mitochondria, and multicolor STED imaging (Figure 1).

Figure 1: Multicolor live-cell imaging of the mitochondrial crest, microfilament protein (SPY650FastAct), and nucleus (SPY505 DNA) of PKMO-labeled HeLa cells

In this study, the cyclooctatetraene (COT) coupling strategy was used to reduce the dye phototoxicity, while precisely manipulating the spectra to make the dye perfectly compatible
with 561nm excitation light and 775nm STED light.
PKMO-stained cells with highly ordered inner laminar crest networks can be observed throughout the cell under STED nanomicroscopy, and dynamic processes such as single mitochondrial crest morphology and mitochondrial division, fusion, and tubular genesis can be captured with optical resolution down to 50 nm
.
PKMO enables 3D STED reconstruction
of individual mitochondria in living cells due to its mild nature and excellent photostability.
In addition to being applied to cancer cells and immortalized cell lines, PKMO can also enable super-resolution imaging
of primary cells and tissues that are extremely sensitive to phototoxicity.
PKMO demonstrated the inner crest structure and its dynamic processes
in COS-7, HeLa, U-2 OS cell lines, as well as primary adipocytes, primary neurons, and primary islet tissues.

The bilayered membrane structure of mitochondria gives rise to mitochondrial subcompartments that serve different purposes
.
Because multicolor nanomicroscopy technology can provide higher resolution and more spatiotemporal information, it will replace traditional biochemical analysis or electron microscopy as a powerful new analytical method
.
As shown in Figure 2, PKMO demonstrates multicolor super-resolution imaging
with mitochondrial IM and mitochondrial DNA (mtDNA), outer mitochondrial membrane, crista junction, tubulin, and endoplasmic reticulum.
Multicolor live cell STED imaging can reveal different mitochondrial localization of biomolecules and mitochondrial interaction networks in the whole cell; Provides similar information to more demanding immunoelectron microscopy techniques and can map more information
about structure and biomolecular dynamics.

Figure 2: Multicolor STED imaging of mitochondrial crest with mtDNA, outer mitochondrial membrane, cristajunction, endoplasmic reticulum, and cytoskeleton

Defects in the inner crest structure are associated with dysfunction of cellular respiration and have been associated
with neurodegenerative diseases or heart disease.
The authors also show differences in the inner crest morphology of different mitochondrial protein-deficient cell lines and wild cell lines
.
In the future, super-resolution imaging of living cells using PKMO is expected to replace traditional time-consuming and laborious electron microscopy preparation and become a daily tool
for mitochondrial structural function studies.

In summary, the authors' team reported a novel mitochondrial probe with high brightness and low phototoxicity compatible with STED imaging of live cells
.
PKMO enables long-term, super-resolution endometrial kinetic recording
in immortalized mammalian cell lines, primary cells, or tissues.
The photostability and phototoxicity of PKMO open the door
to 3D STED microscopy imaging of mitochondria in living cells.
At the same time, PKMO is compatible with green and far-red fluorescent markers to achieve multi-component analysis
of mitochondrial substructures at multicolor superresolution.
Multicolor STED microscopy captures mitochondrial interactions with different cellular components, BAX-induced apoptosis processes, and inner crest phenotypes
in transgenic cells at 100 nm resolution.
Therefore, this work provides a versatile tool for studying the structure and dynamics
of the inner mitochondrial membrane in a multiplexed manner.