Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain

Abstract

Capturing mitochondria’s intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated for live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal strategies are yet to be established, and image acquisition has suffered either from photodamage to the organelles or from rapid photobleaching. Therefore, live-cell nanoscopy of mitochondria has been largely restricted to two-dimensional (2D) single-color recordings of cancer cells. Here, by conjugation of cyclooctatetraene (COT) to a benzo-fused cyanine dye, we report a mitochondrial inner membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability, and markedly reduced phototoxicity. PKMO enables super-resolution (SR) recordings of IM dynamics for extended periods in immortalized mammalian cell lines, primary cells, and organoids. Photostability and reduced phototoxicity of PKMO open the door to live-cell three-dimensional (3D) STED nanoscopy of mitochondria for 3D analysis of the convoluted IM. PKMO is optically orthogonal with green and far-red markers, allowing multiplexed recordings of mitochondria using commercial STED microscopes. Using multi-color STED microscopy, we demonstrate that imaging with PKMO can capture interactions of mitochondria with different cellular components such as the endoplasmic reticulum (ER) or the cytoskeleton, Bcl-2-associated X protein (BAX)-induced apoptotic process, or crista phenotypes in genetically modified cells, all at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial IM architecture and dynamics in a multiplexed manner.

Identifying STEDable BF2-Azadipyrromethene Fluorophores

“…When imaging live in 3D, difficulties may occur due to the increased time taken for acquisition and hence increased light exposure. Challenges may arise such as movement of the cell causing distortion of the final 3D image, photobleaching of the fluorophore, and phototoxicity to the sample [ 39 , 40 ]. Since we are imaging using a DL in the NIR region this should help reduce the phototoxic effects that may occur to a cell when imaged at a shorter wavelength.…”

A Bright, Photostable, and Far-Red Dye That Enables Multicolor, Time-Lapse, and Super-Resolution Imaging of Acidic Organelles

“…Performing linear unmixing − gave spectral separation in both cases, and we ultimately achieved a clear differentiation of lysosomes from either mitochondria (Figure a) or the nucleus (Figure b) in HeLa cells. Linear unmixing also allowed us to perform simultaneous three-color imaging of the nucleus, lysosomes, and mitochondria using SiR-DNA and HMSiR 680 -Me alongside commercially available MitoTracker Orange (Figures c and S4a) and PK Mito Orange (PKMO) (Figures d and S4b, Movie S2). These images clearly demonstrate the applicability of HMSiR 680 -Me for multicolor imaging by leveraging the nontoxic and infrequently used region of the visible spectrum above 650 nm.…”

Deep learning enables fast, gentle STED microscopy

“…Mitochondria labeling in living cells. The HeLa cells were stained with DMEM containing 250 nM PK Mito Orange (Confocal.nl) 31 for 40 min, followed by three washing steps in DMEM. The cells were kept in the incubator for 1hr to remove unbound dyes.…”