Two-color nanoscopy of organelles for extended times with HIDE probes

Abstract

Performing multi-color nanoscopy for extended times is challenging due to the rapid photobleaching rate of most fluorophores. Here we describe a new fluorophore and a bio-orthogonal labeling strategy that enables both super-resolution (STED) and 3D confocal imaging of two organelles simultaneously for extended times using high-density environmentally sensitive (HIDE) probes. Because HIDE probes are small, cell-permeant molecules, they can visualize organelle pairs (ER + mitochondria, ER + plasma membrane) in hard-to-transfect cell lines at super-resolution for up to 7 minutes. The extended time domain possible using these new tools reveal novel dynamic nanoscale targeting between organelles. Super-resolution microscopy ('nanoscopy') can visualize cellular components with resolutionsas high as ~10 nanometers, far below the Abbe diffraction limit 1 . When combined with multicolor labeling strategies, nanoscopy can reveal key features of organelle structure and interactions that remain opaque when visualized using diffraction-limited approaches 2, 3 .However, visualizing organelle dynamics at super-resolution remains challenging because even the most photostable fluorophores bleach within tens to hundreds of seconds under conditions required for STED or SMS nanoscopy 4 .Recently, we reported a set of high-density environmentally sensitive (HIDE) probes that support long time-lapse, single-color nanoscopy of organelles including the ER, mitochondria, Golgi apparatus, and plasma membrane (PM) 5-7 . HIDE probes consist of an organelle-specific lipid or lipid-like small molecule with a reactive trans-cyclooctene (TCO) moiety and a silicon rhodamine-tetrazine (SiR-Tz) 8 reaction partner. These two components undergo a rapid, in situ tetrazine ligation reaction 9, 10 that localizes the SiR dye at high density within the organelle membrane. In this environment, HIDE probes support the acquisition of continuous SMS and

Long-term, super-resolution HIDE imaging of the inner mitochondrial membrane in live cells with a cell-permeant lipid probe

“…When paired with SiR-DBCO and visualized using STED ( Fig. 1d ), MAO-N 3 again improved visualization of discrete IMM cristae structures relative to RhoB-TCO 20 , and supported the acquisition of a 125-frame STED movie showing cristae dynamics. HIDE probes based on MAO-N 3 are versatile, non-toxic, photostable, and completely cell-permeant.…”

“…1d ) and visualized using SMLM, MAO-N 3 distinguished discrete IMM cristae structures that were not resolved using RhoB-HMSiR, a previously reported HIDE probe assembled using RhoB-TCO and HMSiR-Tz 17 . When paired with SiR-DBCO and visualized using STED, MAO-N 3 again improved visualization of discrete IMM cristae structures relative to RhoB-SiR 20 , and supported the acquisition of a 125-frame STED movie showing cristae dynamics.…”

“…HIDE probes have been used for long time-lapse imaging of the ER, Golgi, plasma membrane, mitochondrial matrix, and late endolysosomes 14–21 . They label organelles in model cell lines, primary cells from patient samples 19 , even neurons 18,20 . And because they are assembled from two parts, the emission properties can be altered at will to support multi-color labeling and all established modalities 20 .…”

“…They label organelles in model cell lines, primary cells from patient samples 19 , even neurons 18,20 . And because they are assembled from two parts, the emission properties can be altered at will to support multi-color labeling and all established modalities 20 . One mitochondrial HIDE probe (RhoB-TCO) has been reported 17,20 , but it labels mitochondrial subcompartments with minimal specificity; it is not selective for the IMM and cannot resolve IMM dynamics and fine structure 17,20 .…”

Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

“…They do not require UV irradiation or cytotoxic additives (such as thiol) to induce photoswitching (91,92). High photostability SFs have also been developed, enabling live-cell STED (79,(93)(94)(95). A final regime for live-cell SRM-compatible labelling is based on site-specific conjugation of fluorophores to a target of interest, through genetic code modifications and click chemistry (96-98) (Fig.…”