Long-term Super-resolution Imaging Research Articles

Surface-Functionalized Halo-Tag Gold Nanoprobes for Live-Cell Long-Term Super-Resolution Imaging of Endoplasmic Reticulum Dynamics.

Super-resolution fluorescence microscopy has emerged as a powerful tool for studying endoplasmic reticulum (ER) dynamics in living cells. However, the lack of high-brightness, high-photostability, and stable labeling probes makes long-term super-resolution imaging of the ER still challenging. Herein, we reported a surface-functionalized Halo-tag gold nanofluorescent probe (GNP-Atto565-fR8-CA) that exhibits excellent brightness, photostability, and biocompatibility. GNP-Atto565-fR8-CA can simultaneously load multiple Atto565 dye molecules, significantly improving its brightness. Modifying the cell-penetrating peptide fR8 enables GNP-Atto565-fR8-CA to be efficiently delivered into the cytoplasm, overcoming the challenge of their easy entrapment in vesicles. Fluorescent labeling of ER proteins via Halo tags enables high specificity and stable labeling of GNP-Atto565-fR8-CA to the ER. The SIM super-resolution imaging results showed that GNP-Atto565-fR8-CA can track and observe the long-term dynamic process of the ER, and can also be used for long-term super-resolution imaging of the dynamic interactions between the ER and other organelles. This work offers a practical tool to study live-cell ER ultrastructure and dynamics.

Cell-Impermeable Buffering Fluorogenic Probes for Live-Cell Super-Resolution Imaging of Plasma Membrane Morphology Dynamics.

Super-resolution fluorescence imaging has emerged as a potent tool for investigating the nanoscale structure and function of the plasma membrane (PM). Nevertheless, the challenge persists in achieving super-resolution imaging of PM dynamics due to limitations in probe photostability and issues with cell internalization staining. Herein, we report assembly-mediated buffering fluorogenic probes BMP-14 and BMP-16 exhibiting fast PM labeling and extended retention time (over 2 h) on PM. The incorporation of alkyl chains proves effective in promoting the aggregation of BMP-14 and BMP-16 into nonfluorescent nanoparticles to realize fluorogenicity and regulate the buffering capacity to rapidly replace photobleached probes ensuring stable long-term super-resolution imaging of PM. Utilizing these PM-buffering probes, we observed dynamic movements of PM filopodia and continuous shrinkage, leading to the formation of extracellular vesicles (EVs) using structured illumination microscopy (SIM). Furthermore, we discovered two distinct modes of EV fusion: one involving fusion through adjacent lipids and the other through filamentous lipid traction. The entire process of EV fusion outside the PM was dynamically tracked. Additionally, BMP-16 exhibited a unique capability of inducing single-molecule fluorescence blinking when used for cell membrane staining. This property makes BMP-16 suitable for the PAINT imaging of cell membranes.

Protein proximity ligation probe with wide sensing range resolving dynamics of subcellular ph by super-resolution imaging

Subcellular pH plays a key role to modulate protein structures or specific active sites to impact diverse biological processes. The heterogeneity and high dynamics of intracellular pH require probes that can detect pH in the vicinity of the protein and have a wide pH response range. Here, we reported a protein proximity ligation fluorescent probe for super-resolution imaging of subcellular pH dynamics. Relying on SNAP-tag protein labeling technology, the pH-sensitive fluorophore BG-NDM based on photo-induced electron transfer mechanism was localized to the adjacent positions of the target protein in different subcellular organelles. The equilibrium between the non-fluorescent aggregate and the fluorogenic monomer enabled BG-NDM to have the ability of wash-free super-resolution imaging in live cells. And the wide pH response ranges from pH 6 to pH 12 made the probe have the ability of recognizing pH dynamics. The pH changes around functional proteins during mitophagy, cellular alkalosis and acidosis were revealed. Through long-term super-resolution imaging, we could further record the pH dynamics around mitochondrial proteins COX8A during mitochondrial contacts, fusion and fission at the resolution of a single mitochondria.

BODIPY 493 acts as a bright buffering fluorogenic probe for super-resolution imaging of lipid droplet dynamics

The need for temporal resolution and long-term stability in super-resolution fluorescence imaging has motivated research to improve the photostability of fluorescent probes. Due to the inevitable photobleaching of fluorophores, it is difficult to obtain long-term super-resolution imaging regardless of the self-healing strategy of introducing peroxide scavengers or the strategy of fluorophore structure modification to suppress TICT formation. The buffered fluorogenic probe uses the intact probes in the buffer pool to continuously replace the photobleached ones in the target, which greatly improves the photostability and enables stable dynamic super-resolution imaging for a long time. But the buffering capacity comes at the expense of reducing the number of fluorescent probes in targets, resulting in low staining fluorescence intensity. In this paper, we selected BODIPY 493, a lipid droplet probe with high fluorescence brightness, to explore the dynamic process of lipid droplet staining of this probe in cells. We found that BODIPY 493 only needs very low laser power for lipid droplet imaging due to the high molecular accumulation in lipid droplets and the high brightness, and the spatiotemporal resolution is greatly improved. More importantly, we found that BODIPY 493 also has a certain buffering capacity, which enables BODIPY 493 to be used for super-resolution imaging of lipid droplet dynamics. This work reminds researchers to coordinate the buffering capacity and brightness of fluorogenic probes.

Long-term superresolution imaging and quantification of live-cell plasma membrane biophysical properties with smart exchangeable dyes

The study of biological membranes by fluorescence microscopy is hindered by the resolution limit of conventional microscopy. Superresolution microscopy overcomes this problem, but greatly limits temporal resolution. In the case of STED superresolution microscopy, high photobleaching rates prevent long-term imaging. Here, we employ a new exchangeable dye that reports on the molecular order and dynamics of lipid membranes with no photobleaching-induced signal loss. We demonstrate that this dye is sensitive to lipid packing changes in model and cell membranes and perform simultaneous STED-FCS dynamics and packing measurements.

Development of a multimodal digital micromirror device based microscope for fast label-free imaging and multicolor structured illumination microscopy

Fast, long-term superresolution imaging of live cells can reveal biological processes which occur rarely but with fast dynamics. However, these requirements are difficult to realize simultaneously due to the slow speeds of most superresolution techniques and photobleaching and phototoxicity which are ubiquitous in long-term fluorescence imaging. A possible solution is to perform continuous label-free imaging to monitor a sample for behavior of interest, which then triggers high-speed fluorescent superresolution imaging.

Stable Super-Resolution Imaging of Lipid Droplet Dynamics through a Buffer Strategy with a Hydrogen-Bond Sensitive Fluorogenic Probe.

Although super-resolution imaging offers an opportunity to visualize cellular structures and organelles at the nanoscale level, cellular heterogeneity and unpredictability still pose a significant challenge in the dynamic imaging of live cells. It is thus vital to develop better-performing and more photostable probes for long-term super-resolution imaging. Herein, we report a probe, LD-FG, for imaging lipid droplet (LD) dynamics using structured illumination microscopy (SIM). LD-FG allows wash-free imaging of LDs, owing to a hydrogen-bond sensitive fluorogenic response. The replacement of photobleached LD-FG by intact probe molecules outside the LDs ensures the long-time stability of the fluorescence imaging. With this buffering fluorogenic probe, fast and unpredictable dynamic processes of LDs can be visualized. Using this probe, two LD coalescence modes were discovered. The dynamic imaging also allowed us to propose a new model of LD maturation during adipocyte differentiation, i.e., a fast LD coalescence followed by a slow ripening step. The excellent performance of LD-FG makes the buffer strategy an effective method for designing fluorescent probes for cell dynamic imaging.

Ultrasmall silica nanospheres based blinking nanoprobes for optical super resolution imaging

Single-molecule localization-based super-resolution imaging technology provides a powerful tool for research in the nanoscale field. The properties of organic fluorescent probes have a decisive significance for the quality of super-resolution imaging. Here, we propose an ultrasmall silica nanospheres based blinking nanoprobe for single molecule localization imaging. The blinking nanoprobe is fabricated by attaching fluorophores onto the ultrasmall silica nanospheres and it shows excellent features in three ways. First, it has stable chemical properties and good biocompatibility. Second, it shows a sustainable and excellent fluorescence switching behavior, which is suitable for long-term super-resolution imaging. Third, it possesses a better localization precision (20–25 nm) than the fluorophore itself. To examine the application of the nanoprobe, breast cancer cell (SKBR3) derived exosomes are used as the target. By attaching HER2 aptamers onto the silica nanospheres, super-resolution imaging of the exosomes is realized. Such a new strategy for the preparation of super-resolution nanoprobes has a great potential for further exploring the biological functions of nanoscale biomarkers. • Super resolution nanoprobes based on ultrasmall silica nanospheres are fabricated. • A localization precision of 20–25 nm has been achieved. • The nanoprobes showed better fluorescence switching performance than dyes. • Super-resolution imaging of tumor derived exosomes has been realized.

An assembly-regulated SNAP-tag fluorogenic probe for long-term super-resolution imaging of mitochondrial dynamics

Super-resolution fluorescence microscopy has emerged as a powerful tool for studying mitochondrial dynamics in living cells. However, the lack of photostable and chemstable probe makes long-term super-resolution imaging of mitochondria still a challenging work. Herein, we reported a 4-azetidinyl-naphthliamide derived SNAP-tag probe AN-BG exhibiting excellent fluorogenicity and photostability for long-term super-resolution imaging of mitochondrial dynamics. The azetidinyl group and naphthalimide fluorophore are in a flat conformation which can effectively suppress twisted intramolecular charge transfer and then effectively improve the brightness and photostability. This planarized molecular structure is conducive to the formation of fluorescence-quenched J-aggregates, and the protein labeling process will depolymerize the probes and restore fluorescence. Fluorescent labeling mitochondrial inner membrane proteins via SNAP tags overcomes the shortcomings that variations in mitochondrial inner membrane potential will release probes attached to mitochondria by electrostatic interactions. Therefore, AN-BG realized the stable labeling of mitochondria and the long-term imaging of mitochondrial dynamics under super-resolution microscopy.

Long-term super-resolution imaging of mitochondrial dynamics

Monitoring dynamics of mitochondria has become an essential approach to explore the function of mitochondria in living cells with the emergence of super-resolution fluorescence microscopy. However, long-term super-resolution imaging of mitochondria is still challenging due to the lack of photostable fluorescent probes and stable mitochondria-specific markers which are not affected by the changes of mitochondrial membrane potential. Here, we introduce a method for long-term imaging mitochondrial dynamic through the SNAP-tag fluorogenic probe based on 4-azetidinyl-naphthalimide derivatives. Using structured illumination microscopy (SIM), we observed the fusion and fission of mitochondria over a course of 16 min at 109 nm resolution. Furthermore, the interactions as well as fusion between mitochondria and lysosomes were studied during mitophagy at the nanoscale. Convincingly, the combination of SNAP-tag fluorogenic probes and super-resolution fluorescence microscopy will offer a new way to monitor dynamic mitochondria in living cells.

Long-Term Super-Resolution Imaging of Amyloid Structures using Transient Binding of Standard Amyloid Probes

Oligomeric amyloid structures are crucial therapeutic and diagnostic targets in Alzheimer's disease and other amyloid diseases. However, these oligomers are too small to be resolved by conventional light microscopy. We have developed a new tool to image amyloid structures on a nanometer scale using standard amyloid dyes such as Thioflavin T (ThT), without the need for covalent labeling of the amyloid protein or staining via fluorescently labeled antibodies. Transient amyloid binding (TAB) imaging is compatible with epifluorescence and TIRF microscopies and uses 488 nm cw laser excitation to excite ThT molecules that are bound to amyloid structures. Dynamic binding and unbinding of ThT molecules generate photon bursts (‘blinking’) that are used for single fluorophore localization at nanometer resolution. Thus, photobleaching cannot degrade either the number or brightness of blinking events, which enables us to image the same structure for multiple days without loss of image quality. As proof-of-principle experiments, we imaged oligomeric and fibrillar structures formed during different stages of amyloid-β aggregation as well as the structural remodeling of amyloid fibrils by the anti-amyloid compound epi-gallocatechin gallate (EGCG). TAB promises to directly image native amyloid in cells and tissues using standard probes at nanometer resolution and at the same time record amyloid dynamics over time scales of minutes to days.

Cell-permeable organic fluorescent probes for live-cell long-term super-resolution imaging reveal lysosome-mitochondrion interactions

Characterizing the long-term nanometer-scale interactions between lysosomes and mitochondria in live cells is essential for understanding their functions but remains challenging due to limitations of the existing fluorescent probes. Here, we develop cell-permeable organic fluorescent probes for lysosomes with excellent specificity and high photostability. We also use an existing Atto 647N dye with high brightness and excellent photostability to achieve specific labeling of mitochondria in live cells. Using these probes, we obtain dual-color structured illumination microscopy (SIM) images of dynamic physical lysosome-mitochondrion interactions in live cells at an ~90-nm resolution over a long time course of ~13 min. We successfully record the consecutive dynamic processes of lysosomal fusion and fission, as well as four types of physical lysosome-mitochondrion interactions by super-resolution imaging. Our probes provide an avenue for understanding the functions and the dynamic interplay of lysosomes and mitochondria in live cells.