Single‐Molecule Spectroscopy,Imaging, and Photocontrol: Foundations for Super‐Resolution Microscopy (Nobel Lecture)

The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single‐molecule limit in 1989 using frequency‐modulation laser spectroscopy. In the early 90s, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later super‐resolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super‐resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized.     

SLAP: Small Labeling Pair for Single‐Molecule Super‐Resolution Imaging

Protein labeling with synthetic fluorescent probes is a key technology in chemical biology and biomedical research. A sensitive and efficient modular labeling approach (SLAP) was developed on the basis of a synthetic small‐molecule recognition unit (Ni‐trisNTA) and the genetically encoded minimal protein His_6‐10‐tag. High‐density protein tracing by SLAP was demonstrated. This technique allows super‐resolution fluorescence imaging and fulfills the necessary sampling criteria for single‐molecule localization‐based imaging techniques. It avoids masking by large probes, for example, antibodies, and supplies sensitive, precise, and robust size analysis of protein clusters (nanodomains).     

Subdiffraction‐Resolution Fluorescence Microscopy of Myosin–Actin Motility

Subdiffraction‐resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of ～20 nm with moderate excitation intensities, but have so far been too slow for imaging faster dynamics in biology. Herein, we introduce a novel approach for video‐like subdiffraction microscopy based on rapid and reversible photoswitching of commercially available organic carbocyanine fluorophores. With the present concept, we demonstrate in vitro studies on the motility of fluorophore‐labeled actin filaments along myosin II. Actin filaments were densely labeled with carbocyanine fluorophores, and the gliding velocity adjusted by the concentration of ATP. At imaging frame rates of ～100 Hz, only 100 consecutive frames are sufficient to generate a single high‐resolution image of moving actin filaments with a lateral resolution of ～30 nm. A video‐like sequence is generated from individual reconstructed images by additionally applying a sliding window algorithm. We measured velocities of individual actin filaments of up to ～0.18 μm s^?1, observed strong bending and disruption of filaments as well as locally immobile fragments.     

Minimal Tags for Rapid Dual‐Color Live‐Cell Labeling and Super‐Resolution Microscopy

The growing demands of advanced fluorescence and super‐resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue‐specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine‐functionalized organic dyes by the inverse‐electron‐demand Diels–Alder cycloaddition (SPIEDAC). Furthermore, we fine‐tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus‐like particles (VLPs) with dual‐color super‐resolution microscopy.     

Super‐Resolution Imaging of Plasma Membrane Glycans

Much of the physiology of cells is controlled by the spatial organization of the plasma membrane and the glycosylation patterns of its components, however, studying the distribution, size, and composition of these components remains challenging. A bioorthogonal chemical reporter strategy was used for the efficient and specific labeling of membrane‐associated glycoconjugates with modified monosaccharide precursors and organic fluorophores. Super‐resolution fluorescence imaging was used to visualize plasma membrane glycans with single‐molecule sensitivity. Our results demonstrate a homogeneous distribution of N‐acetylmannosamine (ManNAc)‐, N‐acetylgalactosamine (GalNAc)‐, and O‐linked N‐acetylglucosamine (O‐GlcNAc)‐modified plasma membrane proteins in different cell lines with densities of several million glycans on each cell surface.     

Single‐Molecule Methods to Study Membrane Receptor Oligomerization

Membrane receptors control fundamental cellular processes. Binding of a specific ligand to a receptor initiates communication through the membrane and activation of signaling cascades. This activation process often leads to a spatial rearrangement of receptors in the membrane at the molecular level. Single‐molecule techniques contributed significantly to the understanding of receptor organization and rearrangement in membranes. Here, we review four prominent single‐molecule techniques that have been applied to membrane receptors, namely, stepwise photobleaching, Förster resonance energy transfer, sub‐diffraction localization microscopy and co‐tracking. We discuss the requirements, benefits and limitations of each technique, discuss target labeling, present a selection of applications and results and compare the different methodologies.     

Nanographenes: Ultrastable,Switchable, and Bright Probes for Super‐Resolution Microscopy

Super‐resolution fluorescence microscopy has enabled important breakthroughs in biology and materials science. Implementations such as single‐molecule localization microscopy (SMLM) and minimal emission fluxes (MINFLUX) microscopy in the localization mode exploit fluorophores that blink, i.e., switch on and off, stochastically. Here, we introduce nanographenes, namely large polycyclic aromatic hydrocarbons that can also be regarded as atomically precise graphene quantum dots, as a new class of fluorophores for super‐resolution fluorescence microscopy. Nanographenes exhibit outstanding photophysical properties: intrinsic blinking even in air, excellent fluorescence recovery, and stability over several months. As a proof of concept for super‐resolution applications, we use nanographenes in SMLM to generate 3D super‐resolution images of silica nanocracks. Our findings open the door for the widespread application of nanographenes in super‐resolution fluorescence microscopy.