Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination.

A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 µm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to ∼0.3 µm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.

Compartmentalized Cytoplasmic Flows Direct Protein Transport to the Cell's Leading Edge.

Inside the cell, proteins essential for signaling, morphogenesis, and migration navigate complex pathways, typically via vesicular trafficking or microtubule-driven mechanisms 1-3 . However, the process by which soluble cytoskeletal monomers maneuver through the cytoplasm's ever-changing environment to reach their destinations without using these pathways remains unknown. 4-6 Here, we show that actin cytoskeletal treadmilling leads to the formation of a semi-permeable actin-myosin barrier, creating a specialized compartment separated from the rest of the cell body that directs proteins toward the cell edge by advection, diffusion facilitated by fluid flow. Contraction at this barrier generates a molecularly non-specific fluid flow that transports actin, actin-binding proteins, adhesion proteins, and even inert proteins forward. The local curvature of the barrier specifically targets these proteins toward protruding edges of the leading edge, sites of new filament growth, effectively coordinating protein distribution with cellular dynamics. Outside this compartment, diffusion remains the primary mode of protein transport, contrasting sharply with the directed advection within. This discovery reveals a novel protein transport mechanism that redefines the front of the cell as a pseudo-organelle, actively orchestrating protein mobilization for cellular front activities such as protrusion and adhesion. By elucidating a new model of protein dynamics at the cellular front, this work contributes a critical piece to the puzzle of how cells adapt their internal structures for targeted and rapid response to extracellular cues. The findings challenge the current understanding of intracellular transport, suggesting that cells possess highly specialized and previously unrecognized organizational strategies for managing protein distribution efficiently, providing a new framework for understanding the cellular architecture's role in rapid response and adaptation to environmental changes.

Coumarin as a general switching auxiliary to prepare photochromic and spontaneously blinking fluorophores.

Single-molecule localization microscopy (SMLM) uses activatable or switchable fluorophores to create non-diffraction limited maps of molecular location in biological samples. Despite the utility of this imaging technique, the portfolio of appropriate labels for SMLM remains limited. Here, we describe a general strategy for the construction of "glitter bomb" labels by simply combining rhodamine and coumarin dyes though an amide bond. Condensation of the ortho-carboxyl group on the pendant phenyl ring of rhodamine dyes with a 7-aminocoumarin yields photochromic or spontaneously blinking fluorophores depending on the parent rhodamine structure. We apply this strategy to prepare labels useful super-resolution experiments in fixed cells using different attachment techniques. This general glitter bomb strategy should lead to improved labels for SMLM, ultimately enabling the creation of detailed molecular maps in biological samples.

Using single molecule imaging to explore intracellular heterogeneity.

Despite more than 100 years of study, it is unclear if the movement of proteins inside the cell is best described as a mosh pit or an exquisitely choreographed dance. Recent studies suggest the latter. Local interactions induce molecular condensates such as liquid-liquid phase separations (LLPSs) or non-liquid, functionally significant molecular aggregates, including synaptic densities, nucleoli, and Amyloid fibrils. Molecular condensates trigger intracellular signaling and drive processes ranging from gene expression to cell division. However, the descriptions of condensates tend to be qualitative and correlative. Here, we indicate how single-molecule imaging and analyses can be applied to quantify condensates. We discuss the pros and cons of different techniques for measuring differences between transient molecular behaviors inside and outside condensates. Finally, we offer suggestions for how imaging and analyses from different time and space regimes can be combined to identify molecular behaviors indicative of condensates within the dynamic high-density intracellular environment.

Using Single Molecule Imaging to Explore Intracellular Heterogeneity.

Despite more than 100 years of study, it is unclear if the movement of proteins inside the cell is best described as a mosh pit or an exquisitely choreographed dance. Recent studies suggest the latter. Local interactions induce molecular condensates such as liquid-liquid phase separations (LLPSs) or non-liquid, functionally significant molecular aggregates, including synaptic densities, nucleoli, and Amyloid fibrils. Molecular condensates trigger intracellular signaling and drive processes ranging from gene expression to cell division. However, the descriptions of condensates tend to be qualitative and correlative. Here, we indicate how single-molecule imaging and analyses can be applied to quantify condensates. We discuss the pros and cons of different techniques for measuring differences between transient molecular behaviors inside and outside condensates. Finally, we offer suggestions for how imaging and analyses from different time and space regimes can be combined to identify molecular behaviors indicative of condensates within the dynamic high-density intracellular environment.

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics.

We demonstrate live-cell super-resolution imaging using photoactivated localization microscopy (PALM). The use of photon-tolerant cell lines in combination with the high resolution and molecular sensitivity of PALM permitted us to investigate the nanoscale dynamics within individual adhesion complexes (ACs) in living cells under physiological conditions for as long as 25 min, with half of the time spent collecting the PALM images at spatial resolutions down to approximately 60 nm and frame rates as short as 25 s. We visualized the formation of ACs and measured the fractional gain and loss of individual paxillin molecules as each AC evolved. By allowing observation of a wide variety of nanoscale dynamics, live-cell PALM provides insights into molecular assembly during the initiation, maturation and dissolution of cellular processes.

Organization of the core structure of the postsynaptic density.

Much is known about the composition and function of the postsynaptic density (PSD), but less is known about its molecular organization. We use EM tomography to delineate the organization of PSDs at glutamatergic synapses in rat hippocampal cultures. The core of the PSD is dominated by vertically oriented filaments, and ImmunoGold labeling shows that PSD-95 is a component of these filaments. Vertical filaments contact two types of transmembrane structures whose sizes and positions match those of glutamate receptors and intermesh with two types of horizontally oriented filaments lying 10-20 nm from the postsynaptic membrane. The longer horizontal filaments link adjacent NMDAR-type structures, whereas the smaller filaments link both NMDA- and AMPAR-type structures. The orthogonal, interlinked scaffold of filaments at the core of the PSD provides a structural basis for understanding dynamic aspects of postsynaptic function.

Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes.

Accurate determination of the relative positions of proteins within localized regions of the cell is essential for understanding their biological function. Although fluorescent fusion proteins are targeted with molecular precision, the position of these genetically expressed reporters is usually known only to the resolution of conventional optics ( approximately 200 nm). Here, we report the use of two-color photoactivated localization microscopy (PALM) to determine the ultrastructural relationship between different proteins fused to spectrally distinct photoactivatable fluorescent proteins (PA-FPs). The nonperturbative incorporation of these endogenous tags facilitates an imaging resolution in whole, fixed cells of approximately 20-30 nm at acquisition times of 5-30 min. We apply the technique to image different pairs of proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. For several pairs, we find that proteins that seem colocalized when viewed by conventional optics are resolved as distinct interlocking nano-aggregates when imaged via PALM. The simplicity, minimal invasiveness, resolution, and speed of the technique all suggest its potential to directly visualize molecular interactions within cellular structures at the nanometer scale.

Coupling integrin dynamics to cellular adhesion behaviors.

Visualizing fluorescent proteins is essential for understanding cellular function. While advances in microscopy can now resolve individual molecules, determining whether the labeled molecules report native behaviors and how the measured behaviors can be coupled to cellular outputs remains challenging. Here, we used integrin alpha-beta heterodimers - which connect extracellular matrix (ECM) and the cytoskeleton - to quantify the mobility and conformation of labeled integrins. We found that while unlabeled and labeled integrins all localized to adhesions and support anchorage-dependent cell function, integrin mobility decreased when the beta rather than the alpha subunit was labeled. In contrast to unlabeled and alpha labeled subunits, beta labeled subunits changed cellular behavior; decreasing protrusive activity and increasing adhesion size and the extent of cell spreading. Labeling the beta subunit changed the integrin conformation, extending the molecule and exposing an epitope that is revealed by activation with Mn2+ treatment. Our findings indicate labeling induced changes in dynamic integrin behavior alter molecular conformation as well as cellular adhesion-dependent function to demonstrate a coupling between molecular inputs and distinct cellular outputs.This article has an associated First Person interview with the first author of the paper.

Controlled damage in thick specimens by multiphoton excitation.

Controlled damage by light energy has been a valuable tool in studies of cell function. Here, we show that the Ti:Sapphire laser in a multiphoton microscope can be used to cause localized damage within unlabeled cells or tissues at greater depths than previously possible. We show that the damage is due to a multiphoton process and made wounds as small as 1 microm in diameter 20 microm from the surface. A characteristic fluorescent scar allows monitoring of the damage and identifies the wound site in later observations. We were able to lesion a single axon within a bundle of nerves, locally interrupt organelle transport within one axon, cut dendrites in a zebrafish embryo, ablate a mitotic pole in a sea urchin egg, and wound the plasma membrane and nuclear envelope in starfish oocytes. The starfish nucleus collapsed approximately 1 h after wounding, indicating that loss of compartmentation barrier makes the structure unstable; surprisingly, the oocyte still completed meiotic divisions when exposed to maturation hormone, indicating that the compartmentalization and translocation of cdk1 and its regulators is not required for this process. Multiphoton excitation provides a new means for producing controlled damage deep within tissues or living organisms.

Changes in single-molecule integrin dynamics linked to local cellular behavior.

Recent advances in light microscopy permit visualization of the behavior of individual molecules within dense macromolecular ensembles in live cells. It is now conceptually possible to relate the dynamic organization of molecular machinery to cellular function. However, inherent heterogeneities, as well as disparities between spatial and temporal scales, pose substantial challenges in deriving such a relationship. New approaches are required to link discrete single-molecule behavior with continuous cellular-level processes. Here we combined intercalated molecular and cellular imaging with a computational framework to detect reproducible transient changes in the behavior of individual molecules that are linked to cellular behaviors. Applying our approach to integrin transmembrane receptors revealed a spatial density gradient underlying characteristic molecular density increases and mobility decreases, indicating the subsequent onset of local protrusive activity. Integrin mutants further revealed that these density and mobility transients are separable and depend on different binding domains within the integrin cytoplasmic tail. Our approach provides a generalizable paradigm for dissecting dynamic spatiotemporal molecular behaviors linked to local cellular events.

Super-resolution microscopy for nanosensing.

The recent advances in optical microscopy enable the simultaneous visualization of thousands of structural and signaling molecules as they dynamically rearrange within living cells. Super-resolution microscopy offers an unprecedented opportunity to define the molecular mechanisms of nanosensing through direct observation of protein movement. This technology provides a real-time readout of how genetically targeted molecular perturbations affect protein interactions. As we strive to meet the challenge offered by the opportunity to ask questions about the mechanism of cell that we never thought we could answer, we need to be aware that the new technologies are still evolving. The current limitations of each technique need to be considered when matching them to specific biological questions. In this review, we briefly describe the principles of super-resolution optical microscopy and focus on comparing the characteristics of each technique that are important for their use in studying nanosensing in the cellular microenvironment.

Polymerizing actin fibers position integrins primed to probe for adhesion sites.

Migrating cells extend protrusions, probing the surrounding matrix in search of permissive sites to form adhesions. We found that actin fibers polymerizing along the leading edge directed local protrusions and drove synchronous sideways movement of beta1 integrin adhesion receptors. These movements lead to the clustering and positioning of conformationally activated, but unligated, beta1 integrins along the leading edge of fibroblast lamellae and growth cone filopodia. Thus, rapid actin-based movement of primed integrins along the leading edge suggests a "sticky fingers" mechanism to probe for new adhesion sites and to direct migration.

Distribution of the scaffolding proteins PSD-95, PSD-93, and SAP97 in isolated PSDs.

We compared the distribution of three scaffolding proteins, all belonging to a family of membrane-associated guanylate kinases, thought to have key roles in the organization of the postsynaptic density (PSD). Isolated PSDs readily adhered to treated glass coverslips where they were labeled with immunogold and rotary shadowed for analysis by EM. The distribution of proteins within individual PSDs were measured by counting and mapping individual immunogold particles. PSD-95, as previously described, is distributed evenly throughout the PSD. We find here that PSD-93 has a nearly identical distribution suggesting that PSD-95 and PSD-93 could perform similar roles. SAP97, in contrast, is concentrated near edges of cleft sides of the PSDs, and in small clumps on their cytoplasmic sides. The homogenous distribution of PSD-95 and PSD-93 throughout the PSD is consistent with their being part of a backbone that stabilizes their various binding partners within the PSD. The distribution of SAP97 confirms that this protein is actually an integral component of the PSD, and suggests that it may have a role in inserting or stabilizing its main binding partner, Glu-R1, at the edge of the PSD.