Publications & technical resources

Narrow escape from confinement through a nanochannel is the critical step of complex transport processes including size-exclusion-based separations, oil and gas extraction from the microporous subsurface environment, and ribonucleic acid translocation through nuclear pore complex channels. While narrow escape has been studied using theoretical and computational methods, experimental quantification is rare because of the difficulty in confining a particle into a microscopic space through a nanoscale hole. Here, we studied narrow escape in the context of continuous nanoparticle diffusion within the liquid-filled void space of an ordered porous material. Specifically, we quantified the spatial dependence of nanoparticle motion and the sojourn times of individual particles in the interconnected confined cavities of a liquid-filled inverse opal film. We found that nanoparticle motion was inhibited near cavity walls and cavity escape was slower than predicted by existing theories and random-walk simulations. A combined computational-experimental analysis indicated that translocation through a nanochannel is barrier controlled rather than diffusion controlled.

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing-analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10–20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale organization on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to-mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology.

Protrusions are plasma membrane extensions that are found in almost every cell in the human body. Cancer cell filopodial and lamellipodial protrusions play key roles in the integral processes of cell motility and signaling underlying tumor invasion and metastasis. HER2 (ErbB-2) is overexpressed in diverse types of tumors and regulates PI3K-pathway-mediated protrusion growth. It is known that HER2 resides at breast cancer cell protrusions, but how protrusion-based HER2 spatiotemporal dynamics shape cancer signaling is unclear. Here, we study how HER2 location and motion regulate protrusion signaling and growth using quantitative spatio-temporal molecular imaging approaches. Our data highlight morphologically-segregated features of filopodial and lamellipodial protrusions, in in vitro 2D breast cancer cells and in vivo intact breast tumor. Functional-segregation parallels morphological-segregation, as HER2 and its activated downstream pAKT-PI3K signaling remain spatially-localized at protrusions, provoking new protrusion growth proximal to sites of HER2 activation. HER2 in SKBR3 breast cancer cell filopodia exhibits fast, linearly-directed motion that is distinct from lamellipodia and non-protrusion subcellular regions (~3-4 times greater diffusion constant, rapid speeds of 2-3 um^2 per s). Surprisingly, filopodial HER2 motion is passive, requiring no active energy sources. Moreover, while HER2 motion in lamellipodia and non-protrusion regions show hindered diffusion typical of membrane proteins, HER2 diffuses freely within filopodia. We conclude that HER2 activation, propagation, and functional protrusion growth is a local process in which filopodia have evolved to exploit Brownian thermal fluctuations within a barrier-free nanostructure to transduce rapid signaling. These results support the importance of developing filopodia and other protrusion-targeted strategies for cancer.

Development of single-molecule localization microscopy (SMLM) has sparked a revolution in biological imaging, allowing “super-resolution” fluorescence microscopy below the diffraction limit of light. The past decade has seen an explosion in not only optical hardware for SMLM but also the development or repurposing of fluorescent proteins and small-molecule fluorescent probes for this technique. In this review, written by chemists for chemists, we detail the history of single-molecule localization microscopy and collate the collection of probes with demonstrated utility in SMLM. We hope it will serve as a primer for probe choice in localization microscopy as well as an inspiration for the development of new fluorophores that enable imaging of biological samples with exquisite detail.

We describe a new concept of multimodal super-resolution imaging which combines the cumulant analysis from Super-resolution Optical Fluctuation Imaging (SOFI) with the imprinting of three-dimensional, spectral or other information into peculiar Point-Spread Function (PSF) patterns. This concept allows for encoding multidimensional or multimodal information into a single image plane and to extract this information by an appropriate spatio-temporal correlation analysis of emitter fluctuations. Here, we develop the general theory of this concept, and present proof-of-principle experiments of three-dimensional super-resolution imaging.

We present two-photon fluorescence image scanning microscopy (ISM) with engineered excitation and detection point-spread-functions enabling 3D imaging in a single 2D scan. This demonstration combines excitation using a holographic multispot array of focused femtosecond pulses with a high-efficiency single-helix PSF phase mask detection. Camera detection along with a multiview reconstruction algorithm allows volumetric imaging of biological samples over a depth of field spanning more than 1500 nm with an axial resolution of better than 400 nm. The nonlinear two-photon process improves sectioning and the inherent longer wavelengths increase the penetration depth in scattering samples. Our method extends the performance of 3D ISM towards thicker biological samples.

Metasurfaces are two-dimensional arrangements of antennas that control the propagation of electromagnetic waves with a subwavelength thickness and resolution. Previously, metasurfaces have been mostly used to obtain the function of a single optical element. Here, we demonstrate a plasmonic metasurface that represents the combination of a phase mask generating a double-helix point spread function (DH-PSF) and a metalens for imaging. DH-PSF has been widely studied in three-dimensional (3D) super-resolution imaging, biomedical imaging, and particle tracking, but the current DH-PSFs are inefficient, bulky, and difficult to integrate. The multielement metasurface, which we label as DH-metalens, enables a DH-PSF with transfer efficiency up to 70.3% and an ultrahigh level of optical system integration, three orders of magnitude smaller than those realized by conventional phase elements. Moreover, the demonstrated DH-metalens can work in broadband visible wavelengths and in multiple incident polarization states. Finally, we demonstrate the application of the DH-metalens in 3D imaging of point sources. These results pave ways for realizing integrated DH-PSFs, which have applications in 3D super-resolution microscopy, single particle tracking/imaging, and machine vision.

Super-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of ∼250 nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (∼2–6 μm long and 200–300 nm in diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened, with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10–25 nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, two-color, two-dimensional stimulated emission depletion microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the Smoothened-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses stimulated emission depletion to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest.