Lucien E. Weiss

Flexible electrical recording from cells using nanowire transistor arrays

Semiconductor nanowires (NWs) have unique electronic properties and sizes comparable with biological structures involved in cellular communication, thus making them promising nanostructures for establishing active interfaces with biological systems. We report a flexible approach to interface NW field-effect transistors (NWFETs) with cells and demonstrate this for silicon NWFET arrays coupled to embryonic chicken cardiomyocytes. Cardiomyocyte cells were cultured on thin, optically transparent polydimethylsiloxane (PDMS) sheets and then brought into contact with Si-NWFET arrays fabricated on standard substrates. NWFET conductance signals recorded from cardiomyocytes exhibited excellent signal-to-noise ratios with values routinely >5 and signal amplitudes that were tuned by varying device sensitivity through changes in water gate–voltage potential, Vg. Signals recorded from cardiomyocytes for Vg from −0.5 to +0.1 V exhibited amplitude variations from 31 to 7 nS whereas the calibrated voltage remained constant, indicating a robust NWFET/cell interface. In addition, signals recorded as a function of increasing/decreasing displacement of the PDMS/cell support to the device chip showed a reversible >2× increase in signal amplitude (calibrated voltage) from 31 nS (1.0 mV) to 72 nS (2.3 mV). Studies with the displacement close to but below the point of cell disruption yielded calibrated signal amplitudes as large as 10.5 ± 0.2 mV. Last, multiplexed recording of signals from NWFET arrays interfaced to cardiomyocyte monolayers enabled temporal shifts and signal propagation to be determined with good spatial and temporal resolution. Our modular approach simplifies the process of interfacing cardiomyocytes and other cells to high-performance Si-NWFETs, thus increasing the experimental versatility of NWFET arrays and enabling device registration at the subcellular level.

Cellular Inclusion Bodies of Mutant Huntingtin Exon 1 Obscure Small Fibrillar Aggregate Species

The identities of toxic aggregate species in Huntingtons disease pathogenesis remain ambiguous. While polyQ-expanded huntingtin (Htt) is known to accumulate in compact inclusion bodies inside neurons, this is widely thought to be a protective coping response that sequesters misfolded conformations or aggregated states of the mutated protein. To define the spatial distributions of fluorescently-labeled Htt-exon1 species in the cell model PC12m, we employed highly sensitive single-molecule super-resolution fluorescence imaging. In addition to inclusion bodies and the diffuse pool of monomers and oligomers, fibrillar aggregates ~100 nm in diameter and up to ~1–2 µm in length were observed for pathogenic polyQ tracts (46 and 97 repeats) after targeted photo-bleaching of the inclusion bodies. These short structures bear a striking resemblance to fibers described in vitro. Definition of the diverse Htt structures in cells will provide an avenue to link the impact of therapeutic agents to aggregate populations and morphologies.

Precise Three-Dimensional Scan-Free Multiple-Particle Tracking over Large Axial Ranges with Tetrapod Point Spread Functions

We employ a novel framework for information-optimal microscopy to design a family of point spread functions (PSFs), the Tetrapod PSFs, which enable high-precision localization of nanoscale emitters in three dimensions over customizable axial (z) ranges of up to 20 μm with a high numerical aperture objective lens. To illustrate, we perform flow profiling in a microfluidic channel and show scan-free tracking of single quantum-dot-labeled phospholipid molecules on the surface of living, thick mammalian cells.

Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1

Significance In vertebrates, the transmembrane protein Smoothened (Smo) accumulates in the ciliary membrane when cells receive the Hedgehog (Hh) signal. The presence of Smo in primary cilia at baseline conditions has been postulated, but not directly observed. We used highly sensitive single-molecule imaging in live cells to track and analyze the dynamics of individual Smo molecules in cilia, not only after treatment with pathway agonists but also at low, baseline levels. In both conditions, Smo molecules bind at distinct sites at the bases of cilia, but with different dissociation constants. The results provide mechanistic insight into the Hh signal transduction and highlight the distinct compartmentalization of Smo behavior within cilia, which is normally masked by the bulk distribution in ensemble measurements. Accumulation of the signaling protein Smoothened (Smo) in the membrane of primary cilia is an essential step in Hedgehog (Hh) signal transduction, yet the molecular mechanisms of Smo movement and localization are poorly understood. Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation.

Delayed emergence of subdiffraction-sized mutant huntingtin fibrils following inclusion body formation.

Aberrant aggregation of improperly folded proteins is the hallmark of several human neurodegenerative disorders, including Huntington’s Disease (HD) with autosomal-dominant inheritance. In HD, expansion of the CAG-repeat-encoded polyglutamine (polyQ) stretch beyond ~40 glutamines in huntingtin (Htt) and its N-terminal fragments leads to the formation of large (up to several μm) globular neuronal inclusion bodies (IBs) over time. We report direct observations of aggregating Htt exon 1 in living and fixed cells at enhanced spatial resolution by stimulated emission depletion (STED) microscopy and single-molecule super-resolution optical imaging. Fibrils of Htt exon 1 arise abundantly across the cytosolic compartment and also in neuritic processes only after nucleation and aggregation into a fairly advanced stage of growth of the prominent IB have taken place. Structural characterizations of fibrils by STED show a distinct length cutoff at ~1·5 μm and reveal subsequent coalescence (bundling/piling). Cytosolic fibrils are observed even at late stages in the process, side-by-side with the mature IB. Htt sequestration into the IB, which in neurons has been argued to be a cell-protective phenomenon, thus appears to saturate and over-power the cellular degradation systems and leaves cells vulnerable to further aggregation producing much smaller, potentially toxic, conformational protein species of which the fibrils may be comprised. We further found that exogenous delivery of the apical domain of the chaperonin subunit CCT1 to the cells via the cell medium reduced the aggregation propensity of mutant Htt exon 1 in general, and strongly reduced the occurrence of such late-stage fibrils in particular.

Multicolour localization microscopy by point-spread-function engineering

Super-resolution microscopy has revolutionized cellular imaging in recent years1-4. Methods relying on sequential localization of single point emitters enable spatial tracking at ~10-40 nm resolution. Moreover, tracking and imaging in three dimensions is made possible by various techniques, including point-spread-function (PSF) engineering5-9 -namely, encoding the axial (z) position of a point source in the shape that it creates in the image plane. However, a remaining challenge for localization-microscopy is efficient multicolour imaging - a task of the utmost importance for contextualizing biological data. Normally, multicolour imaging requires sequential imaging10, 11, multiple cameras12, or segmented dedicated fields of view13, 14. Here, we demonstrate an alternate strategy, the encoding of spectral information (colour), in addition to 3D position, directly in the image. By exploiting chromatic dispersion, we design a new class of optical phase masks that simultaneously yield controllably different PSFs for different wavelengths, enabling simultaneous multicolour tracking or super-resolution imaging in a single optical path.

Deep-STORM: super-resolution single-molecule microscopy by deep learning

We present an ultra-fast, precise, parameter-free method, which we term Deep-STORM, for obtaining super-resolution images from stochastically-blinking emitters, such as fluorescent molecules used for localization microscopy. Deep-STORM uses a deep convolutional neural network that can be trained on simulated data or experimental measurements, both of which are demonstrated. The method achieves state-of-the-art resolution under challenging signal-to-noise conditions and high emitter densities, and is significantly faster than existing approaches. Additionally, no prior information on the shape of the underlying structure is required, making the method applicable to any blinking data-set. We validate our approach by super-resolution image reconstruction of simulated and experimentally obtained data.

Robust hypothesis tests for detecting statistical evidence of two-dimensional and three-dimensional interactions in single-molecule measurements.

Experimental advances have improved the two- (2D) and three-dimensional (3D) spatial resolution that can be extracted from in vivo single-molecule measurements. This enables researchers to quantitatively infer the magnitude and directionality of forces experienced by biomolecules in their native environment. Situations where such force information is relevant range from mitosis to directed transport of protein cargo along cytoskeletal structures. Models commonly applied to quantify single-molecule dynamics assume that effective forces and velocity in the x,y (or x,y,z) directions are statistically independent, but this assumption is physically unrealistic in many situations. We present a hypothesis testing approach capable of determining if there is evidence of statistical dependence between positional coordinates in experimentally measured trajectories; if the hypothesis of independence between spatial coordinates is rejected, then a new model accounting for 2D (3D) interactions can and should be considered. Our hypothesis testing technique is robust, meaning it can detect interactions, even if the noise statistics are not well captured by the model. The approach is demonstrated on control simulations and on experimental data (directed transport of intraflagellar transport protein 88 homolog in the primary cilium).

Revealing the nanoscale morphology of the primary cilium using super-resolution fluorescence microscopy

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 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 (SMO), 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 (PSR) 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, 2-color, 2D STimulated Emission Depletion (STED) 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 SMO-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 STED to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest.

Observing DNA in live cells

The structural organization and dynamics of DNA are known to be of paramount importance in countless cellular processes, but capturing these events poses a unique challenge. Fluorescence microscopy is well suited for these live-cell investigations, but requires attaching fluorescent labels to the species under investigation. Over the past several decades, a suite of techniques have been developed for labeling and imaging DNA, each with various advantages and drawbacks. Here, we provide an overview of the labeling and imaging tools currently available for visualizing DNA in live cells, and discuss their suitability for various applications.