Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy.

Rapid, high-resolution volumetric imaging without moving heavy objectives or disturbing delicate samples remains challenging. Pupil-matched remote focusing offers a promising solution for high NA systems, but the fluorescence signal's incoherent and unpolarized nature complicates its application. Thus, remote focusing is mainly used in the illumination arm with polarized laser light to improve optical coupling.

Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy.

The ability to image at high speeds is necessary for biological imaging to capture fast-moving or transient events or to efficiently image large samples. However, due to the lack of rigidity of biological specimens, carrying out fast, high-resolution volumetric imaging without moving and agitating the sample has been a challenging problem. Pupil-matched remote focusing has been promising for high NA imaging systems with their low aberrations and wavelength independence, making it suitable for multicolor imaging.

Multiparatopic antibodies induce targeted downregulation of programmed death-ligand 1.

Programmed death-ligand 1 (PD-L1) drives inhibition of antigen-specific T cell responses through engagement of its receptor programmed death-1 (PD-1) on activated T cells. Overexpression of these immune checkpoint proteins in the tumor microenvironment has motivated the design of targeted antibodies that disrupt this interaction. Despite clinical success of these antibodies, response rates remain low, necessitating novel approaches to enhance performance.

Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase with important roles in many cellular processes as well as in cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. How these dimers relate to higher-order EGFR oligomers seen in cell membranes, however, remains unclear.

Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy.

The ability to image at high speeds is necessary in biological imaging to capture fast-moving or transient events or to efficiently image large samples. However, due to the lack of rigidity of biological specimens, carrying out fast, high-resolution volumetric imaging without moving and agitating the sample has been a challenging problem. Pupil-matched remote focusing has been promising for high NA imaging systems with their low aberrations and wavelength independence, making it suitable for multicolor imaging.

Tolerance to aberration and misalignment in a two-point-resolving image inversion interferometer.

Image inversion interferometry can measure the separation of two incoherent point sources at or near the quantum limit. This technique has the potential to improve upon current state-of-the-art imaging technologies, with applications ranging from microbiology to astronomy. However, unavoidable aberrations and imperfections in real systems may prevent inversion interferometry from providing an advantage for real-world applications.

Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) with important roles in many cellular processes as well as cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. However, it is not clear how these dimers relate to higher-order EGFR oligomers detected at the cell surface.

Automated multi-target super-resolution microscopy with trust regions.

We describe a dedicated microscope for automated sequential localization microscopy which we term Sequential Super-resolution Microscope (SeqSRM). This microscope automates precise stage stabilization on the order of 5-10 nanometers and data acquisition of all user-selected cells on a coverslip, limiting user interaction to only cell selection and buffer exchanges during sequential relabeling. We additionally demonstrate that nanometer-scale changes to cell morphology affect the fidelity of the resulting multi-target super-resolution overlay reconstructions generated by sequential super-resolution microscopy, and that regions affected by these shifts can be reliably detected and masked out using brightfield images collected periodically throughout the experiment.

SMITE: Single Molecule Imaging Toolbox Extraordinaire (MATLAB).

Fluorescence single molecule imaging comprises a variety of techniques that involve detecting individual fluorescent molecules. Many of these techniques involve localizing individual fluorescent molecules with precisions below the diffraction limit, which limits the spatial resolution of (visible) light-based microscopes. These methodologies are widely used to image biological structures at the nanometer scale by fluorescently tagging the structures of interest, elucidating details of the biological behavior observed.

High-precision estimation of emitter positions using Bayesian grouping of localizations.

Single-molecule localization microscopy super-resolution methods rely on stochastic blinking/binding events, which often occur multiple times from each emitter over the course of data acquisition. Typically, the blinking/binding events from each emitter are treated as independent events, without an attempt to assign them to a particular emitter. Here, we describe a Bayesian method of inferring the positions of the tagged molecules by exploring the possible grouping and combination of localizations from multiple blinking/binding events.

Spatiotemporal Clustering of Repeated Super-Resolution Localizations via Linear Assignment Problem.

Many fluorescence super-resolution techniques, such as (d)STORM, PALM, and DNA-PAINT, generate datasets wherein multiple localizations across many camera frames may arise from a single blinking event of an emitter. These repeated localizations not only hinder interpretation and analysis of such datasets, but also represent an incomplete use of the fluorescence photons. Such localizations are typically combined into a single localization either by clustering with hard distance and time thresholds, or by classical hypothesis testing assuming Gaussian localization errors.

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging.

Robust, fiducial-free drift correction for super-resolution imaging.

We describe a robust, fiducial-free method of drift correction for use in single molecule localization-based super-resolution methods. The method combines periodic 3D registration of the sample using brightfield images with a fast post-processing algorithm that corrects residual registration errors and drift between registration events. The method is robust to low numbers of collected localizations, requires no specialized hardware, and provides stability and drift correction for an indefinite time period.

EGFR transactivates RON to drive oncogenic crosstalk.

Crosstalk between different receptor tyrosine kinases (RTKs) is thought to drive oncogenic signaling and allow therapeutic escape. EGFR and RON are two such RTKs from different subfamilies, which engage in crosstalk through unknown mechanisms. We combined high-resolution imaging with biochemical and mutational studies to ask how EGFR and RON communicate.