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Phenotype based screening is a powerful tool to evaluate cellular drug response. Through high content fluorescence imaging of simple fluorescent labels and complex image analysis phenotypic measurements can identify subtle compound-induced cellular changes unique to compound mechanisms of action (MoA). Recently, a screen of 1008 compounds in three cell lines was reported where analysis detected changes in cellular phenotypes and accurately identified compound MoA for roughly half the compounds. However, we were surprised that DNA alkylating agents and other compounds known to induce or impact the DNA damage response produced no measured activity in cells with fluorescently labeled 53BP1—a canonical DNA damage marker. We hypothesized that phenotype analysis is not sensitive enough to detect small changes in 53BP1 distribution and analyzed the screen images with autocorrelation image analysis. We found that autocorrelation analysis, which quantifies fluorescently-labeled protein clustering, identified higher compound activity for compounds and MoAs known to impact the DNA damage response, suggesting altered 53BP1 recruitment to damaged DNA sites. We then performed experiments under more ideal imaging settings and found autocorrelation analysis to be a robust measure of changes to 53BP1 clustering in the DNA damage response. These results demonstrate the capacity of autocorrelation to detect otherwise undetectable compound activity and suggest that autocorrelation analysis of specific proteins could serve as a powerful screening tool.

Involvement of the immune system in tumour progression is at the forefront of cancer research. Analysis of the tumour immune microenvironment has yielded a wealth of information on tumour biology, and alterations in some immune subtypes, such as tumour-associated macrophages (TAM), can be strong prognostic indicators. Here, we use optical tissue clearing and a TAM-targeting injectable fluorescent nanoparticle (NP) to examine three-dimensional TAM composition, tumour-to-tumour heterogeneity, response to colony-stimulating factor 1 receptor (CSF-1R) blockade and nanoparticle-based drug delivery in murine pulmonary carcinoma. The method allows for rapid tumour volume assessment and spatial information on TAM infiltration at the cellular level in entire lungs. This method reveals that TAM density was heterogeneous across tumours in the same animal, overall TAM density is different among separate pulmonary tumour models, nanotherapeutic drug delivery correlated with TAM heterogeneity, and successful response to CSF-1R blockade is characterized by enhanced TAM penetration throughout and within tumours. Tumour-associated macrophages (TAM) can be used as prognostic indicators in cancer. Here, the authors establish a platform for high-throughput 3D microscopy in murine lung carcinoma that allows to visualize TAMs infiltration throughout the entire lung, response to CSF-1R blockade and nanoparticle drug delivery.

Regulation of sodium flux across the cell membrane plays a vital role in the generation of action potentials and regulation of membrane excitability in cells such as cardiomyocytes and neurons. Alteration of sodium channel function has been implicated in diseases such as epilepsy, long QT syndrome, and heart failure. However, single cell imaging of sodium dynamics has been limited due to the narrow selection of fluorescent sodium indicators available to researchers. Here we report, the detection of spatially defined sodium activity during action potentials. Fluorescent nanosensors that measure sodium in real-time, are reversible and are completely selective over other cations such as potassium that were used to image sodium. The use of the nanosensors in vitro was validated by determining drug-induced activation in heterologous cells transfected with the voltage-gated sodium channel Na v 1.7. Spatial information of sodium concentrations during action potentials will provide insight at the cellular level on the role of sodium and how slight changes in sodium channel function can affect the entirety of an action potential.

There have been a variety of nanoparticles created for in vivo uses ranging from gene and drug delivery to tumor imaging and physiological monitoring. The use of nanoparticles to measure physiological conditions while being fluorescently addressed through the skin provides an ideal method toward minimally invasive health monitoring. Here we create unique particles that have all the necessary physical characteristics to serve as in vivo reporters, but with minimized diffusion from the point of injection. These particles, called microworms, have a cylindrical shape coated with a biocompatible porous membrane that possesses a large surface-area-to-volume ratio while maintaining a large hydrodynamic radius. We use these microworms to create fluorescent sodium sensors for use as in vivo sodium concentration detectors after subcutaneous injection. However, the microworm concept has the potential to extend to the immobilization of other types of polymers for continuous physiological detection or delivery of molecules.

The use of fluorescence-polarized microscopy, combined with competitive binding with matched fluorescence companion imaging probes, enable target engagement measurements of covalent and reversible small molecule inhibitors in a single cell. Quantitation of drug target engagement in single cells has proven to be difficult, often leaving unanswered questions in the drug development process. We found that intracellular target engagement of unlabeled new therapeutics can be quantitated using polarized microscopy combined with competitive binding of matched fluorescent companion imaging probes. We quantitated the dynamics of target engagement of covalent BTK inhibitors, as well as reversible PARP inhibitors, in populations of single cells using a single companion imaging probe for each target. We then determined average in vivo tumor concentrations and found marked population heterogeneity following systemic delivery, revealing single cells with low target occupancy at high average target engagement in vivo .

In this article, we present a differential confocal microscopy technique based on pinhole engineering that significantly enhances both lateral resolution and axial sectioning depth. Through simulations and experimental validation with a fluorescent calibration slide, we demonstrated a 1.6-fold improvement in lateral resolution and a two-fold increase in axial sectioning capability. Building on these advancements, we integrated an intensity-weighted lifetime imaging strategy to surpass the diffraction limit in fluorescence lifetime measurements. This approach achieved high spatial resolution and quantitative lifetime data comparable to leading super-resolution FLIM (Fluorescence Lifetime Imaging Microscopy) techniques, yet allows for simpler implementation. We further validated the method in subcellular structure lifetime imaging, demonstrating improved resolution and axial depth enhanced lifetime-based multiplexing capacity. This new method provides an accessible route to high-resolution, multiplexed FLIM for advanced biological imaging.

This work introduces a polymer-free optode nanosensor for ratiometric sodium imaging. Transmembrane ion dynamics are often captured by electrophysiology and calcium imaging, but sodium dyes suffer from short excitation wavelengths and poor selectivity. Optodes, optical sensors composed of a polymer matrix with embedded sensing chemistry, have been translated into nanosensors that selectively image ion concentrations. Polymer-free nanosensors were fabricated by emulsification and were stable by diameter and sensitivity for at least one week. Ratiometric fluorescent measurements demonstrated that the nanosensors are selective for sodium over potassium by ~1.4 orders of magnitude, have a dynamic range centered at 20 mM and are fully reversible. The ratiometric signal changes by 70% between 10 and 100 mM sodium, showing that they are sensitive to changes in sodium concentration. These nanosensors will provide a new tool for sensitive and quantitative ion imaging.

Switching laser mode (SLAM) microscopy is a promising method for achieving super resolution while maintaining compatibility with two photon imaging at depth and . SLAM microscopes typically employ multiple paths for generating the requisite spot-like and donut-like beams; however, having two paths necessitates sub-wavelength-scale alignment which is prone to differential drift, causing degradation of the image quality. Here we demonstrate a single aperture, inline SLAM microscope which makes use of one phase element and polarization switching to generate colinear radially polarized and azimuthally polarized vector beams, which focus to a spot and a donut, respectively. By tailoring the spatial profile of the electric field at the back aperture of the microscope objective, we ensure that the resolution of the spot-like beam is comparable to conventional Gaussian beam imaging. Through subtraction of the two images, we demonstrate a 1.5× narrower focal spot and a resolution of ∼0.28 corresponding to ∼290 nm. Accordingly, this method is of great utility for imaging with sub-diffraction-limited resolution at depth in living tissue.