Extending optical chemical tools and technologies to mice by shifting to the shortwave infrared region.

Fluorescence imaging is an indispensable method for studying biological processes non-invasively in cells and transparent organisms. Extension into the shortwave infrared (SWIR, 1000-2000 nm) region of the electromagnetic spectrum has allowed for imaging in mammals with unprecedented depth and resolution for optical imaging. In this review, we summarize recent advances in imaging technologies, dye scaffold modifications, and incorporation of these dyes into probes for SWIR imaging in mice. Finally, we offer an outlook on the future of SWIR detection in the field of chemical biology.

Multiwell Combinatorial Hydrogel Array for High-Throughput Analysis of Cell-ECM Interactions.

Biophysical cues in the extracellular matrix (ECM) regulate cell behavior in a complex, nonlinear, and interdependent manner. To quantify these important regulatory relationships and gain a comprehensive understanding of mechanotransduction, there is a need for high-throughput matrix platforms that enable parallel culture and analysis of cells in various matrix conditions. Here we describe a multiwell hyaluronic acid (HA) platform in which cells are cultured on combinatorial arrays of hydrogels spanning a range of elasticities and adhesivities. Our strategy utilizes orthogonal photopatterning of stiffness and adhesivity gradients, with the stiffness gradient implemented by a programmable light illumination system. The resulting platform allows individual treatment and analysis of each matrix environment while eliminating contributions of haptotaxis and durotaxis. In human mesenchymal stem cells, our platform recapitulates expected relationships between matrix stiffness, adhesivity, and cell mechanosensing. We further applied the platform to show that as integrin ligand density falls, cell adhesion and migration depend more strongly on CD44-mediated interactions with the HA backbone. We anticipate that our system could bear great value for mechanistic discovery and screening where matrix mechanics and adhesivity are expected to influence phenotype.

Shortwave infrared polymethine fluorophores matched to excitation lasers enable non-invasive, multicolour in vivo imaging in real time.

High-resolution, multiplexed experiments are a staple in cellular imaging. Analogous experiments in animals are challenging, however, due to substantial scattering and autofluorescence in tissue at visible (350-700 nm) and near-infrared (700-1,000 nm) wavelengths. Here, we enable real-time, non-invasive multicolour imaging experiments in animals through the design of optical contrast agents for the shortwave infrared (SWIR, 1,000-2,000 nm) region and complementary advances in imaging technologies. We developed tunable, SWIR-emissive flavylium polymethine dyes and established relationships between structure and photophysical properties for this class of bright SWIR contrast agents. In parallel, we designed an imaging system with variable near-infrared/SWIR excitation and single-channel detection, facilitating video-rate multicolour SWIR imaging for optically guided surgery and imaging of awake and moving mice with multiplexed detection. Optimized dyes matched to 980 nm and 1,064 nm lasers, combined with the clinically approved indocyanine green, enabled real-time, three-colour imaging with high temporal and spatial resolutions.

The response of Escherichia coli to the alkylating agents chloroacetaldehyde and styrene oxide.

DNA damage is ubiquitous and can arise from endogenous or exogenous sources. DNA-damaging alkylating agents are present in environmental toxicants as well as in cancer chemotherapy drugs and are a constant threat, which can lead to mutations or cell death. All organisms have multiple DNA repair and DNA damage tolerance pathways to resist the potentially negative effects of exposure to alkylating agents. In bacteria, many of the genes in these pathways are regulated as part of the SOS reponse or the adaptive response. In this work, we probed the cellular responses to the alkylating agents chloroacetaldehyde (CAA), which is a metabolite of 1,2-dichloroethane used to produce polyvinyl chloride, and styrene oxide (SO), a major metabolite of styrene used in the production of polystyrene and other polymers. Vinyl chloride and styrene are produced on an industrial scale of billions of kilograms annually and thus have a high potential for environmental exposure. To identify stress response genes in E. coli that are responsible for tolerance to the reactive metabolites CAA and SO, we used libraries of transcriptional reporters and gene deletion strains. In response to both alkylating agents, genes associated with several different stress pathways were upregulated, including protein, membrane, and oxidative stress, as well as DNA damage. E. coli strains lacking genes involved in base excision repair and nucleotide excision repair were sensitive to SO, whereas strains lacking recA and the SOS gene ybfE were sensitive to both alkylating agents tested. This work indicates the varied systems involved in cellular responses to alkylating agents, and highlights the specific DNA repair genes involved in the responses.