Perspectives on label-free microscopy of heterogeneous and dynamic biological systems.

Significance: Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging). Aim: This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs. Approach: Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended. Conclusion: Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.

Griddient: a microfluidic array to generate reconfigurable gradients on-demand for spatial biology applications.

Biological tissues are highly organized structures where spatial-temporal gradients (e.g., nutrients, hypoxia, cytokines) modulate multiple physiological and pathological processes including inflammation, tissue regeneration, embryogenesis, and cancer progression. Current in vitro technologies struggle to capture the complexity of these transient microenvironmental gradients, do not provide dynamic control over the gradient profile, are complex and poorly suited for high throughput applications. Therefore, we have designed Griddent, a user-friendly platform with the capability of generating controllable and reversible gradients in a 3D microenvironment. Our platform consists of an array of 32 microfluidic chambers connected to a 384 well-array through a diffusion port at the bottom of each reservoir well. The diffusion ports are optimized to ensure gradient stability and facilitate manual micropipette loading. This platform is compatible with molecular and functional spatial biology as well as optical and fluorescence microscopy. In this work, we have used this platform to study cancer progression.

Metabolic changes to host cells with Toxoplasma gondii infection.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular parasite that infects warm-blooded vertebrates across the world. In humans, seropositivity rates of T. gondii range from 10% to 90%. Despite its prevalence, few studies address how T. gondii infection changes the metabolism of host cells. Here, we investigate how T. gondii manipulates the host cell metabolic environment by monitoring metabolic response over time using non-invasive autofluorescence lifetime imaging of single cells, seahorse metabolic flux analysis, reactive oxygen species (ROS) production, and metabolomics. Autofluorescence lifetime imaging indicates that infected host cells become more oxidized and have an increased proportion of bound NAD(P)H with infection. These findings are consistent with changes in mitochondrial and glycolytic function, decrease of intracellular glucose, fluctuations in lactate and ROS production in infected cells over time. We also examined changes associated with the pre-invasion "kiss and spit" process using autofluorescence lifetime imaging, which similarly showed a more oxidized host cell with an increased proportion of bound NAD(P)H over 48 hours. Glucose metabolic flux analysis indicated that these changes are driven by NADH and NADP+ in T. gondii infection. In sum, metabolic changes in host cells with T. gondii infection were similar during full infection, and kiss and spit. Autofluorescence lifetime imaging can non-invasively monitor metabolic changes in host cells over a microbial infection time-course.

Autofluorescence lifetime imaging classifies human lymphocyte activation and subtype.

New non-destructive tools are needed to reliably assess lymphocyte function for immune profiling and adoptive cell therapy. Optical metabolic imaging (OMI) is a label-free method that measures the autofluorescence intensity and lifetime of metabolic cofactors NAD(P)H and FAD to quantify metabolism at a single-cell level. Here, we investigate whether OMI can resolve metabolic changes between human quiescent versus IL4/CD40 activated B cells and IL12/IL15/IL18 activated memory-like NK cells. We found that quiescent B and NK cells were more oxidized compared to activated cells. Additionally, the NAD(P)H mean fluorescence lifetime decreased and the fraction of unbound NAD(P)H increased in the activated B and NK cells compared to quiescent cells. Machine learning classified B cells and NK cells according to activation state (CD69+) based on OMI parameters with up to 93.4% and 92.6% accuracy, respectively. Leveraging our previously published OMI data from activated and quiescent T cells, we found that the NAD(P)H mean fluorescence lifetime increased in NK cells compared to T cells, and further increased in B cells compared to NK cells. Random forest models based on OMI classified lymphocytes according to subtype (B, NK, T cell) with 97.8% accuracy, and according to activation state (quiescent or activated) and subtype (B, NK, T cell) with 90.0% accuracy. Our results show that autofluorescence lifetime imaging can accurately assess lymphocyte activation and subtype in a label-free, non-destructive manner.

Void sorcerer: an open source, open access framework for mouse uroflowmetry.

Observational and experimental studies of rodent voiding behaviors have greatly contributed to our understanding of lower urinary tract function including the complex social, environmental, and internal stimuli that affect voiding in health and models of disease. Void spot assays (VSA), cystometry (awake or anesthetized), and uroflowmetry are techniques commonly used in rodent models to assess voiding. Uroflowmetry is non-invasive and can be performed multiple times in the same freely moving animals and can be used to generate synchronized video corresponding to each void to characterize micturition patterns (e.g., droplets versus solid stream). However, approaches to evaluate uroflowmetry in rodent models vary widely across laboratories. Most importantly, an open access software to run these tests is not freely available (although complete systems are commercially available), limiting use of this important assay. We developed the Void Sorcerer, an uroflowmetry system for mice for reliable determination of frequency, voided volume, voiding duration, interval times between micturitions, and flow rate. This report provides a detailed description of how to build this system and includes open access software for developing uroflowmetry capability in their laboratories and improve upon it in a cost-effective manner. Our goals are to improve access, increase reproducibility among laboratories, and facilitate standardizing testing procedures.