High-speed fluorescence excitation-scanning hyperspectral imaging microscopy using thin-film tunable filters.

Hyperspectral imaging (HSI) technologies have enabled a range of experimental techniques and studies in the fluorescence microscopy field. Unfortunately, a drawback of many HSI microscope platforms is increased acquisition time required to collect images across many spectral bands, as well as signal loss due to the need to filter or disperse emitted fluorescence into many discrete bands. We have previously demonstrated that an alternative approach of scanning the fluorescence excitation spectrum can greatly improve system efficiency by decreasing light losses associated with emission filtering. Our initial system was configured using an array of thin-film tunable filters (TFTFs, VersaChrome, Semrock) mounted in a tiltable filter wheel (VF-5, Sutter) that required ~150-200 ms to switch between wavelengths. Here, we present a new configuration for high-speed switching of TFTFs to allow rapid time-lapse HSI microscopy. A TFTF array was mounted in a custom holder that was attached to a piezoelectric rotation mount (ThorLabs), allowing high-speed rotation. Switching between adjacent filters was achieved using the internal optics of a DG-4 lightsource (Sutter Instrument), including a pair of off-axis parabolic mirrors and galvanometers. Output light was coupled to a liquid lightguide and into an inverted widefield fluorescence microscope (TI-2, Nikon Instruments). Initial tests indicate that the HSI system provides a 15-20 nm bandwidth tunable excitation band and ~10-20 ms wavelength switch time, allowing for high-speed HSI imaging of dynamic cellular events. This work was supported by NIH P01HL066299, R01HL169522, NIH TL1TR003106, and NSF MRI1725937.

Hyperspectral imaging and dynamic region of interest tracking approaches to quantify localized cAMP signals.

Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger known to orchestrate a myriad of cellular functions over a wide range of timescales. In the last 20 years, a variety of single-cell sensors have been developed to measure second messenger signals including cAMP, Ca2+, and the balance of kinase and phosphatase activities. These sensors utilize changes in fluorescence emission of an individual fluorophore or Förster resonance energy transfer (FRET) to detect changes in second messenger concentration. cAMP and kinase activity reporter probes have provided powerful tools for the study of localized signals. Studies relying on these and related probes have the potential to further revolutionize our understanding of G protein-coupled receptor signaling systems. Unfortunately, investigators have not been able to take full advantage of the potential of these probes due to the limited signal-to-noise ratio of the probes and the limited ability of standard epifluorescence and confocal microscope systems to simultaneously measure the distributions of multiple signals (e.g. cAMP, Ca2+, and changes in kinase activities) in real time. In this review, we focus on recently implemented strategies to overcome these limitations: hyperspectral imaging and adaptive thresholding approaches to track dynamic regions of interest (ROI). This combination of approaches increases signal-to-noise ratio and contrast, and allows identification of localized signals throughout cells. These in turn lead to the identification and quantification of intracellular signals with higher effective resolution. Hyperspectral imaging and dynamic ROI tracking approaches offer investigators additional tools with which to visualize and quantify multiplexed intracellular signaling systems.

Combined hyperspectral imaging, monolayer stress microscopy, and S8 image analysis approaches for simultaneously interrogating cellular signals and biomechanics.

Second messenger signals, e.g., Ca2+ and cyclic nucleotides, orchestrate a wide range of cellular events. The methods by which second messenger signals determine specific physiological responses are complex. Recent studies point to the importance of temporal and spatial encoding in determining signal specificity. Studies also indicate the importance of mechanical stimuli, substrate stiffness, and mechanical responses - the "mechanosome" - in regulating physiology. Hence, approaches that probe both chemical and mechanical signals are needed. Here, we report preliminary efforts to combine hyperspectral imaging for second messenger signal measurements, monolayer stress microscopy for mechanical force measurements, and S8 analysis software for quantifying localized signals - specifically, Ca2+ dynamics and mechanical forces in human airway smooth muscle cells (HASMCs). HASMCs were prepared as confluent monolayers on 11 kPa gels with embedded fluorescent microparticles that serve as fiducial markers as well as smaller microparticles to measure deformation (strain). Imaging was performed using a custom excitation-scanning hyperspectral microscope. Hyperspectral images were unmixed to identify signals from cellular fluorescent labels (e.g., CAL 590-AM) and fluorescent microparticles. Images were analyzed to quantify localized force dynamics through monolayer stress microscopy. S8 software was used to identify, track, and quantify spatially-localized Ca2+ activity. Results indicate that localized and transient cellular signals and forces can be quantified and mapped within cell populations. Importantly, these results establish a method for simultaneous interrogation of cellular signals and mechanical forces that may play synergistic roles in regulating downstream cellular physiology in confluent monolayers. This work was supported by NIH P01HL066299, R01HL137030, R01HL058506, and NSF MRI1725937. Drs. Leavesley and Rich disclose financial interest in a university start-up company, SpectraCyte LLC, to commercialize spectral imaging technologies.

Measurement of agonist-induced Ca2+ signals in human airway smooth muscle cells using excitation scan-based hyperspectral imaging and image analysis approaches.

Ca2+ and cAMP are ubiquitous second messengers known to differentially regulate a variety of cellular functions over a wide range of timescales. Studies from a variety of groups support the hypothesis that these signals can be localized to discrete locations within cells, and that this subcellular localization is a critical component of signaling specificity. However, to date, it has been difficult to track second messenger signals at multiple locations. To overcome this limitation, we utilized excitation scan-based hyperspectral imaging approaches to track second messenger signals as well as labeled cellular structures and/or proteins in the same cell. We have previously reported that hyperspectral imaging techniques improve the signal-to-noise ratios of both fluorescence measurements, and are thus well suited for the measurement of localized Ca2+ signals. We investigated the spatial spread and intensities of agonist-induced Ca2+ signals in primary human airway smooth muscle cells (HASMCs) using the Ca2+ indicator Cal520. We measured responses triggered by three agonists, carbachol, histamine, and chloroquine. We utilized custom software coded in MATLAB and Python to assess agonist induced changes in Ca2+ levels. Software algorithms removed the background and applied correction coefficients to spectral data prior to linear unmixing, spatial and temporal filtering, adaptive thresholding, and automated region of interest (ROI) detection. All three agonists triggered transient Ca2+ responses that were spatially and temporally complex. We are currently analyzing differences in both ROI area and intensity distributions triggered by these agonists. This work was supported by NIH awards P01HL066299, K25HL136869, and R01HL137030 and NSF award MRI1725937.

Novel Hyperspectral imaging approaches allow 3D measurement of cAMP signals in localized subcellular domains of human airway smooth muscle cells.

Studies of the cAMP signaling pathway have led to the hypothesis that localized cAMP signals regulate distinct cellular responses. Much of this work focused on measurement of localized cAMP signals using cAMP sensors based upon FÓ§rster resonance energy transfer (FRET). FRET-based probes are comprised of a cAMP binding domain sandwiched between donor and acceptor fluorophores. Binding of cAMP triggers a conformational change which alters FRET efficiency. In order to study localized cAMP signals, investigators have targeted FRET probes to distinct subcellular domains. This approach allows detection of cAMP signals at distinct subcellular locations. However, these approaches do not measure localized cAMP signals per se, rather they measure cAMP signals at specific locations and typically averaged throughout the cell. To address these concerns, our group implemented hyperspectral imaging approaches for measuring highly multiplexed signals in cells and tissues. We have combined these approaches with custom analysis software implemented in MATLAB and Python. Images were filtered both spatially and temporally, prior to adaptive thresholding (OTSU) to detect cAMP signals. These approaches were used to interrogate the distributions of isoproterenol and prostaglandin-triggered cAMP signals in human airway smooth muscle cells (HASMCs). Results demonstrate that cAMP signals are spatially and temporally complex. We observed that isoproterenol- and prostaglandin-induced cAMP signals are triggered at the plasma membrane and in the cytosolic space. We are currently implementing analysis approaches to better quantify and visualize the complex distributions of cAMP signals. This work was supported by NIH P01HL066299, R01HL058506, and S10RR027535.

Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification.

A variety of FRET probes have been developed to examine cAMP localization and dynamics in single cells. These probes offer a readily accessible approach to measure localized cAMP signals. However, given the low signal-to-noise ratio of most FRET probes and the dynamic nature of the intracellular environment, there have been marked limitations in the ability to use FRET probes to study localized signaling events within the same cell. Here, we outline a methodology to dissect kinetics of cAMP-mediated FRET signals in single cells using automated image analysis approaches. We additionally extend these approaches to the analysis of subcellular regions. These approaches offer a unique opportunity to assess localized cAMP kinetics in an unbiased, quantitative fashion.

Endothelial metabolism in pulmonary vascular homeostasis and acute respiratory distress syndrome.

Capillary endothelial cells possess a specialized metabolism necessary to adapt to the unique alveolar-capillary environment. Here, we highlight how endothelial metabolism preserves the integrity of the pulmonary circulation by controlling vascular permeability, defending against oxidative stress, facilitating rapid migration and angiogenesis in response to injury, and regulating the epigenetic landscape of endothelial cells. Recent reports on single-cell RNA-sequencing reveal subpopulations of pulmonary capillary endothelial cells with distinctive reparative capacities, which potentially offer new insight into their metabolic signature. Lastly, we discuss broad implications of pulmonary vascular metabolism on acute respiratory distress syndrome, touching on emerging findings of endotheliitis in coronavirus disease 2019 (COVID-19) lungs.