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.
Keywords: Ca2+; airway smooth muscle; fluorescence; image cytometry; microscopy; second messengers; spectroscopy.