Leavesley Silas J, Johnson Santina, Paudel Sunita S, Knighten Jennifer, Tambe Dhananjay T, Francis Michael, Gong Na, Taylor Mark S, Rich Thomas C
Department of Chemical and Biomolecular Engineering.
Department of Pharmacology.
Proc SPIE Int Soc Opt Eng. 2023 Jan-Feb;12383. doi: 10.1117/12.2650653. Epub 2023 Mar 15.
Second messenger signals, e.g., Ca 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, Ca 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 Ca 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.
第二信使信号，如钙离子和环核苷酸，协调着广泛的细胞事件。第二信使信号决定特定生理反应的方式很复杂。最近的研究指出了时间和空间编码在决定信号特异性方面的重要性。研究还表明机械刺激、底物硬度和机械反应——“机械体”——在调节生理过程中的重要性。因此，需要能够探测化学和机械信号的方法。在此，我们报告了初步的工作，即将用于第二信使信号测量的高光谱成像、用于机械力测量的单层应力显微镜以及用于量化局部信号的S8分析软件相结合——具体而言，可以测量人气道平滑肌细胞（HASMCs）中的钙离子动态和机械力。将HASMCs制备成在11 kPa凝胶上的汇合单层，凝胶中嵌入荧光微粒作为基准标记以及较小的微粒以测量变形（应变）。使用定制的激发扫描高光谱显微镜进行成像。对高光谱图像进行解混以识别来自细胞荧光标记（如CAL 590-AM）和荧光微粒的信号。通过单层应力显微镜分析图像以量化局部力动态。使用S8软件识别、跟踪和量化空间局部的钙离子活性。结果表明，可以在细胞群体中对局部和瞬时的细胞信号及力进行量化和映射。重要的是，这些结果建立了一种同时探究细胞信号和机械力的方法，这些信号和力可能在调节汇合单层中的下游细胞生理过程中发挥协同作用。这项工作得到了美国国立卫生研究院（NIH）P01HL066299、R01HL137030、R01HL058506以及美国国家科学基金会（NSF）MRI1725937的支持。Leavesley博士和Rich博士披露了在一家大学初创公司SpectraCyte LLC中拥有经济利益，该公司旨在将光谱成像技术商业化。
