Ms. Sunita S. Paudel Profile

Sunita S. Paudel

at Univ of South Alabama

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Publications (2)

Proceedings Article | 15 March 2023 Presentation + Paper

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

Silas Leavesley, Santina Johnson, Sunita Paudel, Jennifer Knighten, Dhananjay Tambe, Michael Francis, Na Gong, Mark Taylor, Thomas Rich

Proceedings Volume 12383, 123830D (2023) https://doi.org/10.1117/12.2650653

KEYWORDS: Hyperspectral imaging, Image analysis, Calcium, Monolayers, Microscopy, Hydrogels, Microspheres, Image visualization, Visualization, Statistical analysis, Fluorescence, Signaling molecules

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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.

Proceedings Article | 15 March 2023 Presentation

An open-source toolkit to visualize cellular morphology, motion, fluorescence, and forces (Conference Presentation)

Dhananjay Tambe, Sunita Paudel, Jessica Bell, Linn Ayers, Thomas Rich, Troy Stevens

Proceedings Volume PC12381, PC123810E (2023) https://doi.org/10.1117/12.2651173

KEYWORDS: Visualization, Luminescence, Visual analytics, Image acquisition, Image analysis, Vascular diseases, Tissue engineering, Signal analyzers, Optical microscopy, Microscopy

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