Vertical Light Sheet Enhanced Side-View Imaging for AFM Cell Mechanics Studies

Beicker, Kellie N.; Falvo, Michael R.; Superfine, R.

doi:10.1038/s41598-018-19791-3

Cited by 40 publications

(42 citation statements)

References 53 publications

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“…Using a custom MATLAB code, force-indentation data were fit to the Hertzian contact mechanics model to determine elastic moduli (stiffness) for each cell (Beicker et al, 2018; Cribb et al, 2016). The force versus indentation data were fit up to indentations of 750nm which corresponded to less than 10% of the cell height.…”

Section: Methodsmentioning

confidence: 99%

Microtubule Acetylation Is Required for Mechanosensation in Drosophila

et al. 2018

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SUMMARY At the cellular level, α-tubulin acetylation alters the structure of microtubules to render them mechanically resistant to compressive forces. How this biochemical property of microtubule acetylation relates to mechanosensation remains unknown, although prior studies have shown that microtubule acetylation influences touch perception. Here, we identify the major Drosophila α-tubulin acetylase (dTAT) and show that it plays key roles in several forms of mechanosensation. dTAT is highly expressed in the larval peripheral nervous system (PNS), but it is largely dispensable for neuronal morphogenesis. Mutation of the acetylase gene or the K40 acetylation site in α-tubulin impairs mechanical sensitivity in sensory neurons and behavioral responses to gentle touch, harsh touch, gravity, and vibration stimuli, but not noxious thermal stimulus. Finally, we show that dTAT is required for mechanically induced activation of NOMPC, a microtubule-associated transient receptor potential channel, and functions to maintain integrity of the microtubule cytoskeleton in response to mechanical stimulation.

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Section: Methodsmentioning

confidence: 99%

Microtubule Acetylation Is Required for Mechanosensation in Drosophila

et al. 2018

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“…Our combined atomic force microscope and side-view light sheet fluorescence microscope ( Figure 1A) allows visualization of the dynamics of cellular deformations in the plane of applied force while simultaneously monitoring the force response of the cell . We have previously used this tool to show the existence of separate elastic moduli correlated with whole-cell and nuclear deformations (Beicker et al 2018). We built on this previous work by studying the dynamics of nuclear morphology and the correlated force response under compression by AFM.…”

Section: Combined Afm and Side-view Light Sheet Microscopy Show Straimentioning

confidence: 99%

Correlating nuclear morphology and external force with combined atomic force microscopy and light sheet imaging separates roles of chromatin and lamin A/C in nuclear mechanics

Hobson

Kern

Stephens

et al. 2020

Preprint

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Nuclei are constantly under external stressbe it during migration through tight constrictions or compressive pressure by the actin capand the mechanical properties of nuclei govern their subsequent deformations. Both altered mechanical properties of nuclei and abnormal nuclear morphologies are hallmarks of a variety of disease states. Little work, however, has been done to link specific changes in nuclear shape to external forces. Here, we utilize a combined atomic force microscope and light sheet microscope (AFM-LS) to show SKOV3 nuclei exhibit a two-regime force response that correlates with changes in nuclear volume and surface area, allowing us to develop an empirical model of nuclear deformation. Our technique further decouples the roles of chromatin and lamin A/C in compression, showing they separately resist changes in nuclear volume and surface area respectively; this insight was not previously accessible by Hertzian analysis. A two-material finite element model supports our conclusions. We also observed that chromatin decompaction leads to lower nuclear curvature under compression, which is important for maintaining nuclear compartmentalization and function. The demonstrated link between specific types of nuclear morphological change and applied force will allow researchers to better understand the stress on nuclei throughout various biological processes.

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“…5). For both methods we introduced a small (180 µm) right-angle reflective prism (8531-607-1, Precision Optics) attached to a 6 degree-offreedom mount, and lowered the mirror adjacent to the cell of interest [25]. For HLS imaging, the LBS reflected off the mirror and illuminated a single x-y plane.…”

Section: Horizontal and Vertical Lsfm Of Highlights Organization Of Hmentioning

confidence: 99%

“…Transitioning from HLS to VLS is straightforward, fast, and can be done entirely through software. It is important to note that resolution is not affected when the imaging plane is rotated by the mirror [25]. Because of LSFM's ability to illuminate a single plane, we obtained images with low background and high signal-to-noise of orthogonal planes of the same nucleus.…”

Section: Horizontal and Vertical Lsfm Of Highlights Organization Of Hmentioning

confidence: 99%

“…immersion objectives are almost precluded from LSFM applications due to the geometry configurations and the physical dimensions of the objectives. Both our research group and others have implemented LSFM in a single-objective system [22][23][24][25]; here we present how we can combine single-objective LSFM into a microscope design that allows for other FM techniques.…”

Section: Introductionmentioning

confidence: 99%

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VIEW-MOD: A Versatile Illumination Engine With a Modular Optical Design for Fluorescence Microscopy

Liu

Hobson

Pimenta

et al. 2019

Preprint

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We developed VIEW-MOD (Versatile Illumination Engine With a Modular Optical Design): a compact, multi-modality microscope, which accommodates multiple illumination schemes including variable angle total internal reflection, point scanning and vertical/horizontal light sheet. This system allows combining and flexibly switching between different illuminations and imaging modes by employing three electrically tunable lenses and two faststeering mirrors. This versatile optics design provides control of 6 degrees of freedom of the illumination source (3 translation, 2 tilt, and beam shape) plus the axial position of the imaging plane. We also developed standalone software with an easy-to-use GUI to calibrate and control the microscope. We demonstrate the applications of this system and software in biosensor imaging, optogenetics and fast 3D volume imaging. This system is ready to fit into complex imaging circumstances requiring precise control of illumination and detection paths, and has a broad scope of usability for a myriad of biological applications. the detection pathway. Through using some or all of the modules available, our system can implement a variety of imaging modalities. Here, we outline the theory and purpose of each module, with the reader directed to Figure 1 for the optical layout.

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Vertical Light Sheet Enhanced Side-View Imaging for AFM Cell Mechanics Studies

Cited by 40 publications

References 53 publications

Microtubule Acetylation Is Required for Mechanosensation in Drosophila

Microtubule Acetylation Is Required for Mechanosensation in Drosophila

Correlating nuclear morphology and external force with combined atomic force microscopy and light sheet imaging separates roles of chromatin and lamin A/C in nuclear mechanics

VIEW-MOD: A Versatile Illumination Engine With a Modular Optical Design for Fluorescence Microscopy

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