VIEW-MOD: a versatile illumination engine with a modular optical design for fluorescence microscopy

Kern

et al. 2020

Sci Rep

the central goals of mechanobiology are to understand how cells generate force and how they respond to environmental mechanical stimuli. A full picture of these processes requires high-resolution, volumetric imaging with time-correlated force measurements. Here we present an instrument that combines an open-top, single-objective light sheet fluorescence microscope with an atomic force microscope (AfM), providing simultaneous volumetric imaging with high spatiotemporal resolution and high dynamic range force capability (10 pN-100 nN). With this system we have captured lysosome trafficking, vimentin nuclear caging, and actin dynamics on the order of one second per single-cell volume. to showcase the unique advantages of combining Line Bessel light sheet imaging with AfM, we measured the forces exerted by a macrophage during fcɣR-mediated phagocytosis while performing both sequential two-color, fixed plane and volumetric imaging of F-actin. This unique instrument allows for a myriad of novel studies investigating the coupling of cellular dynamics and mechanical forces. Cells interact mechanically with their environment by generating and responding to forces. A focus on the mechanical dynamics of cell phenomena such as motility, division and phagocytosis is essential for understanding stem cell fate 1 , cancer progression 2 and innate immunity 3. These mechanical processes are inherently three-dimensional (3D) and are regulated both by very local (nm) interactions as well as whole-cell scale (μm) biochemical and mechanical signaling. Obtaining a more complete picture of a cell's mechanical interaction with its environment requires monitoring local and global structures in 3D while simultaneously measuring associated forces. These goals necessitate integrating high spatial and temporal resolution volumetric imaging methods with high resolution force acquisition. Light sheet florescence microscopy (LSFM) enables acquisition of volumetric, multicolor time series at a high resolution and frame rate with low background fluorescence and low phototoxicity 4-6. Among single cell force methods 7 , Atomic Force Microscopy (AFM) 8 is unique in combining a large force range (10-11-10-6 N) that enables molecular-scale to tissue-level mechanics measurements, with high bandwidth temporal resolution (μs) and sub-nanometer spatial control. LSFM provides sufficient spatiotemporal resolution for studying cell-wide protein specific dynamics that AFM imaging cannot. Therefore, by using the AFM for force spectroscopy and LSFM for imaging we have exploited the strengths of each technique resulting in a system that is greater than its parts. Combining LSFM with AFM (AFM-LS) imposes significant geometrical constraints to the optical system design, and demands low vibration operation to accommodate sensitive force measurements. To address this challenge, we used a single-objective selective plane microscopy (soSPIM) technique integrated with, and time-synchronized to, an AFM. As described in a report on our first generation system 9 , we gene...

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Combined Atomic Force Microscope and Volumetric Light Sheet System for Correlative Force and Fluorescence Mechanobiology Studies

Nelsen

Kern

et al. 2020

Sci Rep

“…We find this is an acceptable tradeoff given our systems other advantages. These issues can be fixed by employing horizontal light sheet illumination by reflecting the light sheet off the prism, with the imaging focus of the objective at the plane of the horizontal light sheet 23 .…”

Section: Discussionmentioning

confidence: 99%

“…Volume scanning during phagocytosis. Our protocol for initialization of volume scanning is described in our prior report 23 . Otherwise, the set up for the volume imaging of phagocytosis experiment was similar to the fixed light sheet.…”

Section: Fixed Light Sheet Phagocytosis Experimentmentioning

confidence: 99%

See 1 more Smart Citation

Combined Atomic Force Microscope and Volumetric Light Sheet System for Mechanobiology

Nelsen

Kern

et al. 2019

Preprint

The central goals of mechanobiology are to understand how cells generate force and how they respond to environmental mechanical stimuli. A full picture of these processes requires high-resolution, volumetric imaging with time-correlated force measurements. Here we present an instrument that combines an open-top, single-objective light sheet fluorescence microscope with an atomic force microscope (AFM), providing simultaneous volumetric imaging with high spatiotemporal resolution and high dynamic range force capability (10 pN – 100 nN). With this system we have captured lysosome trafficking, vimentin nuclear caging, and actin dynamics on the order of one second per volume. To showcase the unique advantages of combining Line Bessel light sheet imaging with AFM, we measured the forces exerted by a macrophage during FcɣR-mediated phagocytosis while performing both sequential two-color, fixed plane and volumetric imaging of F-actin. This unique instrument allows for a myriad of novel studies investigating the coupling of cellular dynamics and mechanical forces.

Combined Selective Plane Illumination Microscopy and FRAP Maps Intranuclear Diffusion of NLS-GFP

Falvo

Superfine

2020

Biophysical Journal

Self Cite

doi: bioRxiv preprint techniques require minute-long acquisition times; SPIM-FRAP remedies both of these issues by generating FRAP-based diffusion maps in 4 seconds. This technique can easily be expanded to 3D by photobleaching a single plane and performing light sheet volumetric imaging, which has the benefits of minimal photobleaching and phototoxicity for studying long-term protein turnover. Furthermore, SPIM-FRAP of slowly-recovering structures enables characterization of spatial distortions to measure intracellular stresses.