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

Abstract

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.

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

“…We constructed an axisymmetric FEA model featuring a stiff, spherical, polystyrene indenter and an ellipsoidal nucleus ( Figure 5A and Supplemental Movie S3). Previous research has examined FEA models of AFM ( Chen and Lu, 2012 ; Liu et al. , 2019 ), yet to our knowledge none have examined the relationship of nuclear morphology and force.…”

“…The intricate details of our AFM-LS system, both regarding the optical design and integration of the atomic force microscope, are described in our previous work ( Liu et al. , 2019 ; Nelsen et al.…”

“…A vertical light sheet propagates out of the objective lens and an electrically tunable lens (ETL) was used to ensure the waist of the light sheet was in the cell. A second ETL is used in the detection path to dynamically adjust focus without moving the objective lens ( Liu et al. , 2019 ).…”

Nuclear Deformation Causes DNA Damage by Increasing Replication Stress

“…To further validate that deformation of the nucleus is sufficient to cause DNA damage, we compressed parts of individual nuclei approximately to a height of 2 mm using an atomic force microscope (AFM) cantilever with a spherical tip (6 mm in diameter) while monitoring nuclear deformation and formation of DNA damage on a light-sheet (LS) microscope ( Figure 2E). The AFM-LS system allows for high-resolution, 3D imaging of the whole nucleus throughout the compression application, [39][40][41] thereby enabling us to observe the spatiotemporal dynamics of DNA damage (Video S4). Experiments in which the spherical tip was brought in contact with the cell without inducing nuclear compression served as controls (Video S4).…”

Nuclear deformation causes DNA damage by increasing replication stress

“…2E). The AFM-LS system allows for high-resolution, 3-D imaging of the whole nucleus throughout the compression application (Gavara, 2017;Liu et al, 2019;Nelsen et al, 2020), thereby enabling us to observe the spatio-temporal dynamics of DNA damage (Suppl. Video 4).…”