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 fast-steering 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.
Highlights
Fluorescence microscopy (FM) is one of most powerful techniques in biological and biomedical research
We have demonstrated the first microscope design capable of vaTIRF, point-scanning, and Light sheet fluorescence microscopy (LSFM), the first localized photoactivation combined with vaTIRF, and the first single-objecive LSFM imaging of orthogonal planes of a single cell nucleus
Optical systems are often designed around a single microscopy technique and even though such systems have drastically advanced our ability to investigate single cell dynamics, the focus on individual techniques can limit the scope of available research questions
Summary
Fluorescence microscopy (FM) is one of most powerful techniques in biological and biomedical research. LSFM requires two objective lenses, one for illumination and one for detection, placed at a 90-degree angle relative to each other such that the light sheet from the illumination source coincides with the imaging plane of the detection objective [14,15,16,17,18,19,20,21] Such systems can be less user-friendly than conventional microscopes. Other groups have previously developed multi-modality microscopes that combine two separate super resolution methods [26], multiple light sheets [27], or epifluorescence and TIRF [28,29,30] These systems are not adaptable to all the illumination techniques we have outlined. The microscope is operated with an open-source software package (available upon request) to calibrate and control the system, which is independent of the microscope automation software
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