Simultaneous multiple-level magnification selective plane illumination microscopy (sMx-SPIM) imaging system

We report an optical-based (microscopy) imaging technology – Simultaneous Multiple-level Magnification Selective Plane Illumination Microscopy (sMx-SPIM) Imaging System – that addresses a longstanding (technological) challenge of obtaining images, specifically of the biological specimen non-destructively, at different fields of view (FOV) and spatial resolutions (or magnification powers) simultaneously in real-time. Thus, this imaging system provides not only 3D images but also time-resolved sequential images with temporal resolution msecs. Magnification powers (or FOVs) of the individual images can be controlled independently that can be achieved by housing two separate detection arms, in SPIM imaging system, fitted with objective lenses of different magnification powers. These unique features hold promises to observe and study of: (i) sub-microscopic details and entire structure of biological specimen side-by-side simultaneously and (ii) spatio-temporal dynamics of functional activities of biological specimen. For validation study of robustness of the proposed sMx-SPIM imaging system, experiments are conducted in various biological samples (zebrafish embryo, Drosophila melanogaster, and Allium cepa root). Experimental results demonstrate that the study is of significant impacts from two aspects (technological and biological applications).

μSPIM: A Software Platform for Selective Plane Illumination Microscopy

Selective Plane Illumination Microscopy (SPIM) is a fluorescence imaging technique that allows volumetric imaging at high spatio-temporal resolution to monitor neural activity in live organisms such as larval zebrafish. A major challenge in the construction of a custom SPIM microscope is the control and synchronization of the various hardware components. Here we present a control toolset, μSPIM, built around the open-source MicroManager platform that has already been widely adopted for the control of microscopy hardware. Installation of μSPIM is relatively straightforward, involving a single C++ executable and a Java-based extension to Micro-Manager. Imaging protocols are defined through the μSPIM extension to Micro-Manager. The extension then synchronizes the camera shutter with the galvanometer mirrors to create a light-sheet that is scanned in the z-dimension, in synchrony with the imaging objective, to produce volumetric recordings. A key advantage of μSPIM is that a series of calibration procedures optimizes acquisition for a given set-up making it relatively independent of the optical design of the microscope, or the hardware used to build it. Two laser illumination arms can be used while also allowing for the introduction of illumination masks. μSPIM allows imaging of calcium activity throughout the brain of larval zebrafish at rates of 100 planes per second with single cell resolution as well as slower imaging to reconstruct cell populations, for example, in the cleared brains of mice.

Combined Selective Plane Illumination Microscopy and FRAP maps intranuclear diffusion of NLS-GFP

Since its initial development in 1976, fluorescence recovery after photobleaching (FRAP) has been one of the most popular tools for studying diffusion and protein dynamics in living cells. Its popularity is derived from the widespread availability of confocal microscopes and the relative ease of the experiment and analysis. FRAP, however, is limited in its ability to resolve spatial heterogeneity. Here, we combine selective plane illumination microscopy (SPIM) and FRAP to create SPIM-FRAP, wherein we use a sheet of light to bleach a 2D plane and subsequently image the recovery of the same image plane. This provides simultaneous quantification of diffusion or protein recovery for every pixel in a given 2D slice, thus moving FRAP measurements beyond these previous limitations. We demonstrate this technique by mapping intranuclear diffusion of NLS-GFP in live MDA-MB-231 cells; SPIM-FRAP proves to be an order of magnitude faster than fluorescence correlation spectroscopy (FCS) based techniques for such measurements. We observe large length-scale (> ~500 nm) heterogeneity in the recovery times of NLS-GFP, which is validated against simulated data sets. 2D maps of recovery times were correlated with fluorescence images of H2B to address conflicting literature on the role of chromatin in diffusion of small molecules. We observed no correlation between histone density and diffusion. We developed a diffusion simulation for our SPIM-FRAP experiments to compare across techniques; our measured diffusion coefficients are on the order of previously reported results, thus validating the quantitative accuracy of SPIM-FRAP relative to well-established methods. With the recent rise of accessibility of SPIM systems, SPIM-FRAP is set to provide a simple and quick means of quantifying the spatial distribution of protein recovery or diffusion in living cells.Statement of SignificanceWe developed selective plane illumination microscopy combined with fluorescence recovery after photobleaching (SPIM-FRAP) to perform simultaneous FRAP measurements for each pixel in a 2D slice. This technique has the potential to be implemented on almost any light sheet microscope with minimal software development. FRAP studies were previously unable to resolve spatial heterogeneity and FCS 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.

Image quality guided smart rotation improves coverage in microscopy

Fluorescence microscopy is an essential tool for biological discoveries. There is a constant demand for better spatial resolution across a larger field of view. Although strides have been made to improve the theoretical resolution and speed of the optical instruments, in mesoscopic samples, image quality is still largely limited by the optical properties of the sample. In Selective Plane Illumination Microscopy (SPIM), the achievable optical performance is hampered by optical degradations encountered in both the illumination and detection. Multi-view imaging, either through sample rotation or additional optical paths, is a popular strategy to improve sample coverage. In this work, we introduce a smart rotation workflow that utilizes on-the-fly image analysis to identify the optimal light sheet imaging orientations. The smart rotation workflow outperforms the conventional approach without additional hardware and achieves a better sample coverage using the same number of angles or less and thereby reduces data volume and phototoxicity.