Light sheet fluorescence microscopy (LSFM) uses a thin sheet of light to excite only fluorophores within the focal volume. Light sheet microscopes (LSMs) have a true optical sectioning capability and, hence, provide axial resolution, restrict photobleaching and phototoxicity to a fraction of the sample and use cameras to record tens to thousands of images per second. LSMs are used for in-depth analyses of large, optically cleared samples and long-term three-dimensional (3D) observations of live biological specimens at high spatio-temporal resolution. The independently operated illumination and detection trains and the canonical implementations, selective/single plane illumination microscope (SPIM) and digital scanned laser microscope (DSLM), are the basis for many LSM designs. In this Primer, we discuss various applications of LSFM for imaging multicellular specimens, developing vertebrate and invertebrate embryos, brain and heart function, 3D cell culture models, single cells, tissue sections, plants, organismic interaction and entire cleared brains. Further, we describe the combination of LSFM with other imaging approaches to allow for super-resolution or increased penetration depth and the use of sophisticated spatio-temporal manipulations to allow for observations along multiple directions. Finally, we anticipate developments of the field in the near future. Light sheet fluorescence microscopy (LSFM) is a technique that uses a thin sheet of light for illumination, allowing optical sectioning of the sample. In this Primer, Stelzer et al. outline the fundamental concepts behind LSFM, discuss the different experimental set-ups for light sheet microscopes and detail steps for processing LSFM images. The Primer also describes the range of applications for this technique across the biological sciences and concludes by discussing advances for enhancing imaging depth and resolution.

Light sheet fluorescence microscopy

The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective; (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition; (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. Our instrument achieves a resolution of 450 nm laterally and 2 μm axially over an imaging volume of 3,000 × 800 × 300 μm. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophila egg chamber development, zebrafish whole-brain activity and zebrafish embryonic development - up to nine embryos at a time. 

/pdf/daxi-high-resolution-large-imaging-volume-and-multi-view-2e800g6a.pdf

DaXi—high-resolution, large imaging volume and multi-view single-objective light-sheet microscopy

Light-sheet fluorescence microscopy has transformed our ability to visualize and quantitatively measure biological processes rapidly and over long time periods. In this review, we discuss current and future developments in light-sheet fluorescence microscopy that we expect to further expand its capabilities. This includes smart and adaptive imaging schemes to overcome traditional imaging trade-offs, i.e., spatiotemporal resolution, field of view and sample health. In smart microscopy, a microscope will autonomously decide where, when, what and how to image. We further assess how image restoration techniques provide avenues to overcome these tradeoffs and how "open top" light-sheet microscopes may enable multi-modal imaging with high throughput. As such, we predict that light-sheet microscopy will fulfill an important role in biomedical and clinical imaging in the future. 

/pdf/light-sheets-and-smart-microscopy-an-exciting-future-is-1t2kv05t.pdf

Light-sheets and smart microscopy, an exciting future is dawning

Light microscopy allows observing cellular features and objects with sub-micrometer resolution. As such, light microscopy has been playing a fundamental role in the life sciences for more than a hundred years. Fueled by the availability of mass-produced electronics and hardware, publicly shared documentation and building instructions, open-source software, wide access to rapid prototyping and 3D printing, and the enthusiasm of contributors and users involved, the concept of open microscopy has been gaining incredible momentum, bringing new sophisticated tools to an expanding user base. Here, we will first discuss the ideas behind open science and open microscopy before highlighting recent projects and developments in open microscopy. We argue that the availability of well-designed open hardware and software solutions targeting broad user groups or even non-experts, will increasingly be relevant to cope with the increasing complexity of cutting-edge imaging technologies. We will then extensively discuss the current and future challenges of open microscopy.

Open microscopy in the life sciences: Quo Vadis?.

Light-sheet fluorescence microscopy is a rapidly growing technique that has gained tremendous popularity in the life sciences owing to its high-spatiotemporal resolution and gentle, non-phototoxic illumination. In this protocol, we provide detailed directions for the assembly and operation of a versatile light-sheet fluorescence microscopy variant, referred to as axially swept light-sheet microscopy (ASLM), that delivers an unparalleled combination of field of view, optical resolution and optical sectioning. To democratize ASLM, we provide an overview of its working principle and applications to biological imaging, as well as pragmatic tips for the assembly, alignment and control of its optical systems. Furthermore, we provide detailed part lists and schematics for several variants of ASLM that together can resolve molecular detail in chemically expanded samples, subcellular organization in living cells or the anatomical composition of chemically cleared intact organisms. We also provide software for instrument control and discuss how users can tune imaging parameters to accommodate diverse sample types. Thus, this protocol will serve not only as a guide for both introductory and advanced users adopting ASLM, but as a useful resource for any individual interested in deploying custom imaging technology. We expect that building an ASLM will take ~1-2 months, depending on the experience of the instrument builder and the version of the instrument. 

Isotropic imaging across spatial scales with axially swept light-sheet microscopy

We rapidly determine the 3-D position of a glass-pipette micromanipulator using color-coded illumination and optical microscopy, and demonstrate its potential for autonomous visual servoing and multi-manipulator systems for highly parallelized cell manipulation.

3-D Localization of Micromanipulators Using Microscopy for Autonomous Visual Servoing

/pdf/pycro-manager-open-source-software-for-customized-and-58bug18bl2.pdf

Pycro-Manager: open-source software for customized and reproducible microscope control.

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3-D Localization of Micromanipulators Using Microscopy for Autonomous Visual Servoing

Pycro-Manager: open-source software for customized and reproducible microscope control.