imaging set-up and volumetric scans for cytoskeleton network visualization

Customizable Fluorescence Microscope to Visualize Cytoskeleton Networks

Light-sheet fluorescence microscopy (LSFM) represents a family of high-resolution fluorescence imaging techniques in which the excitation light is shaped into a sheet1,2, including selective plane illumination microscopy (SPIM), swept confocally-aligned planar excitation (SCAPE), and oblique-plane microscopy (OPM)3,4,5,6,7. Unlike other microscopy modalities such as epi-fluorescence, total internal reflection fluorescence microscopy (TIRFM), or confocal microscopy, phototoxicity is minimal in LSFM and samples can be imaged over longer timescales because only the plane of the sample being actively imaged is illuminated8,9,10. Therefore, LSFM techniques are extremely useful for imaging 3D samples over extended time periods, notably even those too thick for confocal microscopy techniques. Due to these reasons, since its original development in 2004, LSFM has become the imaging technique of choice for many physiologists, developmental biologists, and neuroscientists for the visualization of entire organisms such as live zebrafish and Drosophila embryos3,4,6,11. In these last two decades, the advantages of LSFM have been leveraged to visualize structure and dynamics at progressively smaller scales, including the tissue11,12, cellular, and subcellular scales, both in vivo and in vitro13,14,15,17.
Despite the reports of successful use cases in the literature, the high cost of commercial LSFM systems (~0.25 million USD as of the time of writing)18,19 prevents the widespread use of the technique. To make DIY builds a feasible alternative for researchers, multiple build guides have been published8,13,20,21, including the open-access effort OpenSPIM22. However, to date, researchers with minimal optics experience can only use earlier LSFM designs, which are incompatible with traditional slide-mounted samples (Figure 1A). Recent single-objective, light-sheet (SOLS) implementation use a single objective for both the excitation and detection (Figure 1C), thereby overcoming the limitation related to compatibility5,6,8,13,20. However, the cost for the versatility of the SOLS design is a substantial increase in the complexity of the build due to the requirement of two additional objectives to relay, de-tilt, and reimage the object plane onto the camera for imaging (Figure 1D). To facilitate access to the complex SOLS-style setups, this paper presents step-by-step guide on the design, build, alignment process, and use of a slide-compatible SOLS system, which would be useful to researchers with knowledge of only an entry-level optics course.
Although the protocol itself is succinct, readers must refer to other resources during the preparation steps to learn more about particular parts of the design or hardware considerations. However, if a reader intends to follow the specifications of this design, it may not be necessary to understand how to select particular optical components.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65411/65411fig01.jpg" /> 
Figure 1: Characteristics of different LSFM configurations. (A) The setup with two orthogonal objectives common in early LSFM designs. In this configuration, a capillary tube or a cylinder of gel is used to contain the sample, which is not compatible with traditional slide mounting techniques. (B) A schematic of an SOLS light-sheet design showing the following: (C) the single objective used for both the excitation and emission collection at the sample plane (O1); this allows a traditional slide to be mounted on top, and (D) the relay objective system in the SOLS emission path. O2 collects the emission light and demagnifies the image. O3 images the plane at the correct tilt angle onto the camera sensor. Abbreviations: LSFM = light-sheet fluorescence microscopy; SOLS = single-objective light-sheet; O1-O3 = objectives. <a href="https://www.jove.com/files/ftp_upload/65411/65411fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Advancing Knowledge in Far-From-Equilibrium Materials Through Light-Sheet Microscopy

Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks

Our work aims to advance knowledge on the relationship between trans work dynamics, local structure, and mechanical properties in far-from-equilibrium materials. Visualizing far-from-equilibrium materials such as cytoskeleton networks requires an ability to image dense, three-dimensional samples with comparable image quality throughout the volume of the sample, while causing minimal damage via photobleaching, a technically challenging task that fluorescence light-sheet microscopy is best suited for. Fluorescence light-sheet microscope systems provide excellent optical sectioning capabilities, which allows for imaging of these thick, three-dimensional samples for long time scales with minimal photobleaching.In particular, the single objective light-sheet system we will present is compatible with traditional slide-mounted samples, making it a very versatile tool. The overwhelming majority of single objective light-sheet guides are written for users with extensive optics experience. To make the complex single objective light-sheet style setup more accessible, this detailed guide provides steps that a user with an entry-level optics course depth of knowledge can follow.

Imaging Set-Up and Volumetric Scans for Cytoskeleton Network Visualization

imaging-set-up-volumetric-scans-for-cytoskeleton-network

Research

JoVE Journal

Biology

216 visualizações.  Scripps College. Para começar, certifique-se de que o layout do microscópio seja preparado na superfície da mesa óptica com todas as distâncias medidas com precisão. Em seguida, monte o laser de excitação sobre a mesa. Ajuste duas íris para a altura pretendida do laser e use essas íris para garantir que o feixe esteja nivelado e centralizado.Posicione o estágio de translação, ou TS1, sob a localização do espelho 1 ou M1. Use o par de íris ajustado à altura exata para definir o caminho ...

Vídeo: Configuração de imagens e varreduras volumétricas para visualização da rede do citoesqueleto

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the dynamics of these 3D, dense networks calls for optical sectioning, which is often associated with fluorescence confocal microscopes. However, recent developments in light-sheet fluorescence microscopy (LSFM) have established it as a cost-effective and, at times, superior alternative. To make LSFM accessible to cytoskeleton researchers less familiar with optics, we present a step-by-step beginner's guide to building a versatile light-sheet fluorescence microscope from off-the-shelf components. To enable sample mounting with traditional slide samples, this LSFM follows the single-objective light-sheet (SOLS) design, which utilizes a single objective for both the excitation and emission collection. We describe the function of each component of the SOLS in sufficient detail to allow readers to modify the instrumentation and design it to fit their specific needs. Finally, we demonstrate the use of this custom SOLS instrument by visualizing asters in kinesin-driven microtubule networks.

This protocol describes in detail how to build a single-objective, light-sheet fluorescence microscope and its usage for visualizing cytoskeleton networks.

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the ...