A versatile, multi-laser twin-microscope system for light-sheet imaging.
ABSTRACT: Light-sheet microscopy offers faster imaging and reduced phototoxicity in comparison to conventional point-scanning microscopy, making it a preferred technique for imaging biological dynamics for durations of hours or days. Such extended imaging sessions pose a challenge, as it reduces the number of specimens that can be imaged in a given day. Here, we present a versatile light-sheet imaging instrument that combines two independently controlled microscope-twins, built so that they can share an ultrafast near-infrared laser and a bank of continuous-wave visible lasers, increasing the throughput and decreasing the cost. To permit a wide variety of specimens to be imaged, each microscope-twin provides flexible imaging parameters, including (i) operation in one-photon and/or two-photon excitation modes, (ii) delivery of one to three light-sheets via a trio of orthogonal excitation arms, (iii) sub-micron to micron imaging resolution, (iv) multicolor compatibility, and (v) upright (with provision for inverted) detection geometry. We offer a detailed description of the twin-microscope design to aid instrument builders who wish to construct and use similar systems. We demonstrate the instrument's versatility for biological investigation by performing fast imaging of the beating heart in an intact zebrafish embryo, deep imaging of thick patient-derived tumor organoids, and gentle whole-brain imaging of neural activity in behaving larval zebrafish.
Project description:Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies (EBs) enlarge the scope of investigations to multi-cellular phenomena. The highest level of complexity, brain organoids that-in many aspects-recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that uses planar two-photon (2P) excitation. Its particularity is that-unlike two- or three-lens light-sheet microscopes-it uses a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petrá? spinning-disk scheme for achieving wide-field excitation. However, unlike the Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This new design, advantageous for 2P excitation, circumvents problems arising with the tandem disk from the large wavelength difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited by the light sheet is collected with the same objective and imaged onto a fast sCMOS camera. We demonstrate 3-D imaging of TO-PRO3-stained EBs and of brain organoids, uncleared and after rapid partial transparisation with triethanolamine formamide (RTF) and we compare the performance of our instrument to that of a confocal laser-scanning microscope (CLSM) having a similar numerical aperture. Our large-field 2P-spinning disk microscope permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.
Project description:Light sheet fluorescence microscopy enables fast, minimally phototoxic, three-dimensional imaging of live specimens, but is currently limited by low throughput and tedious sample preparation. Here, we describe an automated high-throughput light sheet fluorescence microscope in which specimens are positioned by and imaged within a fluidic system integrated with the sheet excitation and detection optics. We demonstrate the ability of the instrument to rapidly examine live specimens with minimal manual intervention by imaging fluorescent neutrophils over a nearly 0.3 mm3 volume in dozens of larval zebrafish. In addition to revealing considerable inter-individual variability in neutrophil number, known previously from labor-intensive methods, three-dimensional imaging allows assessment of the correlation between the bulk measure of total cellular fluorescence and the spatially resolved measure of actual neutrophil number per animal. We suggest that our simple experimental design should considerably expand the scope and impact of light sheet imaging in the life sciences.
Project description:In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM.
Project description:Dendritic signals play an essential role in processing visual information in the retina. To study them in neurites too small for electrical recording, we developed an instrument that combines a multi-photon (MP) microscope with a through-the-objective high-resolution visual stimulator. An upright microscope was designed that uses the objective lens for both MP imaging and delivery of visual stimuli to functionally intact retinal explants or eyecup preparations. The stimulator consists of a miniature liquid-crystal-on-silicon display coupled into the optical path of an infrared-excitation laser-scanning microscope. A pair of custom-made dichroic filters allows light from the excitation laser and three spectral bands ('colors') from the stimulator to reach the retina, leaving two intermediate bands for fluorescence imaging. Special optics allow displacement of the stimulator focus relative to the imaging focus. Spatially resolved changes in calcium-indicator fluorescence in response to visual stimuli were recorded in dendrites of different types of mammalian retinal neurons.
Project description:Digital-scanned light-sheet microscopy (DSLM) illuminates a sample in a plane and captures single-photon-excitation fluorescence images with a camera from a direction perpendicular to the light sheet. This method is potentially useful for observing biological specimens, because image acquisition is relatively fast, resulting in reduction of phototoxicity. However, DSLM cannot be effectively applied to high-scattering materials due to the image blur resulting from thickening of the light sheet by scattered photons. However, two-photon-excitation DSLM (2p-DSLM) enables collection of high-contrast image with near infrared (NIR) excitation. In conventional 2p-DSLM, the minimal excitation volume for two-photon excitation restricts the field of view. In this study, we achieved wide-field 2p-DSLM by using a high-pulse energy fiber laser, and then used this technique to perform intravital imaging of a small model fish species, medaka (Oryzias latipes). Wide fields of view (>700 μm) were achieved by using a low-numerical aperture (NA) objective lens and high-peak energy NIR excitation at 1040 nm. We also performed high-speed imaging at near-video rate and successfully captured the heartbeat movements of a living medaka fish at 20 frames/sec.
Project description:Selective plane illumination microscopy (SPIM) allows rapid 3D live fluorescence imaging on biological specimens with high 3D spatial resolution, good optical sectioning capability and minimal photobleaching and phototoxic effect. SPIM gains its advantage by confining the excitation light near the detection focal plane, and its performance is determined by the ability to create a thin, large and uniform excitation light sheet. Several methods have been developed to create such an excitation light sheet for SPIM. However, each method has its own strengths and weaknesses, and tradeoffs must be made among different aspects in SPIM imaging. In this work, we present a strategy to select the excitation light sheet among the latest SPIM techniques, and to optimize its geometry based on spatial resolution, field of view, optical sectioning capability, and the sample to be imaged. Besides the light sheets discussed in this work, the proposed strategy is also applicable to estimate the SPIM performance using other excitation light sheets.
Project description:Coherent anti-Stokes Raman scattering (CARS) microscopy was applied to image myelinated fibers in different regions of a mouse brain. The CARS signal from the CH2 symmetric stretching vibration allows label-free imaging of myelin sheath with 3D sub-micron resolution. Compared with two-photon excited fluorescence imaging with lipophilic dye labeling, CARS microscopy provides sharper contrast and avoids photobleaching. The CARS signal exhibits excitation polarization dependence which can be eliminated by reconstruction of two complementary images with perpendicular excitation polarizations. The capability of imaging myelinated fibers without exogenous labeling was used to map the whole brain white matter in brain slices and to analyze the microstructural anatomy of brain axons. Quantitative information about fiber volume%, myelin density, and fiber orientations was derived. Combining CARS with two-photon excited fluorescence allowed multimodal imaging of myelinated axons and other cells. Furthermore, in vivo CARS imaging on an upright microscope clearly identified fiber bundles in brain subcortex white matter. These advances open up new opportunities for the study of brain connectivity and neurological disorders.
Project description:The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space.
Project description:The central goals of mechanobiology are to understand how cells generate force and how they respond to environmental mechanical stimuli. A full picture of these processes requires high-resolution, volumetric imaging with time-correlated force measurements. Here we present an instrument that combines an open-top, single-objective light sheet fluorescence microscope with an atomic force microscope (AFM), providing simultaneous volumetric imaging with high spatiotemporal resolution and high dynamic range force capability (10 pN - 100 nN). With this system we have captured lysosome trafficking, vimentin nuclear caging, and actin dynamics on the order of one second per single-cell volume. To showcase the unique advantages of combining Line Bessel light sheet imaging with AFM, we measured the forces exerted by a macrophage during Fc?R-mediated phagocytosis while performing both sequential two-color, fixed plane and volumetric imaging of F-actin. This unique instrument allows for a myriad of novel studies investigating the coupling of cellular dynamics and mechanical forces.
Project description:In the last decade light sheet fluorescence microscopy techniques, such as selective plane illumination microscopy (SPIM), has become a well established method for developmental biology. However, conventional SPIM architectures hardly permit imaging of certain tissues since the common sample mounting procedure, based on gel embedding, could interfere with the sample morphology. In this work we propose an inverted selective plane microscopy system (iSPIM), based on non-linear excitation, suitable for 3D tissue imaging. First, the iSPIM architecture provides flexibility on the sample mounting, getting rid of the gel-based mounting typical of conventional SPIM, permitting 3D imaging of hippocampal slices from mouse brain. Moreover, all the advantages brought by two photon excitation (2PE) in terms of reduction of scattering effects and contrast improvement are exploited, demonstrating an improved image quality and contrast compared to single photon excitation. The system proposed represents an optimal platform for tissue imaging and it smooths the way to the applicability of light sheet microscopy to a wider range of samples including those that have to be mounted on non-transparent surfaces.