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.
Highlights
Most of what we recognize as the phenomena of life are not properties of stationary structures but emerge from dynamic interactions among many elements over time
We present the design and construction of the flex-SPIM, an instrument with two independently controlled light-sheet microscope-twins sharing the same multi-laser source, dramatically cutting the cost of the system
We demonstrate instrument versatility and application-specific customization by imaging a variety of specimens
Summary
Most of what we recognize as the phenomena of life are not properties of stationary structures but emerge from dynamic interactions among many elements over time. A key development was to create light-sheets by dynamically scanning a focused Gaussian beam, generated via a low-numericalaperture (NA) lens, across the plane (Fig. 1).18 This scanned Gaussian-beam light-sheet approach ( termed digital scanned laser light-sheet fluorescence microscopy; DSLM) provides better spatial illumination uniformity, higher light throughput, and more precise spatial control over the selected plane of interest compared to a static 2D light-sheet at the cost of replacing the simple cylindrical lens with the expense and complexity of a scanning galvanometer (galvo) mirror and associated optics and electronics. We describe the flex-SPIM, which combines two independently controlled light-sheet microscope-twins that share an ultrafast NIR laser and a bank of continuous-wave (CW) visible lasers This permits two specimens to be imaged simultaneously for far less than the cost of two multi-laser microscopes. Beam steering mirrors shared by both the ultrafast and CW lasers (illumination-scanning optics) have protected silver coatings, whereas those used by the ultrafast or CW lasers alone have broadband dielectric coatings
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