A versatile, multi-laser twin-microscope system for light-sheet imaging

Abstract

Recent developments in light-sheet microscopy techniques has led to its cutting-edge application across a range of fields, from developmental biology to behavioral neuroscience. Its low photodamage propensity allows biological dynamics to be imaged for a duration of several hours up to days; however, a tradeoff exists between such extended imaging sessions and imaging throughput, which in practice directly limits analysis workflows and the resulting time to biological discovery. Here, we present an economical instrument that can image multiple sample types simultaneously. Unlike any existing light-sheet microscope, our instrument, the flex-SPIM, shares both an ultrafast laser and a bank of continuous-wave lasers between two independently controlled light-sheet microscope-twins. Harnessing the power of modern lasers yields a substantial reduction in the cost of the light source over standard dual systems, and a doubling in imaging throughput relative to a single system. Each microscope-twin (i) operates in both one-photon and two-photon excitation modes, (ii) delivers one to three light-sheets via a trio of orthogonal excitation arms, (iii) provides sub-micron to micron imaging resolution, (iv) is multicolor compatible, and (v) permits upright and/or inverted lightsheet detection. We offer a detailed description of the flex-SPIM design to aid instrument builders who wish to construct and use a similar system. We then 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.

Lightsheet Microscopy

“…A sophisticated wealth of material (Garbellotto & Taylor, 2018; Girstmair et al., 2016; Gualda et al., 2013; Keomanee‐Dizon, Fraser, & Truong, 2020; Voigt et al., 2019), including a full parts list, costings, and construction guide is available with step‐by‐step instructions for building a microscope. The final cost is very much dependent upon the quality of the camera, but it is in the range of $20,000‐$40,000.…”

Extended depth-of-field light-sheet microscopy improves imaging of large volumes at high numerical aperture

“…Sub-diffractive fluorescent beads (175 ± 5 nm diameter; PS-Speck Microscope Point Source Kit, P7220, Molecular Probes) were diluted (1:1000) and embedded in a mixture of 2% agarose, using the caddy-dive bar system described in Ref. [16]. The beads were then submerged in an imaging chamber filled with 60 mL of distilled water.…”

“…Fish were embedded in 2% agarose as described in Ref. [16]. To measure photobleaching rates, cranial vasculature 4-5-dpf Tg(kdrl:eGFP) larvae were used.…”

“…Imaging was performed over a 400 × 800 µm 2 region of the same specimen with 3.5 ms exposure (∼ 141 frames per second) and ∼ 150 µW of laser power for all modalities. To cover a 200-µm zdepth of nearly the entire zebrafish brain, the z-scanning galvo was synchronized with the objective piezoelectric collar [16], and 25 image slices spaced 8 µm apart were recorded. We did not observe any appreciable signs of fluorescence photobleaching or phototoxicity over 10 minutes of recording from any of the modalities.…”

“…Propagation-invariant beams, such as Bessel-Gauss [6][7][8], Airy-Gauss beams [9,10], or bound two-dimensional (2D) optical lattices partially improve this tradeoff, by providing a thin (∼1 µm FWHM) light-sheet profile over a ∼ 50-µm FOV [11,12]. However, to image dynamic systems having volumetric dimensions of hundreds of microns or more with cellular resolution [13][14][15][16], low-NA illumination is needed to produce light sheets that span a sufficinetly large FOV. But low-NA illumination also produces thicker light sheets (∼4-5 µm or more), which excites fluorescence outside the narrow DOF of the high-NA detection objective.…”