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.

Quantity as a Fish Views It: Behavior and Neurobiology

“…The parallelized 2D image collection enables a longer signal integration time for each voxel, thus requiring a much-reduced peak laser intensity compared with 2p-PSM. Recent implementations of 2p-SPIM, as applied to brain-wide functional imaging of zebrafish (Wolf et al, 2015(Wolf et al, , 2017Keomanee-Dizon et al, 2020;de Vito et al, 2022), have achieved imaging frame rates of 20-150 Hz and volumetric rates spanning ∼1-5 Hz. Importantly, in these studies, the upper limit of the imaging rate is not dictated by the imaging hardware but rather by the putative threshold of photodamage, which starts to appear as the laser power is increased.…”

“…We highlight some of our preliminary results in Figure 4. We used the microscopy platform described in Keomanee-Dizon et al (2020) to record the whole-brain activity map of awake zebrafish larvae, which were transgenically labeled with fluorescent calcium indicators. A projector system was used to present visual numerosity stimuli to the zebrafish (Figure 4A).…”

First steps into the cloud: Using Amazon data storage and computing with Python notebooks

“…To help users enter cloud computing practice, an example dataset and code to process it is provided as supplemental data. Neural activity data presented here is collected using a custom two-photon light-sheet microscope based on previous publication [ 5 ]. Total laser power of 300mW was used at 920nm wavelength to collect spontaneous neural activity data from larval zebrafish expressing pan-neuronal nuclear-localized calcium indicator GCaMP6s.…”

Open-Source Thermometer, Temperature Controller, and Light Meter for Use in Animal Facilities and During Experiments

“…With a single Arduino device, a temperature sensor can be bundled together with a heater to create a control device to keep conditions at a given temperature during microscopy sessions (Figure 3A). We applied a simple non-linear proportional control process (Code Snippet 5) to regulate the temperature of a 50-mL imaging sample chamber (40 mm x 60 mm x 20 mm, Figure 3A) in a custom light-sheet microscope similar to a previously published design [9]. We used a commercially available ceramic heating element and the thermometer described in the previous section (Arduino-based thermometer).…”