Hybrid multiphoton volumetric functional imaging of large-scale bioengineered neuronal networks

Abstract

Planar neural networks and interfaces serve as versatile in vitro models of central nervous system physiology, but adaptations of related methods to three dimensions (3D) have met with limited success. Here, we demonstrate for the first time volumetric functional imaging in a bio-engineered neural tissue growing in a transparent hydrogel with cortical cellular and synaptic densities, by introducing complementary new developments in nonlinear microscopy and neural tissue engineering. Our system uses a novel hybrid multiphoton microscope design combining a 3D scanning-line temporal-focusing subsystem and a conventional laser-scanning multiphoton microscope to provide functional and structural volumetric imaging capabilities: dense microscopic 3D sampling at tens of volumes/sec of structures with mm-scale dimensions containing a network of over 1000 developing cells with complex spontaneous activity patterns. These developments open new opportunities for large-scale neuronal interfacing and for applications of 3D engineered networks ranging from basic neuroscience to the screening of neuroactive substances.

Microfluidic Chip for Site‐Specific Neuropharmacological Treatment and Activity Probing of 3D Neuronal “Optonet” Cultures

“…In summary, we have presented a novel microfl uidic device for controlled, reversible, and site-specifi c drug administration unto neuronal "optonets"-a potentially powerful tool for studying the functional and structural effects of drugs, growth factors, and toxins in a developing 3D model neural network with brain-like properties. [ 10 ] The device's design provides spatial specifi city (Figures 1 A and 2 D)-the specifi city of druginduced neuromodulation was verifi ed by imaging network activity under both the administration site and a distant drug free channel (Figure 3 A,B). While devices for studying chemotactic gradient effects are typically designed for multihour relaxation times (e.g., [ 20 ] functional drug effects in our device can be seen within a few minutes (Figure 3 A,B), allowing to use the same network both as a drug-free control and to compare the effect of multiple drugs in parallel (to avoid sample variations).…”

“…[ 23 ] Importantly, however, the microfl uidic fl ow-chambers in such devices are not specifi cally designed to provide site-specifi c dynamic drug administration, nor do they offer or pursue the ability to probe the embedded network's activity as a drug response marker. Here we address these challenges through the design, fabrication, and application of a compact multichannel microfl uidic device that allows compartmentalized drug stimulation of 3D neural "optonets" [ 10 ] -optically accessible, optogentically probed neural cultures embedded in an ECM-like hydrogel. Specifi cally, we demonstrate that the device supports normal development of the optonet cultures, and that it can be used for eliciting spatially localized, reversible pharmacological effects upon network activity that can be simultaneously monitored with single cell resolution using state-of-the-art genetically encoded calcium indicators (GECIs).…”

“…[3][4][5] While current systems are mostly based on in vitro models with neurons cultured on 2D substrates (primarily due to the ease of culturing and handling), [6][7][8] recent advances in neural tissue engineering facilitate the encapsulation of neurons in extracellular matrix (ECM)-like systems, where they form spatially organized 3D in vitro neural tissue models. [9][10][11] 3D neural cultures offer significant advantages over 2D models. [ 9,12 ] To name a few, i) the substrate surrounding the neurons in 3D models closely mimics the chemical and mechanical properties of nervous systems, whereas a substrate used for 2D cultures is orders of magnitude stiffer than typical in vivo environments; ii) neurons in 3D cultures get access to soluble factors similar to tissue environments and also establish outgrowth and networks in all spatial directions; iii) In vivo-like cell-cell and cell-matrix interactions can be achieved in 3D cultures that are known to infl uence gene expression and cellular metabolism and behavior.…”

“…[ 10 ] To optically record network activity, neurons were transfected with GCaMP6m, a modern fl uorescent calcium indicator of neuronal spiking activity, [ 24 ] and three planes with 40 µm depth intervals were functionally imaged at a rate of 4 volumes s −1 (Figure 2 C).…”

In vivo volumetric imaging of biological dynamics in deep tissue via wavefront engineering

“…To study the orchestrated cellular signaling and intercellular interplays in vivo, one needs 3D imaging methods that can sample near the natural spatiotemporal resolutions. So far, several wide-field fluorescence imaging methods, such as light-sheet microscopy [3], light-field microscopy [4], and temporal focusing multiphoton microscopy [5,6], have been developed towards such a goal. The parallel detection scheme of these methods benefits high-speed imaging.…”