ADVANCED 3D GLASS CHIP FOR PATTERNED STIMULATION AND RECORDING OF NEURONAL ACTIVITY IN SINGLE CELL RESOLUTION

Microfluidic technology is unrivaled in its ability to apply soluble chemical stimuli with high spatiotemporal precision.Analogous, light-sheet microscopy is unmatched in its ability of low phototoxic but fast volumetric in vivo imaging with single cell resolution.Due to their optical translucency during the larval stages, zebrafish (Danio rerio) are an ideal model to combine both techniques [1]; yet, thus far this required light-sheet microscopes, which were in most cases custom-built and adapted to the available softlithographic chip technology.The development of a microfluidic perfusion system that can be operated in combination with light sheet imaging places much higher demands on the design when commercial microscopes are to be used.In particular restrictions regarding the light sheet coupling via two opposing and closely spaced mirrors require unique solutions for optical interrogation windows in the chip.Therefore, a monolithic thin glass chip was developed which offers substantially improved conditions for the in and out coupling of light, but poses high demands on microfabrication compared to relatively easy to fabricate PDMS microdevices, which are suitable in combination with tailored custom built light sheet microscopes.[2] Our aim was to use a commercial light-sheet microscope to illuminate a microfluidic chip from two opposite lateral directions and to record images with the detection objective placed orthogonally above the chip.Deep tissue penetration can be achieved by superimposing beams from opposite directions to form a single light sheet.But a microfluidic chip that allows a) targeted stimulus application in a closed microenvironment, b) interference-free incoupling of excitation light from two directions and c) outcoupling of fluorescence in the perpendicular direction through an optically perfect cover glass was not known until now.Here, we present a novel monolithic glass chip [3] with the required plane-parallel sidewalls, constructed by femtosecond laser ablation, thermal bonding and surface smoothing processes (Figure 1).The 3D shape of a fish fixator unit was tailored to match the body shape of a zebrafish larva to ensure stable positioning during wholebrain recording.Compared to the earlier NeuroExaminer 1.0 [4], decisive changes have been made in the design and manufacturing to allow a breakthrough in imaging quality particular at deeper brain levels (Figure 2).The measured point spread functions proved a resolution capability for recording single cell responses.This was possible by implementing two light sheet ports using an appropriate femtosecond laser ablation technique.With hydrodynamic focusing a targeted partial exposure of the larva's head to chemical stimuli and fast position switching (in less than 10 seconds) was possible (Figure 3).The gentle fluidic manipulation and agarose-free immobilization allows for monitoring brain activity of zebrafish over longer time scales, enabling detailed studies that would not be possible using traditional methods.With the capabilities of this unique monolithic glass chip and its up-scalable wafer-level fabrication process, the new NeuroExaminer is prone to become an excellent addition to neurobiology laboratories already equipped with highquality commercial light sheet microscopes.