Abstract
.Significance: Light-sheet fluorescence microscopy (LSFM) is a powerful technique for high-speed volumetric functional imaging. However, in typical light-sheet microscopes, the illumination and collection optics impose significant constraints upon the imaging of non-transparent brain tissues. We demonstrate that these constraints can be surmounted using a new class of implantable photonic neural probes.Aim: Mass manufacturable, silicon-based light-sheet photonic neural probes can generate planar patterned illumination at arbitrary depths in brain tissues without any additional micro-optic components.Approach: We develop implantable photonic neural probes that generate light sheets in tissue. The probes were fabricated in a photonics foundry on 200-mm-diameter silicon wafers. The light sheets were characterized in fluorescein and in free space. The probe-enabled imaging approach was tested in fixed, in vitro, and in vivo mouse brain tissues. Imaging tests were also performed using fluorescent beads suspended in agarose.Results: The probes had 5 to 10 addressable sheets and average sheet thicknesses for propagation distances up to in free space. Imaging areas were as large as in brain tissue. Image contrast was enhanced relative to epifluorescence microscopy.Conclusions: The neural probes can lead to new variants of LSFM for deep brain imaging and experiments in freely moving animals.
Highlights
New methods in optogenetics[1,2,3] and, especially, the advent of fluorescent reporters of neuronal activity, have opened many novel approaches for actuating and recording neural activity en masse, through the use of powerful free-space single-photon and multi-photon microscopy methods.[4,5,6,7,8] existing approaches to functional imaging of the brain have significant limitations
Single-photon (1P) epifluorescence imaging readily lends itself to high frame-rate wide-field microscopy, but, in its simplest implementations, image contrast is hampered by out-of-focus background fluorescence, and the depth of imaging is restricted by the optical attenuation in the tissue
Non-digitally scanned 1P-Light-sheet fluorescence microscopy (LSFM) is inherently faster than point- or line-scan methods; and since the illumination is restricted to a plane, photobleaching, phototoxicity, and out-of-focus background fluorescence are reduced compared to epifluorescence microscopy
Summary
New methods in optogenetics[1,2,3] and, especially, the advent of fluorescent reporters of neuronal activity, have opened many novel approaches for actuating and recording neural activity en masse, through the use of powerful free-space single-photon and multi-photon microscopy methods.[4,5,6,7,8] existing approaches to functional imaging of the brain have significant limitations. Single-photon (1P) epifluorescence imaging readily lends itself to high frame-rate wide-field microscopy, but, in its simplest implementations, image contrast is hampered by out-of-focus background fluorescence, and the depth of imaging is restricted by the optical attenuation in the tissue. Confocal imaging improves the contrast by optical sectioning, and out-of-focus light is rejected using a pinhole; a laser beam must be scanned across each point of the tissue and this significantly slows the image acquisition rate.[9] Multi-photon microscopy is inherently a point or line scanning method, but because it uses infrared excitation (which provides a longer optical attenuation length5), the imaging depth in brain tissue can be extended to ∼1 mm and the focus of the light beam can be rastered in three-dimensions to achieve volumetric imaging.[5,10,11,12]. An LSFM variant called swept confocally aligned planar excitation (SCAPE) microscopy, which requires only a single objective, removes these constraints.[6,19] While in vivo calcium neural imaging has been demonstrated using SCAPE in mice,[6] miniaturization of the system to be compatible with freely moving animal experiments remains challenging due to the additional optics required
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
