Abstract
Imaging of neuronal activity with fluorescent indicators is an important technique in neuroscience. However, it remains challenging to record volumetric image data at fast frame rates and good resolution. One promising technique to achieve this goal is light sheet microscopy (LSM), but the right angle configuration of the excitation and imaging system limits its application. Oblique plane microscopy (OPM), a variant of LSM, circumvents this limitation by exciting oblique planes and detecting the image through the same microscope objective lens. So far, these techniques have relied on the use of high numerical aperture (NA) detection objective lenses, which limits their field of view. Here we present an OPM technique that allows for the use of low NA objective lenses by redirecting the light with the help of a diffraction grating. The microscope maintains a micrometer-scale lateral resolution over a large addressable imaging volume of 3.3×3.0×1.0 mm3. We demonstrate its practicality by imaging the whole brain of larval and juvenile zebrafish.
Highlights
Optical recording of neural activity has become an essential part of neuroscientific research
One way to guarantee optical sectioning in linear microscopy is to break the collinearity of emission and excitation point spread functions (PSFs) as in light sheet microscopy or selective plane illumination microscopy (LSM/SPIM) techniques
To enable the reimaging of an oblique plane formed by a low-numerical aperture (NA) objective lens, we introduced a reflective blazed grating at the intermediate image plane and aligned it to be coplanar with the image of the oblique fluorescence plane [Figs. 1(a) and 1(c)]
Summary
Optical recording of neural activity has become an essential part of neuroscientific research. Other optical techniques achieve volumetric recordings by capturing a single snapshot on a camera sensor and thereby circumvent the limitations of sequential scanning schemes This is achieved by multi-aperture imaging combined with computational reconstruction, as in light field microscopy [6,7,8,9,10] or by multiplexing and focusing different focal planes onto different sensor areas with the help of diffractive optics [11,12,13]. These camera-based techniques employ one-photon excitation, since the requirements on the total power of the light source as well as on the maximal permissive energy flux into the specimen can be more met. This requires the specimen to be accessible and optically transparent on both the imaging side as well as on the perpendicular excitation side, which is often not possible due to the cranium or other anatomical features of the organism of interest, such as the eyes in the case of fish
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