Oblique Plane Microscopy (OPM)

The orthogonal illumination and detection geometry adopted by most LSFMs is accompanied by several underappreciated disadvantages. For example, this geometry is incompatible with hardware-based focus systems that reduce thermal and mechanical drift, standard imaging dishes, multi-well plates, microfluidics that establish chemotactic gradients, or parallel plate flow chambers that deliver controlled levels of shear stress. They are also accompanied by high rates of reagent consumption owing to large imaging chambers (~8.5mL for LLSM), which can be prohibitive for chemogenetic  and pharmacological perturbations. Importantly, the use of high numerical aperture water dipping objectives compromises the sterility of the specimen. Thus, evaluation of slow biological phenomena that take place over ~24 hours are highly challenging. In contrast, because oblique plane microscopy illuminates the specimen with an obliquely launched light-sheet and collects the fluorescence with the same objective (referred to as the primary objective) and uses aberration-free remote focusing (with a secondary and tertiary objective) to reorient and image the fluorescence on a scientific camera, it can operate in a more-convenient inverted format. Here, we will disseminate a highly optimized laser-scanning variant of OPM that is achieves a raw resolution of 299x336x731nm throughout a large field of view.The orthogonal illumination and detection geometry adopted by most LSFMs is accompanied by several underappreciated disadvantages. For example, this geometry is incompatible with hardware-based focus systems that reduce thermal and mechanical drift, standard imaging dishes, multi-well plates, microfluidics that establish chemotactic gradients, or parallel plate flow chambers that deliver controlled levels of shear stress. They are also accompanied by high rates of reagent consumption owing to large imaging chambers (~8.5mL for LLSM), which can be prohibitive for chemogenetic  and pharmacological perturbations. Importantly, the use of high numerical aperture water dipping objectives compromises the sterility of the specimen. Thus, evaluation of slow biological phenomena that take place over ~24 hours are highly challenging. In contrast, because oblique plane microscopy illuminates the specimen with an obliquely launched light-sheet and collects the fluorescence with the same objective (referred to as the primary objective) and uses aberration-free remote focusing (with a secondary and tertiary objective) to reorient and image the fluorescence on a scientific camera, it can operate in a more-convenient inverted format. Here, we will disseminate a highly optimized laser-scanning variant of OPM that is achieves a raw resolution of 299x336x731nm throughout a large field of view.