Miniscope3D: optimized single-shot miniature 3D fluorescence microscopy

Miniature fluorescence microscopes are a standard tool in systems biology. However, widefield miniature microscopes capture only 2D information, and modifications that enable 3D capabilities increase the size and weight and have poor resolution outside a narrow depth range. Here, we achieve the 3D capability by replacing the tube lens of a conventional 2D Miniscope with an optimized multifocal phase mask at the objective’s aperture stop. Placing the phase mask at the aperture stop significantly reduces the size of the device, and varying the focal lengths enables a uniform resolution across a wide depth range. The phase mask encodes the 3D fluorescence intensity into a single 2D measurement, and the 3D volume is recovered by solving a sparsity-constrained inverse problem. We provide methods for designing and fabricating the phase mask and an efficient forward model that accounts for the field-varying aberrations in miniature objectives. We demonstrate a prototype that is 17 mm tall and weighs 2.5 grams, achieving 2.76 μm lateral, and 15 μm axial resolution across most of the 900 × 700 × 390 μm3 volume at 40 volumes per second. The performance is validated experimentally on resolution targets, dynamic biological samples, and mouse brain tissue. Compared with existing miniature single-shot volume-capture implementations, our system is smaller and lighter and achieves a more than 2× better lateral and axial resolution throughout a 10× larger usable depth range. Our microscope design provides single-shot 3D imaging for applications where a compact platform matters, such as volumetric neural imaging in freely moving animals and 3D motion studies of dynamic samples in incubators and lab-on-a-chip devices.

Geometrical Optics

Discuss the limits imposed by the paraxial approximation. Define the sign convention based on the cartesian coordinate system, the foiundation of analytic geometery. Demonstrate ray tracing technique to derive the ABCD maxtrix which will generate both the gaussian and Newtonian form of the thin lens equation and the lens maker’s equation. The cardinal points of a lens are also derived. The ABCD matrix is used to explore the methods used in ray tracing to locate the aperture stop of a Cooke’s triplet lens system. In the problem set, the student is asked to use the aperture stop to locate the entrance and exit pupil of a Cooke’s triplet.

A model of the entrance pupil of the human eye

ABSTRACTThe aperture stop of the iris is subject to refraction by the cornea, and thus an outside observer sees a virtual image: the “entrance pupil” of the eye. When viewed off-axis, the entrance pupil has an elliptical form. The precise appearance of the entrance pupil is a consequence of the anatomical and optical properties of the eye, and the relative positions of the eye and the observer. This paper presents a ray-traced model eye that provides the parameters of the entrance pupil ellipse for an observer at an arbitrary location. The model is able to reproduce empirical measurements of the shape of the entrance pupil with good accuracy. I demonstrate that accurate specification of the entrance pupil of a stationary eye requires modeling of corneal refraction, the misalignment of the visual and optical axes, and the non-circularity of the aperture stop. The model, including a three-dimensional ray-tracing function through quadric surfaces, is implemented in open-source MATLAB code.