High-contrast multifocus microscopy with a single camera and z-splitter prism

Abstract

We present a multifocus imaging strategy based on the use of a simple z-splitter prism that can be assembled from off-the-shelf components. Our technique enables a widefield image stack to be distributed onto a single camera and recorded simultaneously. We exploit the volumetric nature of our image acquisition by further introducing a novel extended-volume 3D deconvolution strategy to suppress far-out-of-focus fluorescence background to significantly improve the contrast of our recorded images, conferring to our system a capacity for quasi optical sectioning. By swapping in different z-splitter configurations, we can prioritize high speed or large 3D field-of-view imaging depending on the application of interest. Moreover, our system can be readily applied to a variety of imaging modalities in addition to fluorescence, such as phase-contrast and darkfield imaging. Because of its simplicity, versatility, and performance, we believe our system will be a useful tool for general biological or biomedical imaging applications.

High-speed multifocus phase imaging in thick tissue

“…In summary, we have demonstrated fast multifocus phase imaging by combining OBM [31] with a z-splitter-based microscope [29]. The main advantage of OBM is that it enables phase imaging in arbitrarily thick, scattering samples, which, by definition, are volumetric.…”

“…To enable fast volumetric imaging of 3D phase information, we combine here the principle of OBM with a recently developed multifocus imaging strategy based on a z-splitter prism [29]. Our experimental setup is shown in Fig.…”

“…1 (a), which is similar to the multifocus microscope described in Ref. [29] but with illumination delivered through off-axis optical light guides. In brief, illumination light from LEDs (Thorlabs, M617L3) is coupled into 1 mm diameter plastic light guides (Edmund Optics, #02-536), and delivered into the sample at a 2.5 mm offset distance from the optical axis, and at a roughly 45°angle relative to the sample surface.…”

“…The key to our multifocus imaging system is the z-splitter prism. In our case, this was obtained from Artifex, though alternatively it could be assembled entirely from off-the-shelf beamsplitters (BS's) and right angle prisms (RAPs) [29]. The functions of these is to split the detection path into multiple paths of increasing optical pathlength, so that each path, when projected onto the camera, is conjugate to a different focal plane in the sample.…”

Autofocusing technologies for whole slide imaging and automated microscopy

“…With the image captured by the focusing sensor, a certain focus measure and fitting model can be used to infer the optimal focus position. In additional to autofocusing, this scheme can also be modified for real‐time multiplane microscopy [116–119], which finds important applications in volumetric imaging of biological samples.…”