Pupil engineering for extended depth-of-field imaging in a fluorescence miniscope

Abstract

Significance: Fluorescence head-mounted microscopes, i.e., miniscopes, have emerged as powerful tools to analyze in-vivo neural populations but exhibit a limited depth-of-field (DoF) due to the use of high numerical aperture (NA) gradient refractive index (GRIN) objective lenses.Aim: We present extended depth-of-field (EDoF) miniscope, which integrates an optimized thin and lightweight binary diffractive optical element (DOE) onto the GRIN lens of a miniscope to extend the DoF by 2.8× between twin foci in fixed scattering samples.Approach: We use a genetic algorithm that considers the GRIN lens' aberration and intensity loss from scattering in a Fourier optics-forward model to optimize a DOE and manufacture the DOE through single-step photolithography. We integrate the DOE into EDoF-Miniscope with a lateral accuracy of 70 μm to produce high-contrast signals without compromising the speed, spatial resolution, size, or weight. Results:We characterize the performance of EDoF-Miniscope across 5-and 10-μm fluorescent beads embedded in scattering phantoms and demonstrate that EDoF-Miniscope facilitates deeper interrogations of neuronal populations in a 100-μm-thick mouse brain sample and vessels in a whole mouse brain sample.Conclusions: Built from off-the-shelf components and augmented by a customizable DOE, we expect that this low-cost EDoF-Miniscope may find utility in a wide range of neural recording applications.

E2E-BPF microscope: extended depth-of-field microscopy using learning-based implementation of binary phase filter and image deconvolution

“…The DoF defines the depth range of an object that can be sharply imaged by a given optical imaging system, and is determined by the operating wavelength, effective focal length and aperture size of the imaging lens. In many biomedical imaging applications, such as in cytometry 1,2 , histology 3 , and endoscopy [4][5][6] , high-resolution imaging over a large spatial scale is often desired; for instance, a pathological examination is typically performed with a high numerical-aperture (NA) objective to visualize cellular and subcellular features of tissue specimens, but it is accompanied by limited fieldof-view (FoV) and DoF. Therefore, to image large-area pathological/cytology slides, either objects or imaging optics should be scanned and refocused repetitively, which is costly and labor-intensive.…”