3D differential phase contrast microscopy

We demonstrate 3D phase and absorption recovery from partially coherent intensity images captured with a programmable LED array source. Images are captured through-focus with four different illumination patterns. Using first Born and weak object approximations (WOA), a linear 3D differential phase contrast (DPC) model is derived. The partially coherent transfer functions relate the sample's complex refractive index distribution to intensity measurements at varying defocus. Volumetric reconstruction is achieved by a global FFT-based method, without an intermediate 2D phase retrieval step. Because the illumination is spatially partially coherent, the transverse resolution of the reconstructed field achieves twice the NA of coherent systems and improved axial resolution

Computational structured illumination for high-content fluorescent and phase microscopy

High-content biological microscopy targets high-resolution imaging across large fields-of-view (FOVs). Recent works have demonstrated that computational imaging can provide efficient solutions for high-content microscopy. Here, we use speckle structured illumination microscopy (SIM) as a robust and cost-effective solution for high-content fluorescence microscopy with simultaneous high-content quantitative phase (QP). This multi-modal compatibility is essential for studies requiring cross-correlative biological analysis. Our method uses laterally-translated Scotch tape to generate high-resolution speckle illumination patterns across a large FOV. Custom optimization algorithms then jointly reconstruct the sample's super-resolution fluorescent (incoherent) and QP (coherent) distributions, while digitally correcting for system imperfections such as unknown speckle illumination patterns, system aberrations and pattern translations. Beyond previous linear SIM works, we achieve resolution gains of 4x the objective's diffraction-limited native resolution, resulting in 700 nm fluorescence and 1.2 um QP resolution, across a FOV of 2x2.7 mm^2, giving a space-bandwidth product (SBP) of 60 megapixels

Structured illumination microscopy with unknown patterns and a statistical prior

Structured illumination microscopy (SIM) improves resolution by down-modulating high-frequency information of an object to fit within the passband of the optical system. Generally, the reconstruction process requires prior knowledge of the illumination patterns, which implies a well-calibrated and aberration-free system. Here, we propose a new \textit{algorithmic self-calibration} strategy for SIM that does not need to know the exact patterns {\it a priori}, but only their covariance. The algorithm, termed PE-SIMS, includes a Pattern-Estimation (PE) step requiring the uniformity of the sum of the illumination patterns and a SIM reconstruction procedure using a Statistical prior (SIMS). Additionally, we perform a pixel reassignment process (SIMS-PR) to enhance the reconstruction quality. We achieve 2$\times$ better resolution than a conventional widefield microscope, while remaining insensitive to aberration-induced pattern distortion and robust against parameter tuning

3D computational microscopy of dynamic samples

This talk will describe computational imaging methods for fast capture and reconstruction of 3D and 4D images in a commercial microscope. Our experimental setups employ inexpensive illumination-side and detection-side coding of angle (Fourier) space with simple hardware. The result is high-resolution intensity and phase images that span a large field-of-view, breaking the diffraction limit of the objective lens used and achieving high space- bandwidth-time product. We demonstrate real-time Gigapixel microscopy for in vitro biological cells and extend our methods to 3D imaging and algorithmic self-calibration. Through an end-to-end design of both the optical system and the computational algorithms, we achieve real-time 3D and phase recovery with digital aberration correction and mitigation of multiple scattering effect

Three-dimensional scanless holographic optogenetics with temporal focusing (3D-SHOT).

Optical methods capable of manipulating neural activity with cellular resolution and millisecond precision in three dimensions will accelerate the pace of neuroscience research. Existing approaches for targeting individual neurons, however, fall short of these requirements. Here we present a new multiphoton photo-excitation method, termed three-dimensional scanless holographic optogenetics with temporal focusing (3D-SHOT), which allows precise, simultaneous photo-activation of arbitrary sets of neurons anywhere within the addressable volume of a microscope. This technique uses point-cloud holography to place multiple copies of a temporally focused disc matching the dimensions of a neurons cell body. Experiments in cultured cells, brain slices, and in living mice demonstrate single-neuron spatial resolution even when optically targeting randomly distributed groups of neurons in 3D. This approach opens new avenues for mapping and manipulating neural circuits, allowing a real-time, cellular resolution interface to the brain

2D and 3D structured illumination microscopy with unknown patterns and a statistical prior

Structured illumination microscopy (SIM) is one of the most widely applied super-resolution microscopy techniques in bioimaging. It improves resolution by down-modulating a sample’s high spatial frequency information to fit within the passband of the optical system. Normally, the reconstruction process requires prior knowledge of the illumination patterns. Aberrations from the optical system or from the sample itself will distort the patterns and degrade performance. Here, we propose a new algorithmic self-calibration strategy for both 2D and 3D SIM that does not need to know the exact patterns a priori, but only their covariance. The algorithm, termed PE-SIMS, includes a pattern-estimation (PE) step requiring the uniformity of the sum of the illumination patterns and a SIM reconstruction procedure using a statistical prior (SIMS). We achieve 2x better resolution than a conventional widefield microscope, without needing to know the illumination patterns and while remaining insensitive to aberration-induced pattern distortion. Please click Additional Files below to see the full abstract

Computational illumination for high-speed in vitro Fourier ptychographic microscopy

We demonstrate a new computational illumination technique that achieves large space-bandwidth-time product, for quantitative phase imaging of unstained live samples in vitro. Microscope lenses can have either large field of view (FOV) or high resolution, not both. Fourier ptychographic microscopy (FPM) is a new computational imaging technique that circumvents this limit by fusing information from multiple images taken with different illumination angles. The result is a gigapixel-scale image having both wide FOV and high resolution, i.e. large space-bandwidth product (SBP). FPM has enormous potential for revolutionizing microscopy and has already found application in digital pathology. However, it suffers from long acquisition times (on the order of minutes), limiting throughput. Faster capture times would not only improve imaging speed, but also allow studies of live samples, where motion artifacts degrade results. In contrast to fixed (e.g. pathology) slides, live samples are continuously evolving at various spatial and temporal scales. Here, we present a new source coding scheme, along with real-time hardware control, to achieve 0.8 NA resolution across a 4x FOV with sub-second capture times. We propose an improved algorithm and new initialization scheme, which allow robust phase reconstruction over long time-lapse experiments. We present the first FPM results for both growing and confluent in vitro cell cultures, capturing videos of subcellular dynamical phenomena in popular cell lines undergoing division and migration. Our method opens up FPM to applications with live samples, for observing rare events in both space and time