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

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