We demonstrate an extended FOV imaging concept which relies on exchanging the real and the Fourier spaces relative to the structured illumination microscopy (SIM). While SIM achieves Fourier space extension by structuring illumination in the real space, we show that effective detector area of an imaging system can be extended by structuring in the Fourier space. Structuring in Fourier space is achieved by a designer phase mask in the Fourier plane of the imaging system and the extended FOV can be obtained in single shot image record. A PSF design for extended FOV and corresponding experimental results will be described.

We design and implement a novel imaging technique that integrates bimodal phase and 3D fluorescence capabilities through aperture segmentation. This approach involves capturing four distinct fluorescence images, mirroring the principles of the Fourier light field microscope and the multi-view reflector microscope, enabling accurate 3D sample reconstruction. Additionally, four brightfield images are acquired for quantitative phase and amplitude reconstruction based on the Kramers-Kronig relations. By combining the strengths of phase imaging, such as digital refocusing, extended depth of field, and non-invasiveness, with the specificity of fluorescence imaging, this method offers a unique imaging solution. Imaging maize roots highlights its exceptional depth of field extension, while imaging a mixture of bacterial cells with and without fluorescent protein tags demonstrates its unique bimodal capabilities.

Optical Coherence Tomography (OCT) is a valuable tool for label-free imaging with micrometer resolution. However, conventional approaches to OCT typically image a sample from a single direction or angle, which limits its ability to image structures placed behind strongly attenuating material or deep inside the object. Here we introduce a solution to this problem by adding a small chamber to a spectral-domain OCT setup. Acoustic actuation enables a contact-free levitation and stepwise reorientation of samples such as zebra-fish larvae and tumor spheroids in a controlled and reproducible manner. We further developed a model-based compressive algorithm, which is able to exploit the diverse multi-angle OCT volumes for a 3D-reconstruction with isotropic resolution and estimation of refractive index values. We demonstrate and validate our approach on zebrafish larvae. We believe that our approach represents a powerful enabling tool for developmental biology and for organoid and cancer spheroid research.

The chromatic confocal microscope is an innovative method to acquire axial information simultaneously but suffers from limited imaging depth. We designed an 800um chromatic shift chromatic objective and manufactured it by precision diamond turning. By utilizing this objective, our chromatic confocal system can capture a large depth axis information simultaneously without stage scanning, while maintaining 780um lateral resolution. Biology tissues are visualized under this microscope to evaluate its performance.

Obtaining high resolution images in scattering tissue has been a long-standing challenge in microscopy. This challenge is exacerbated when high frame rates are required, or when 3D imaging is required over extended volumes. I will present several tools we have developed to address this challenge. These can be applied to a variety of microscopy modalities ranging from multiplane imaging with speckle, to multi-focus confocal imaging with targeted illumination, and finally to multiphoton imaging. Our main applications are in-vivo imaging in the mouse brain, where we demonstrate high contrast kHz-rate imaging of neuronal electrical activity and blood flow deep in tissue.

Fourier light field microscopy (FLFM) captures a sample 3D volume in a single snapshot, providing game-changing imaging speed for various bio-imaging applications. Existing FLFM platforms have often been designed as single-purpose implementations, with a fixed performance in resolution and field of view, restricting widespread applications. Here, to democratize FLFM toward broader adoption for biomedical research, we describe a multi-purpose implementation of FLFM that enables synchronous volumetric imaging across a wide range of resolution and field of view. With our single instrument, we demonstrate a variety of bio-imaging applications across scales, including sub-cellular dynamics, tissue cellular dynamics, and whole-brain neural activity.

Light from a dipole source (e.g., a single molecule) has long been known to possess a spatially varying polarization state, but no current method exists to emulate this response. Simulations of an engineered scattering element in a photonic integrated circuit have demonstrated the concept of a synthetic dipole. Through the AIM Photonics Foundry, photonic integrated circuits with synthetic dipoles were fabricated and characterized under a custom SWIR microscope setup. The polarization response of designed transverse dipoles was characterized using quantitative imaging and rotating quarter-wave plate polarimetry.

Camera-based time-correlated single photon counting (TCSPC) is a method where the position and the arrival time of the photons are recorded simultaneously. This has some advantages for fluorescence lifetime imaging (FLIM) with certain types of microscopy. It also allows FLIM with a macroscopic field of view. We report on nanosecond fluorescence lifetime imaging (FLIM) combined with total internal reflection fluorescence (TIRF) microscopy based on a 40 mm diameter crossed delay line anode detector. It has a few 100 picoseconds time resolution, and is read out via three standard TCSPC boards. We apply this wide-field TCSPC detector to identify Förster Resonance Energy Transfer (FRET) in cell membrane proteins in TIR-FLIM microscopy. In addition, we use a TCSPC single photon avalanche diode (SPAD) array as a macroscopic camera to visualise varnish removal on paintings.

Most two photon and confocal microscopes are limited by the sequential detection of pixels. I present recent work using low-cost silicon photomultiplier (SiPM) technology to build arrays of single-photon sensitive detectors. I present both spatially multiplexed imaging in highly scattering tissue and hyperspectral detection using 16 parallel SiPMs in two detector configurations. These enable 200 MP/s imaging through 4 spectral channels or hyperspectral read out at 50 MP/s and 20 nm spectral resolution at very low cost.

Understanding the dynamic interaction between fluorescence-labeled and label-free structures is important. The two types of signals are conventionally collected by fluorescence and phase microscopy, respectively. Recently, we developed a one-photon bi-functional microscopy system for simultaneous imaging of fluorescence and refractive index. However, it cannot reconstruct the refractive index of highly scattering samples with dense fluorophores. To solve this challenge, we develop new two-photon multimodal microscopy that reconstructs the 3D refractive index and fluorescence from multiple two-photon fluorescence images with a multi-slice model. We validated this method using a sample consisting of a mixture of fluorescence and glass beads.

Image Scanning Microscopy (ISM) enables super-resolution at an excellent signal-to-noise ratio thanks to a detector array. The microscope collects a confocal-like image for each detector element, generating a large dataset that requires tailored processing tools to be converted into a single super-resolved image. We propose a novel algorithm to fuse the dataset into an image with enhanced optical sectioning and resolution. Our method exploits the information inherently contained in the dataset to reject out-of-focus contributions and reconstruct an image with a smaller pixel size and a better resolution. The proposed method requires minimal user inputs and outperforms existing reconstruction methods.

Refractive index matching is typically achieved with combinations of silicone oils in Scanning Laser Optical Tomography. This leads to destruction of some types of samples such as silicone waveguides. A correction method was developed which allows image acquisition with high quality without perfectly matching the refractive index of medium and sample. This enables the use of water as the immersion medium instead of silicone oil. Promising results with high image quality are presented.

Deep Ultraviolet (UV) microscopy enables high-resolution, molecular imaging and typically yields 2D images representing the axial projection of a sample’s 3D absorption onto a plane. In this work, we present a tomographic imaging approach based on multispectral UV microscopy, to visualize complex 3D structural features in samples. We aim to employ through-focus intensity images captured with varying partially coherent and asymmetric illumination patterns to extract the 3D absorption and refractive index (RI) distributions of the sample at distinct UV wavelengths. The recovery procedure relies on solving the inverse scattering problem using the 3D optical transfer function of our microscope.

The neonatal mouse heart in contrast to the adults has the ability to regenerate after myocardial infarction. The investigation of cardiac morphogenesis is critical to uncover the underlying mechanism of cardiac regeneration. Hence, we have developed a light-sheet microscope (LSM) along with the tissue-clearing method to investigate the 3-dimensional (3D) architecture of the intact neonatal mouse heart. We improved our imaging system by incorporating an axially swept remote focusing arm with a voice coil actuator for isotropic imaging. Our LSM offers a lateral resolution of 2.08 ± 0.11 μm and an axial resolution of 2.84 ± 0.19 μm. Using this strategy, we can take optical sections throughout the intact heart and enabling us to reveal the cardiac structure of the neonatal mouse.

Optical techniques enable the non-contact surface measurement of three-dimensional objects at sub-micrometer resolution. However, they typically require the acquisition of multiple images at different axial positions, a time-consuming task that strongly limits imaging speed. Here, we propose Encoded Search Focal Scan (ESFS), a method able to reconstruct sample height maps from an order-of-magnitude reduced set of images. We demonstrate ESFS measurements over axial ranges exceeding 100 µm at sub-micrometric resolution while using only 8 input images. This leads to striking reconstruction rates of 67 topographies per second, opening the door to the real-time characterization of large and rapidly moving systems.

We introduce an innovative MCAM architecture using a 6x8 array of 48 lenses and sensors for simultaneous 0.624 gigapixel imaging within a few centimeters, delivering near-cellular resolution. This enables 3D video recording and radiometric fluorescence imaging of organisms using stereoscopic capture and appropriate filters. Such a feature proves advantageous when conducting combined investigations into organism behavior and functional fluorescence measurements. Moreover, the MCAM is equipped to perform birefringent imaging by incorporating suitable polarizers. We demonstrate the multimodal imaging capacity of this system using a variety of specimens, notably Drosophila, and zebrafish.

Multispectral fluorescence lifetime imaging microscopy (λFLIM) is a powerful optical technique to investigate biological processes, which generally requires long acquisition time. Single Pixel Camera (SPC) is an imaging architecture base on Compressive Sensing (CS) techniques which allows to strongly reduce the acquisition time while preserving the information content at the cost of an increased computational time. In this work we present a λFLIM microscope based on CS-SPC architecture. We have tested the multiscale capability of the system by merging SPC zooming with data fusion and proposed a fast fitting framework, which runs in parallel with the acquisition, allowing a fast visualization.

HiLo microscopy is a widefield optical sectioning technique that involves computational reconstruction from two images, one with structured illumination and the other with uniform illumination. A variety of methods, including speckle and periodic grids, can be employed to achieve structured illumination. In this study, we introduce a novel HiLo strategy that utilizes an off-the-shelf holographic diffuser and a low-coherence LED source to generate random caustic patterns. This method offers several benefits over existing ones, such as simplicity and cost-effectiveness. We achieve 4.5 µm optical sectioning capability with a 20x 0.75 NA objective and demonstrate the performance of our method by imaging a 400 µm thick, highly scattering brain section. We anticipate that our caustic-based structured illumination approach will augment the versatility of HiLo microscopy and extend to various imaging applications.

In widefield fluorescence imaging, out-of-focus and scattered light emanating from the cell body frequently obscures nearby dim fibers and degrades their contrast. Scanning techniques can ameliorate this issue but are hindered by a slower imaging speed and higher cost. We dramatically reduce stray light in widefield imaging by directing illumination primarily to neuron fibers. We identify fibers through real-time iterative image processing and pattern illumination onto these fibers using a digital micromirror device in a standard widefield microscope. By illuminating bright cell bodies with minimal light and in-focus fibers with high light intensity, we diminish the background and enhance the fibers' visibility. This methodology retains a high imaging speed and remains cost-effective. Employing this targeted illumination strategy, we have achieved confocal quality imaging of complex neurons in anesthetized C. elegans, ex vivo mouse brain slices, and restrained zebrafish larvae.

It is challenging to study behavior of and track freely-moving model organisms using conventional 3D microscopy techniques. To overcome motion artifacts and prevent the organism from leaving the field of view (FOV), existing techniques require paralyzing or otherwise immobilizing the organism. Here, we demonstrate hemispheric Fourier light field tomography, featuring a parabolic objective that enables synchronized multi-view fluorescence imaging over ~2pi steradians at up to 120 fps and across multi-millimeter 3D FOVs. Our method is not only able to track the 6D pose of freely-moving zebrafish and fruit fly larvae, but also other properties such as heartbeat, fin motion, jaw motion, and muscle contractions. We also demonstrate simultaneous multi-organism imaging.

We have devised an innovative SIM imaging system to addresses these issues and achieves the following: (1) Aberration free super-Resolution Imaging: We introduce a hybrid adaptive optics system, TACO, correcting both illumination and excitation aberrations. This results in high-quality SIM super-resolution imaging even in highly aberrative environments. (2) Scalable multicolor Imaging: A dispersion compensation grating module was designed to correct the divergence of different excitation wavelength beams, enabling effortless increasement of color channels number with minimal realignment. (3) Multiplane 3D Imaging: Simultaneous multiplane imaging is achieved through a specialized multi-plane beam splitter. A deformable mirror facilitates rapid z-scan and aberration correction, ensuring fast, aberration-free volumetric imaging. (4) Automated Structure Decomposition: Our system adapts a convolutional neural network model with a novel architecture for automated decomposition of multiple cellular structures in a single channel. This innovation significantly broadens the application of SIM in complex high-dimensional data acquisition tasks.