Measuring single molecules’ 3D orientations simultaneously with their 3D spatial localization is currently a topic of intense efforts in optical design. We have developed different methods based on polarized imaging, that are capable to report both 3D spatial localization and 3D orientation (including wobbling) from single molecules. We present how the design of such schemes can be adapted to account from experimental constraints versus accuracy and precision of the estimation of both orientation and localization parameters. We illustrate such performances on actin structural STORM imaging in complex and dense meshworks in fixed cells.

FMCW depth imaging is a coherent 3D imaging modality analogous to SS-OCT. Due to constraints of mechanical steering mirrors, meter-scale FMCW depth cameras typically suffer from low data rates (<1Hz 3D map rate). Here, we describe the design and construction of a high-speed FMCW depth camera that employs a grating for beam steering and a telescope for angular FOV magnification. Our camera produces 3D depth maps at 33Hz, each consisting of 475x500 pixels, spanning a depth range of 32.8cm with sub-millimeter depth localization accuracy. Our FMCW depth camera is suitable for room-scale real-time 3D imaging applications, particularly computer vision applications.

Phasor domain clustering of Fluorescence Lifetime data has gained a lot of attention because of fit-free algorithm and the ability to resolve heterogeneity of the sample. Despite the popularity, the optimal modulation frequency has not been discussed yet. Especially, an optimal condition must be considered where the two clusters are too close to discriminate, or it is challenging to acquire enough photons. In this study, we found optimal modulation frequency to resolve two clusters in the phasor domain. We theoretically derived the Davies-Bouldin index (DBI) of two clusters in the phasor domain and predicted the optimal frequency based on it. We verified the theoretical prediction via two different fluorescence droplets experiments.

For increased efficiency and standardization, automated urine screening is highly desirable. We explored the capabilities of quantitative phase imaging (QPI) by digital holographic microscopy (DHM) for the characterization and classification of urine sediments based on biophysical parameter sets. Digital off-axis holograms from a liquid control for urine analysis were acquired with a modular DHM system attached to a commercial optical microscope. From the retrieved quantitative phase images, various particle morphology parameters were extracted to differentiate and quantify particles contained in the test samples. Our results show that DHM represents a robust and promising tool for t label-free characterization of urine sediments with enhanced throughput.

Cerebral microhemorrhages (CMHs) occur due to ruptures in cerebral microvessels that cause deposits of blood in the brain. Hypertension (HTN) is a major risk factor for CMHs, which have been associated with cognitive decline and ischemic strokes. Despite the clinical significance of CMHs, our understanding of CMH formation remains limited. To address this gap, our group has employed a perfusion-based vascular label with tissue clearing to enable three-dimensional visualization of CMHs with the surrounding microvasculature in HTN mice. Vessel diameters surrounding a CMH were approximately 4.22±0.81 µm. Vessel density in CMH positive tissue regions was approximately 0.083±0.017 µm-1.

Conventional microscopy usually has a single function aiming to measure one type of signal, such as fluorescence microscopy or phase microscopy. Here, we develop a new computational hybrid imaging method, based on a multi-slice multiple scattering model, that reconstructs both 3D fluorescence and 3D RI by solving an inverse problem from a single dataset of dozens fluorescence images captured at a single image plane. This multi-functional system not only bridges the gap between fluorescence and RI imaging, but also can digitally correct multiple scattering effects in the fluorescence images by using the phantom structure recovered by the reconstructed 3D RI.

Quantitative measurements of the polarization state of light at various points within a photonic integrated circuit is a challenging but important problem in the packaging and testing of photonic systems. We analyze and experimentally test polarimetric microscopy of several types of engineered subwavelength scatterers designed for use in a silicon photonics foundry process.

Saturated-excitation (SAX) improves the spatial resolution of laser scanning microscopy in three dimensions by inducing nonlinear fluorescence signals that localize within a focus spot. However, the spatial resolution of SAX microscopy is practically limited by the signal-to-noise ratio (SNR). In this research, we introduce image scanning microscopy (ISM) to improve the SNR of SAX microscopy. The improvement of the SNR by ISM enables the detection of weak nonlinear signal components and contributes to the improvement of the spatial resolution of SAX microscopy in practice.

Image Scanning Microscopy (ISM) super-resolution microscopy has gained momentum for its almost instantaneous improved resolution capabilities. Multiphoton ISM utilizes the shorter emission wavelength and confocal acquisition by exploiting each pixel on the camera as a pinhole, and numerical enhancement to achieve sub-diffraction-limit resolution. We present the use of a multiplexed approach for signal acquisition using a regular EMCCD camera. A spatiotemporal modulation scheme is employed to direct the ultrafast laser pulses to select foci within a field-of-view. Combined with a novel image acquisition method, we show that only 49 images are required to achieve a resolution of 100 nm.

This talk will provide an overview of the local description of polarization for nonparaxial fields, for which all three Cartesian components of the vector field are significant. The polarization of light at each point is characterized by a 3x3 polarization matrix, as opposed to the 2x2 matrix used in the study of polarization for paraxial light. For nonparaxial light, concepts like the degree of polarization, the Stokes parameters and the Poincare sphere have generalizations that are neither unique nor trivial. This presentation aims to clarify some of these discrepancies and provide a framework that highlights the similarities and differences with the standard description for the paraxial regime, emphasizing geometric interpretations. Finally, the application of these concepts to super-resolution fluorescence microscopy will be presented.

Hyperpsectral fluorescence imaging has been gaining its popularity in life-science field for its simultaneous multiplexing capability of multiple fluorescent labels. Traditional diffraction grating-based hyperspectral acquisition has limited photon-throughput due to the loss at the diffractive optics. The uniform spectral sampling using multiple narrow spectral bands also limits the detectable photons for each channel, which limits the imaging speed as longer exposure is required to achieve sufficient signal to noise ratios. Here we present a Fourier transform-based spectral sampling strategy based on high efficiency dichroic mirrors, enabling video-speed snapshot acquisition with the capability of multiplexing more than five fluorescent signatures.