Liver-on-a-chip is a 3D in vitro hepatic microphysiological system aiming to recreate the conditions of liver tissue on a microscopic scale. CN Bio microphysiological system (CN Bio Innovations, UK) is one of the advanced liver-on-a-chip models. In this study, a multimodal optical imaging platform incorporating nonlinear optical imaging techniques such as multiphoton microscopy (MPM), fluorescence lifetime imaging microscopy (FLIM), coherent anti-Stokes Raman scattering (CARS) microscopy, and simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used for characterizing the structural and functional changes associated with inflammation, lipid accumulation and drug uptake in the CNBio liver-on-a-chip model.

We introduce FOCALS (Fast Optical Coherence, Autofluorescence Lifetime imaging, and Second harmonic generation) microscopy capable of generating NAD(P)H fluorescence lifetime, second harmonic generation (SHG), and polarization-sensitive optical coherence microscopy (OCM) images simultaneously and equipped with closed-loop sensorless adaptive optics (AO). Multimodal imaging generates quantitative metabolic and morphological characterization of biological samples and the complementary contrasts improve the overall specificity of our setup. We highlight the versatility of our setup to image seconds-scale dynamics of in vitro mammalian cells, ex vivo tumor and neural environments, and in vivo skin imaging with sub-micron resolution simultaneously.

Time multiplexing is a super resolution technique that sacrifices time to overcome the resolution reduction obtained because of diffraction. In structured illumination super-resolved imaging, high resolution and time changing patterns are projected on top of an object and a set of low resolution images are captured with the low quality imaging system. The set of low resolution images are digitally decoded with decoding patterns that are based on the high resolution projected encoding patterns. In conventional structured-illuination approaches, the projected encoding patterns need to be a priori known in order to be used in the decoding process. In this presentation, we will describe an enhancement of structured illumination approach towards label free imaging while obtaining the super resolved result without the need of requiring the a priori knowledge on the projected encoding patterns.

Optical imaging techniques with physical tissue sectioning have become indispensable tools. However, acquiring volumetric anatomy of multiple organs and statistical studies remain a difficult challenge due to light scattering and long data acquisition period. Here, we propose a novel protocol for the high-throughput and quantitative analysis of 3D mouse organs using Coherence gating imaging (CGI) with tissue clearing. For statistical analysis, we also applied deep learning and outcomes were compared with computed tomography. Our preliminary results can improve imaging depth as well corresponding acquisition time, which would be promising tool for 3D digital histopathology.

There is an urgent need for predictive platforms for response to immunotherapy in patients. In vivo phenotyping of tumor-immune microenvironment (TiME) for predicting response to immunotherapy was evaluated using non-invasive reflectance confocal microscopy (RCM) in skin cancer patients. Phenotypes were correlated with underlying biology and response to topical immunotherapy. Using both inflammation and vasculature features, four major phenotypes were observed. The VaschiInfhi phenotype correlated with high immune activation, exhaustion, and vascular signatures while VaschiInflo with endothelial anergy and immune exclusion. Highest response to immunotherapy was seen in VascloInfhi phenotype. This study establishes proof-of-concept for in vivo TiME phenotyping in patients.

Partial Wave Spectroscopy (PWS) is a nanoscale sensitive imaging technique, emerging as a potent modality for minimally invasive and cost-efficient early risk stratification for colorectal cancer. Feature engineering on the PWS map of nuclear chromatin structures, we developed 50 features extracted from the map as potentially representative of future colorectal cancer development risk. Along with feature extraction utilizing convolutional neural networks, classification model is developed with 10-fold cross validation from a multicenter data consisting of a total of 187 patients. Our newly developed feature engineering results in improved performance of the early risk stratification model for colorectal cancer.

Brillouin microscopy is a noncontact all-optical method for biomechanical imaging. Whilst spontaneous Brillouin scattering microscopy has been widely used in biology, stimulated Brillouin scattering (SBS) microscopy has been mainly used for the examination of materials. Recently, SBS microscopy has been demonstrated useful for the mechanical imaging of live Caenorhabditis elegans nematodes. Here, we will present SBS microscopy of live NIH/3T3 cells, with no apparent photodamage for ~260mW optical power on the samples. We will discuss the importance of multicomponent spectral analysis of the SBS spectra acquired along with the high spectral resolution of the SBS microscope using information theoretic measures.

Label-free intravital optical imaging, and imaging of fresh, unstained, resected tissue specimens, offer a wealth of new biomarkers for revealing the true colors of cancer and for diagnosing disease. Using new fiber-laser-pumped photonic crystal fiber source technology and nonlinear optics to generate new excitation wavelengths and manipulate the light stimulus in new ways, Simultaneous Label-free Auto-fluorescence Multi-harmonic (SLAM) microscopy can achieve fast simultaneous visualization of not only architectural features, but also the rich intrinsic molecular and metabolic features within tissues. Quantitative machine/deep learning analyses of these multi-dimensional datasets can be used to identify selective biomarkers for cancer, such as cancer-associated extracellular vesicles. The clinical demonstration of these optical biomedical imaging technologies offers new paradigms for point-of-procedure diagnosis and guidance.

We report the development of a novel point-scanning frequency-domain (FD) fluorescence lifetime imaging (FLIM) system. The system, which was customized for label-free metabolic and biochemical imaging of oral epithelial tissue, is capable of simultaneous excitation at 375nm and 445nm, emission detection across 4 spectral bands, real-time fluorescence modulation and phase lifetime calculation, and low-cost data acquisition implementation using an Abaco FMC104 digitizer, Xilinx Zedboard FPGA, and Python software on the host computer. In this work we discuss our challenges and workflow with the goal of helping fellow researchers develop versatile and cost-effective FLIM systems for biomedical and clinical imaging applications.

Intravital multiphoton microscopy of the metabolic co-enzymes NAD(P)H and FAD (optical metabolic imaging, or OMI) provides label-free imaging of metabolic changes in vivo. Since the metabolism of tumor and immune cells is associated with cancer progression, we aim to study metabolic changes during a triple-combination immunotherapy regimen that cures murine melanoma tumors. Our results demonstrate that intravital OMI can capture tumor and T cell autofluorescence intensity and lifetime changes during immunotherapy. Overall, this technology enables analysis of single cell metabolic changes in vivo to provide insight for immunotherapy development.

Gelatin-methacrylate (GelMA) yields tunable hydrogels that are used in studies involving drug delivery and tissue engineering. Methods to validate their physicochemical properties however are typically destructive or require labeling, and there is a need for non-invasive techniques capable of evaluating dynamic changes in hydrogel properties. Here we demonstrate the sensitivity of label-free fluorescence lifetime imaging (FLIM) to alterations in GelMA cross-linking and pH levels. Increases in cross-linking shorten long-lifetime (τ2) species, while increases in pH shorten the short-lifetime (τ1) components of green autofluorescence. These findings demonstrate a non-destructive, label-free method with broad applicability in evaluating hydrogel properties.

Near-Infrared Photoacoustic Microscopy (NIR-PAM) has a unique potential to label-free image lipids with high-resolution, -sensitivity, and -speed, which constitute essential biohallmarks to investigate biological functionalities and pathophysiological processes. To empower NIR-PAM for accessing the intrinsic vibrational absorption of lipids, we demonstrate a novel stimulated Raman scattering (SRS) fiber amplifier that delivers optical excitation at ~1200 nm, >200 kHz repetition rates, <2 ns pulse durations, and 5 µJ pulse energies. We apply NIR-PAM to resolve intrinsic lipid contrast in biomedical specimens ranging from single-cells to lipid-rich tissue with subcellular resolution.

Full Field Optical Transmission Tomography (FFOTT) relies on forward scattering using the Gouy’s phase shift modulation that is achieved close to the focus of a microscope objective. FFOTT principle is different than Full Field OCT (FFOCT) that relies on backscattering of light, This new type of endogenous cell imaging technique offers structural and metabolic contrasts and is particularly well suited for imaging cell culture on glass slide or Petri dishes avoiding fringes that mask cells in FFOCT as well as biological structures such as biofilms. The sectioning ability is close to confocal microscopy but no contrast agent is required.

Being a label-free technique, iSCAT has emerged as a powerful tool complementary to fluorescence microscopy because its superb spatio-temporal resolution holds great promise in biological investigations ranging from detection of nm-sized proteins to real-time dynamics of organelles in vivo. Here we present the polarization-selective iSCAT (psiSCAT) microscopy to detect the orientation and anisotropy of nano-objects. We also succeeded in developing the remote-focusing iSCAT (RF-iSCAT) to realize fast, vibration-free, 3D imaging over a long z-tracking range (< 5 μm). We envisage that our achievement would greatly advance the field of single-particle tracking into new dimensions with superb precision and sensitivity.

We present a non-invasive (label-free) and 3-D method for anti-cancer drug response evaluation of human originated tumor spheroids. Our method is a combination of an OCT microscope and a statistical analysis framework of the OCT signal temporal fluctuations induced by the intra-cellular motions. This method allows the visualization of the 3-D metabolic activity of tumor spheroids and its alteration induced by the drug applications. Two human originated tumor spheroid’s drug responses were evaluated including breast adenocarcinoma (MCF7 cell-line) and colorectal carcinoma (HT-29 cell-line). The drug response was not only visualized as an images, but also quantitative analysis has been performed.

We demonstrate volumetric phase contrast imaging by using optical coherence tomography (OCT). In general, the randomness of the scatterers’ distribution prohibits the volumetric measurement of a meaningful phase in a scattering mode. Our method uses complex numerical manipulation of an en-face complex OCT and gives a transversally differential phase image similar to a differential interference contrast microscope (DIC). Not like the DIC, our method can arbitrarily select the amount and direction of the shear after the OCT acquisition. In addition it provides DIC-like images at arbitrary depths. This method is validated by using a 840-nm spectral domain OCT system. A zebrafish sample is measured over a 1-mm × 1-mm transversal scanning range.

A polarization sensitive Jones matrix optical coherence tomography (JM-OCT) prototype was utilized to investigate in vivo wildtype zebrafish in different development stages. JM-OCT is a functional extension of conventional intensity-based OCT to visualize polarization properties of samples. We demonstrated simultaneous structural and functional imaging of in vivo wildtype zebrafish at the age of eight-days, one and two-months post-fertilization. The scattering and polarization characteristics of the anatomical features, such as the eye, and the muscles, were investigated and quantified non-invasively and in three-dimensions. Furthermore, the setup was able to visualize dynamic processes like the blood flow in a label-free way.

We introduce the cargo-localization iSCAT microscopy, label-free, live-cell imaging technique, that visualizes the traffic of cargos in a massively parallel fashion and hence uncovers the actively-used cytoskeletal network from cargos’ traces. By applying a particle localization algorithm to over a hundred cargos moving in a lamellipodium, the fine architecture of the cytoskeletal highway could be reconstructed. We also discuss a host of interesting kinetic events associated with cargo transport such as intermittent pausing, turnover, and frequent jamming of cargos due to physical collision with other obstacles or at the intersection of the highway.

Typically, tomographic reconstruction of dynamic 3D samples is limited by sample movement speed and illumination scanning speed. However, using the six-pack holography technique that we have developed, 6 off-axis holograms of different illumination angles can be acquired in a single camera exposure, without sacrificing individual image resolution or field of view, and without requiring sample sparsity. Using these 6 holograms we reconstruct the 3D refractive index tomogram of a dynamic sample from each video frame using the optical diffraction tomography algorithm. We demonstrate our technique experimentally on flowing microbeads and swimming microalgae.

Label-free microscopy methods have been developed to measure isotropic (phase) and anisotropic components of the uniaxial permittivity tensor (uPT) separately. These methods are now broadly used to analyze biological architecture. Few methods have combined these measurements, but they do not provide complete measurement of uPT considering diffraction. Here we report a computational microscopy method, termed uniaxial permittivity tensor imaging (uPTI) for diffraction-aware measurements of uPT. The invisible uPT is converted into visible intensity variations using add-on microscopy modules for asymmetric illumination and polarization-sensitive detection. We develop a vectorial partially coherent imaging model that describes the intensities in terms of uPT. We retrieve the uPT with a multi-channel deconvolution algorithm. We demonstrate the multi-modal high-resolution imaging of biological specimens with uPTI.

Disorder strength is a metric that is used to measure cell stiffness. Through calculating disorder strength from cell phase images reconstructed from various QPI systems, we have discovered that this parameter differs by system used for acquisition. With our model, we demonstrate that disorder strength is highly dependent on image resolution. When corrected with our simulation model, disorder strength measurements from different QPI systems can be directly compared and enable cross-system analysis. Cell disorder strength infers important biophysical information on cellular stiffness of normal cells, carcinogen-exposed cells, and cancer cells.

Interferometric microscopy techniques have been recently developed to detect small particles at the nanoscale without the need for specific labeling. Here, we introduce a highly sensitive, label-free approach on a common path interferometric set-up working on a transmission that amplifies weak scattering signals coming from small particles. Using single-particle tracking analysis, we can track and differentiate viruses and other biotic particles in aquatic environment. Furthermore, we have developed a fast assay based on antibody recognition of targeted virus in solution. We associate changes in diffusion and in the interferometric signal of the detected particles with the immune reaction between antibodies and surface proteins of the virus.

Digital holographic microscopy (DHM) has been used in numerous successful studies in materials and life sciences. It is well known that the light source coherence, useful to generate high-quality interference patterns that encode the phase in an extremely accurate manner, generates coherent noise (CN), which precludes to adequately address some important applications, especially when single-shot fast recording is required. We propose an original approach, called polychromatic DHM, which thanks to the reconstruction of several holograms acquired at different wavelengths provides quasi-CN-free optical path difference images. Preliminary results concerning materials and life sciences will be presented.

Functional composite materials that can change their spectral properties in response to external stimuli have a plethora of applications in fields ranging from sensors to biomedical imaging. One of the most promising types of materials used to design spectrally active composites are fluorescent single-walled carbon nanotubes (SWCNTs), non-covalently functionalized by synthetic amphiphilic polymers. These coated SWCNTS can exhibit modulations in their fluorescence spectra in response to their interaction with target analytes. Hence, identifying new amphiphiles with interchangeable building blocks that can form unique coronae around the SWCNT, tailored for a specific application is of great interest. This study presents highly modular amphiphilic polymer-dendron hybrids, composed of hydrophobic dendrons and hydrophilic polyethylene glycol (PEG) that can be synthesized with high degree of structural freedom, for suspending SWCNTs in aqueous solution. Taking advantage of the high molecular precision of these PEG-dendrons, we show that precise differences in the chemical structure of the hydrophobic end-groups of the dendrons can be used to control the interactions of the amphiphiles with the SWCNT-surface. These interactions can be directly related to differences in the intrinsic near-infrared fluorescence emission of the various chiralities in a SWCNT sample. Utilizing the susceptibility of the PEG-dendron towards enzymatic degradation, we demonstrate the ability to monitor enzymatic activity by changes in the SWCNT fluorescent signal. These findings open new avenues for a rational design of SWCNT functionalization, and optical sensing of enzymatic activity in the near infra-red spectral range.

We present a minimally invasive, needle-injectable, wirelessly-powered diffuse optical spectroscopy sensor implant that fits within a 12G breast biopsy needle. The sensor uses red and near-infrared lasers to measure tumor hemoglobin concentration changes. It contains two photodiodes controlled by an analog front end integrated circuit with an integrated transimpedance amplifier. This ultralow power sensor is wirelessly powered using near-field resonant inductive coupling and communicates via backscattered load shift keying. Characterization in liquid tissue mimicking phantoms showed a response to absorption concentrations consistent with estimated tumor hemoglobin concentrations. After implantation in murine breast cancer models, tissue stainings revealed no adverse effects.

Currently there are no biomarkers for ovarian cancer. We discover an ovarian cancer biomarker using mass spectrometry and use microtoroid resonators for label-free ultra-sensitive detection of this marker in ovarian cancer tumor-implanted mice. After characterizing the sensor response with standard solutions, we then perform a quantitative analysis to investigate the concentration of the biomarker in several mouse vaginal lavage samples. The results show that the biomarker levels increase with time, revealing the progression of tumors implanted in mice.