In this work, we propose a deep-UV LED-based imaging modality for generating AF images on human prostate paraffin sections. The achieved lateral resolution is around 1 µm. Moreover, a virtual staining framework is used to style transfer the AF images into clinical standard H&E images with an MS-SSIM > 0.65 between the virtually stained images and their corresponding ground truth images. The high similarity between the two domains is recognized by medical professionals, illustrating the high accuracy of the virtual staining algorithm and the clinical importance of this work for the post-operative histopathology workflow.

The efficient transition of hPSCs to definitive endoderm (DE) progeny is an essential step toward disease modeling and the manufacturing of a wide range of cellular therapeutics in medical relevant quantities. Two-photon excited fluorescence (TPEF) imaging, as a non-invasive, non-destructive, label-free modality for metabolic studies, reveals the distinct metabolic switches during DE differentiation in a real-time monitoring mode. Since metabolic pathways orchestrate important regulatory mechanisms that influence and determine cell fate decisions, TPEF imaging serves as an important enabling technology in hPSC-based tissue engineering applications affording non-invasive determination of metabolic biomarkers and informing optimizations of hPSCs differentiation processes.

Real-time visualization of burn severity and hypoxic response is important in understanding wound healing outcomes. Herein we propose a novel approach to macroscopically image burn induced hypoxia through endogenous PpIX delayed fluorescence. The approach is validated in-vivo using athymic nude mice with induced full-thickness burns. PpIX delayed fluorescence imaging was performed longitudinally for 3 days post injury. The localization of PpIX signals was also correlated to Indocyanine Green fluorescence imaging, a technique for blood perfusion and tissue injury in the burn wound. The current results highlight the capability of the approach for real-time in-vivo quantification of hypoxic response to burn wounds.

We report a fiber-based imaging system that combines autofluorescence and reflectance spectroscopy for rapid and multidimensional characterization of tissues. The system utilizes dual wavelength excitation and time-resolved fluorescence data to assess tissue properties in real-time. Initial experiments using human cancer specimens demonstrated the system's effectiveness in providing multiparametric tissue characterization, which could improve diagnostic and therapeutic approaches. The integration of the system with a commercial endoscope further emphasizes its clinical applicability and viability. This work showcases a potential pathway for translating this technology into clinical settings, enabling objective and quantitative evaluation of patients in real-time for diagnostic and follow-up.

We demonstrate optical redox ratio and fluorescence lifetime imaging microscopy (FLIM) of intrinsic metabolic co-factors NAD(P)H and FAD to quantify single cell metabolism and function. This approach is attractive because it does not require cell surface labels or transfection, enabling rapid assessment of dynamic events. Multiphoton microscopy provides near infrared excitation of these autofluorescent molecules, thereby maximizing cell viability. Newly trained neural networks automatically segment single cells for analysis of heterogeneity within and between patients. Overall, this approach is attractive for both basic research and patient management in cancer and immunology.

Label-free nonlinear microscopy allows for high-resolution and three-dimensional imaging of live biological specimens without the need for exogenous labels. The integration of multiple modalities further enhances molecular specificity and visualization diversity for metabolic and structural mapping of heterogeneous tissue architectures. In this work, we introduce high-speed simultaneous label-free autofluorescence-multiharmonic (hSLAM) microscopy, where a high-peak-power adaptive fiber source based on multimode fiber (MMF) is employed with a nonlinear fiber piano. We will also talk about how the higher speed SLAM enables multicellular dynamics in living tissues with higher spectral flexibility and peak power, providing new possibilities for bioimaging.

Efficient cell line development is crucial for optimizing biopharmaceutical production. We demonstrate the potential of SLAM and FLIM microscopy to optimize this process by correlating metabolism-related features with measured productivity in early CHO cell passages. Eight CHO cell lines were imaged using SLAM and FLIM microscopy, and a pipeline was developed to classify the cells. A linear SVM achieved 95% accuracy in predicting productivity. Important features and their channel affiliations were identified, revealing optical metabolic characteristics from NAD(P)H and FAD associated with productivity. SLAM features correlated with growth and viability, while FLIM features correlated with protein production, highlighting the importance of multimodal label-free imaging.

Induced pluripotent stem cells (iPSC) can be differentiated into cardiomyocytes (CM) for disease modeling and drug screening. Batch variability in differentiated cells frequently occurs due to heterogeneity in commercial culture substrates. We performed optical metabolic imaging (OMI) on synthetic hydrogels of varying stiffnesses and compositions and observed expected metabolic shifts as iPSCs differentiated. OMI revealed metabolic differences between cells cultured on heterogenous commercial substrates and synthetic hydrogels and can be used to monitor cell sensitivity to the microenvironment. These data demonstrate OMI is a powerful tool for identifying iPSC differentiation and maturation conditions, crucial in stem cell manufacturing.

Our past contribution was to introduce a Gouy phase Full Field Optical Transmission technique applied to detect and characterise nanoparticles such as virus, vesicles and nano plastics in terms of size and refractive index. More recently we have adapted this interferometric approach to achieve optical tomography in cells and tissues. We will show improvements in sensitivity and resolution obtained with 20 nm virus as well as with biological tissues using both a static mode (morphology) and dynamic one (metabolic contrast). We discuss the importance of the illumination spatial coherence and compare the results with those obtained with Full Field OCT.

Cerebral microvascular alterations are increasingly recognized as important contributors to neurodegenerative diseases, such as Alzheimer's Disease (AD) and chronic traumatic encephalopathy (CTE). To characterize these changes, we used a custom serial-sectioning, polarization-sensitive optical coherence tomography system to collect volumetric images of brain tissue volumes from the dorsolateral frontal lobe of normal controls and subjects with pathologically confirmed AD and CTE. We performed vasculature segmentation on each tissue volume and then analyzed the geometric properties of the vascular networks. The preliminary results suggest reduced microvascular density in AD compared to both CTE and NC.

The structural and functional imaging of ophthalmic tissues in cellular level play an important role in the understanding and evaluation of the physiology and pathology of ophthalmic diseases. In this study, we developed a dual-mode full-field optical coherence tomography (FFOCT) that is capable of acquiring label-free cellular images of freshly excised ophthalmic tissues, achieving static contrasts gained from structural refractive index gradients and dynamic contrast induced by endogenous cell motility related to cell functions. Through imaging experiments on both normal and pathological ophthalmic tissues, we show that while the static FFOCT images better reveal the relative stationary cellular structures like nerve fibers and collagens, the dynamic FFOCT images show enhanced contrast of various transparent cells with active intracellular metabolic motions, offering complementary information of major corneal and retinal layers. Our study has shown the dual-mode FFOCT system is a straightforward promising technique for cellular imaging exploration and pathological analysis of ophthalmic tissues.

This presentation aims at classifying, mathematically modeling, and numerically simulating the intratissue activities for better understanding the dynamic optical coherence tomography (D-OCT) imaging. The intracellular motilities are classified into six types, and each type of motility is modeled by mono-direction translation, random ballistic, diffusion motions and their combination. The intracellular scatterer dynamic and D-OCT signals including logarithmic intensity variance (LIV) and complex variance were numerically simulated. These D-OCT signals increase within the velocity range of 4.5 to 270 nm/s and become almost constant for larger velocities. In addition, it was found that the shorter wavelength gives higher LIV and complex variances.

We investigate wavelength and resolution dependency on dynamic optical coherence tomography (D-OCT) signal using two OCT systems. One is 840-nm spectral domain OCT. The other is 1310-nm swept-source OCT. We kept the resolution roughly the same between the two systems by computationally reducing the axial resolution of 840-nm system. In addition, we performed numerical simulation by modeling the intracellular scatterer dynamics by a diffusion. Experimental results showed that longer wavelengths correspond to lower D-OCT, while lower resolutions lead to slightly lower D-OCT. Simulation results confirm the wavelength dependency of D-OCT, with resolution variations having non-significant impact.

Whispering gallery mode microtoroid optical resonators are one of the most sensitive and rapid sensors in existence. However, the need for a tapered optical fiber to couple light into these sensors hinders translation out of laboratory as the fiber is fragile and suffers from mechanical vibration. Here, we developed a free space coupling system together with a compatible fluidic platform which allows monitoring of molecular interactions in real time. We obtained Q-factors as high as 1.6×10^8 with this approach. The large effective coupling area, ~10 μm in diameter for a numerical aperture of 0.14, provides high coupling stability during biosensing experiments.

Esophageal cancer’s increasing prevalence coupled with a 5-year average survival rate below 20% due largely to late detection indicates a significant need for improved imaging tools that can detect and localize early, unseen lesions and be incorporated into endoscopy for screening and evaluation of early symptoms. While white light imaging or virtual chromoendoscopy contrast-enhancement techniques like narrow-band imaging have largely seen commercialization, there remain emerging label-free imaging-based techniques that show promise for improving diagnosis and biopsy guidance. Among them we investigate the clinical potential of hyperspectral (HSI) and autofluorescence imaging (AFI) which lend themselves well to implementation in an endoscopic system. We performed ex-vivo imaging on esophageal biopsies suspicious for carcinoma (N=11) and/or Barrett’s esophagus (N=6) and adjacent normal appearing squamous mucosa in the same patient as controls. Our results indicate AFI and HSI are both promising imaging modalities for detecting and localizing morphological and metabolic changes associated with esophageal cancer.

In this presentation we will present an all-optical pump-probe approach for non-contact photo-acoustic sensing and imaging. We will show various configurations for enhancing the obtainable spatial and temporal resolution as well as usage of the proposed sensing technique for various important applications. The presented pump-probe approach allows localized and direct technique for performing photoacoustic imaging while the focused pump beam excites the acoustic signal, and a focused probe beam does the spatial-temporal analysis of the diffracted speckle patterns. This analysis is done in one configuration by analyzing time changing contrast of the time varying speckle patterns and in another sensing configuration, where the speckle pattern do not vary but rather laterally move, by tracking the position of the correlation peak. Due to its advantage of performing localized photo-acoustic sensing, the proposed concept can allow sensing in solutions or other mediums typically problematic for conventional transducers-based photo-acoustic sensing.

The detection and quantification of circulating tumor cells (CTCs) in a patient’s bloodstream is a promising minimally invasive avenue for improving cancer diagnostics and prognostics. However, clinical utilization of CTC-based diagnostics has been limited due to the arduous nature of currently employed CTC detection strategies which largely involve tedious staining and purification procedures. We report the use of photoacoustic remote sensing microscopy to enable the label-free visualization of peripheral blood mononuclear cells for the purpose of categorizes white blood cell populations and identifying putative circulating tumor cells present in the blood stream. Image analysis of cellular characteristics is performed, and identification of cell phenotype is validated by comparison to histological staining.

Collagen has hierarchical structure and often undergoes remodeling at the tumor margin [1]. Polarimetric second harmonic generation (SHG) microscopy can be used to investigate changes in the collagen structure and provide insight into the metastatic progression of cancer. In this work, we apply double Stokes-Mueller polarimetry (DSMP) to investigate the disorder of collagen at the tumor margin. The disorder is analyzed at several levels of spatial organization – texture analysis (gray level co-occurrence matrix (GLCM) method) is applied at the microscopic tissue organizational level, while ultrastructure is analyzed with DSMP to obtain nonlinear susceptibility tensor for each image voxel. This allows to comprehensively investigate the changes occurring in collagen during tumor progression. [1] Winkler, J. et al., “Concepts of extracellular matrix remodelling in tumour progression and metastasis”, Nat Commun 11, 5120 (2020).

Vascular imaging is an essential tool for understanding tissue health in the diagnosis and treatment of disease, especially for applications in cancer and ophthalmology. This work explores the application of Photon Absorption Remote Sensing (PARS) microscopy to several established in vivo vasculature models including chicken embryo and mouse ear. Significant system improvements are presented which have enabled the repeatable acquisition of images that are larger, require less scan time, and capture detailed absorption contrast of both radiative and non-radiative relaxation. The presented work demonstrates PARS as a promising platform for future clinical evaluation of disease pathology and treatment efficacy.

The latest innovations in label-free virtual-histology using Photon Absorption Remote Sensing (PARS) are presented. PARS captures endogenous absorption (radiative and non-radiative) and scattering contrasts, enabling label-free biomolecule specific imaging. Combined with innovations in AI and signal processing, PARS provides several label-free virtual histochemical stains (e.g., H&E, T-blue) directly from a single scan of an unprocessed tissue specimen. Results validated through clinical efficacy studies indicate that PARS virtual staining is diagnostically indistinguishable from gold standard chemical histology. This represents a crucial milestone in deploying PARS as an alternative to standard histopathology.

This presentation will highlight the unique opportunities to assess metabolic and mitochondrial function and/or dysfunction afforded by label-free, two photon imaging. The high resolution of two-photon images enables unique assessments of the spatiotemporal heterogeneity of metabolic and mitochondrial dysfunction, which is recognized increasingly as a significant driver of events that determine the development and/or treatment response of numerous diseases. The combined use of readouts from fluorescence intensity and lifetime images yields sensitivity and specificity to metabolic function changes. Examples to highlight such measurements in the context of cancer, vitiligo, and neurodegeneration will be presented.

Nonlinear microscopy encompasses a range of imaging techniques that leverage laser technology to reveal the chemical composition and structure of a sample. Nonlinear microscopes exploit femtosecond laser pulses to target intrinsic biomolecules of cells and tissues. Fiber lasers have limited bandwidth and reduced wavelength tunability, leading to long pulse durations and limited molecular applications. Supercontinuum generation solves this problem, enabling <50 fs pulses and a larger range of molecular excitation. This paper presents nonlinear microscopy with supercontinuum generation from a Yttrium Aluminum Garnet (YAG) crystal enabling simultaneous label-free autofluorescence multi-harmonic (SLAM) microscopy with high resolution and specificity in biological tissues.

A 2D dispersion spectrometer based on a virtually imaged phased array (VIPA) etalon and a diffraction grating has been built and characterized for Brillouin and low frequency Raman (LFR) scattering detection. With the spectral resolution offered by the VIPA etalon, the 2D dispersion spectrometer can detect Brillouin and LFR scattering with a resolution less than 500 MHz (0.017 cm-1) and a minimum of 100 cm-1 spectral range. Here we demonstrated detections of Brillouin and LFR scattering from multiple materials. This dual-functional spectrometer can acquire mechanical properties and probe the intermolecular interactions of a sample.

We demonstrate a rapid deep ultraviolet transmission and scattering microscopy system which utilizes a single 266 nm nanosecond pulsed laser source to generate virtual histology images. By detecting both the transmitted and back-scattered 266 nm radiation, distinct contrasts of the tissue section are produced. Maximally realistic virtual histology images are then generated using each contrast as separate inputs to a CycleGAN deep neural network. This work demonstrates virtual histology images of mm-scale areas at fine resolutions (< 500 nm) within minutes. This system greatly simplifies the optics and alignment required for imaging, and could provide future utility for clinical studies.

Accurate and reliable characterization of solids and liquids is essential in medical applications as well as in pharmaceutical and industrial production sites. Vibrational microspectroscopy techniques such as quantum cascade laser based (QCL-IR), Fourier Transform IR-microscopes or near field scanning imagers have been established. Here we demonstrate how modern IR- spectroscopy and imaging from micro to nanoscale can provide a deep insight on the chemical composition. Here we use a dental application as an example for highlighting the potential of combining different techniques. The different results achieved by using complementary systems are presented.

This talk will describe label-free microscopy protocols which optimize the (quantum) information acquired from the imaging interaction between the probing optical field and the imaged sample. These protocols are useful in high throughput or low dose applications, where image formation benefits from efficient use of each probing photon. We illustrate these methods with proof-of-concept demonstrations of flow cytometry, multi-photon Raman imaging and fluorescence lifetime imaging.

We have developed Polychromatic Digital Holographic Microscopy (P-DHM), providing quasi coherent noise-free OPD images, but with some limitations regarding the acquisition and reconstruction time of the multi-wavelength holograms. Our recent work, enabling a fully automated multi-wavelength hologram reconstruction associated with an increased acquisition speed, achieving a frame rate between 6 and 15 fps, while preserving OPD high sensitivity, have led to a P-DHM efficient for live-cell imaging. In addition to these technical developments, appealing applications such as the exploration of intracellular trafficking, cell migration, and neurite evolution achieved with P-DHM will be presented.

High throughput quantitative phase imaging show immense potential for clinical applications in various disease detection, however, traditional interferometric setups on optical benchtops are bulky and expensive by design. We present a low-cost, compact interferometric chamber system that would consist of merely three lenses and one beam splitter. The presented system can provide high throughput single-cell level screening of million cell sample population. In combination with customized dataset refinement algorithm and machine learning, we developed a novel technique that would allow for detection of RBC morphology-altering diseases such as sickle cell disease.

Quantitative Oblique Back-Illumination Microscopy (qOBM) enables 3D quantitative phase imaging (QPI) in thick scattering samples. Recently, we developed a deep learning assisted single-capture qOBM (SC-qOBM) method to achieve fast quantitative phase tomography in vivo. This novel approach paves the way for a number of in-vivo applications that were previously out of reach for QPI. Here, we showcase two applications of SC-qOBM: (1) in-vivo imaging of blood flow in mouse brains for studying blood diseases and (2) non-invasive imaging of blood flow in humans for hematological and in-vivo flow cytometry analysis. This work highlights SC-qOBM’s potential in various biomedical applications.

Embryo quality is a crucial factor affecting live birth outcomes. However, an accurate diagnostic for embryo quality remains elusive in the in vitro fertilization clinic. Determining physical parameters of the embryo may offer key information for this purpose. Here, we demonstrate that digital holographic microscopy (DHM) can rapidly and non-invasively assess the refractive index of mouse embryos. Murine embryos were cultured in either low- or high-lipid containing media and digital holograms recorded at various stages of development. We showed that DHM can detect spatio-temporal changes in refractive index during embryo development that are reflective of its lipid content. As accumulation of intracellular lipid is known to compromise embryo health, DHM may prove beneficial in developing an accurate, non-invasive, multimodal diagnostic.

Digital holographic microscopy (DHM) offers a label-free and high-throughput imaging approach for biomedical applications. In this study, we present a protocol utilizing DHM to quantify the characteristic morphological changes of neutrophils during Neutrophil Extracellular Trap formation (NETosis). Our method provides a non-invasive and cost-effective solution, eliminating the need for time-consuming sample preparation required for traditional blood smears or fluorescence flow cytometry. By harnessing the unique capabilities of DHM, we aim to provide a tool for the understanding of various inflammatory diseases and to support clinical intervention decisions at the point-of-care.

Brain organoids offer immense potential for studying brain development, neurological disorders, and evaluating drug responses. However, their exploitation is hindered by several challenges, including the lack of a label-free and non-destructive monitoring technique for studying their development and response to the external perturbations. To address this, we propose a non-invasive Raman Spectroscopy approach, enabling label-free discrimination of maturation stages without disruption. Using a custom high-throughput multi-modal Raman microscope equipped with a 785 nm laser source, we collected Raman Spectroscopy (RS) data from cortical organoids at various developmental stages and employing machine learning techniques, we extracted crucial features linked to each stage. This label-free methodology facilitates observing dynamic changes in organoids without compromising their growth, enabling longitudinal studies for deeper insights into their development and drug responses.

We demonstrate the capability of linear and non-linear optical microspectroscopy techniques such as fluorescence lifetime imaging (FLIM), Raman spectroscopy (RS), second-harmonic generation (SHG), and two-photon fluorescence (TPF), to monitor structural, biochemical and biomechanical alterations in collagenous tissues upon both collagen digestion and cross-linking using genipin as cross-linker. Thanks to a unique combination of optical and force microscopy techniques in a correlative manner, we obtained detailed information about the biochemical, structural, and biomechanical properties of collagenous tissues upon both digestion and cross-linking treatment.

Our study introduces a label-free imaging and quantitative analysis approach for investigating lipofuscin aggregates in human brain tissue. Leveraging the colocalization of lipofuscin with cell soma, our novel method accurately identifies and counts cells, especially large neurons. Achieving an impressive 92% accuracy at submicron resolution, our label-free approach outperforms the commonly used Nissl stain. We develop a robust segmentation technique for lipofuscin aggregates, revealing layered structures in the cortical gray matter, potentially associated with cell distribution. Furthermore, we validate our results using state-of-the-art techniques, including fluorescence lifetime imaging microscope and sub-micron resolution two photon imaging. Our findings contribute valuable insights into neurodegenerative diseases and hold promise for future diagnostic advancements.