We developed photoacoustic tomography (PAT) to peer deep into biological tissue. PAT provides in vivo omniscale functional, metabolic, molecular, and histologic imaging across the scales of organelles through organisms. We also developed compressed ultrafast photography (CUP) to record 70 trillion frames per second, orders of magnitude faster than commercially available camera technologies. CUP can record in real time the fastest phenomenon in nature, namely, light propagation, and can be slowed down for slower phenomena such as neural conduction. PAT physically combines optical and ultrasonic waves. Conventional high-resolution optical imaging of scattering tissue is restricted to depths within the optical diffusion limit (~1 mm). PAT beats this limit and provides deep penetration at high ultrasonic resolution and high optical contrast by sensing molecules. Broad applications include early-cancer detection and brain imaging. The annual conference on PAT has become the largest in SPIE’

Infrared spectroscopic imaging combines the ability to record molecular content with the ability to visualize chemistry in its spatial diversity. Given the need to record a significantly larger quantity of data than a typical microscopy image (MB vs. GB) and the extensive bandwidth of the spectra (~10 m), trade-offs often have to be made between the closely related considerations of signal to noise ratio, spatial-spectral coverage, resolution and optical arrangements. Here, we present a path from rigorous theory to modeling and design to realizing the advantages offered by new ideas on fundamentally changing these trade-offs. We first describe a new microscope design for increased speed and rapid coverage that is useful for biomedical and clinical tissue imaging. Next, we describe a configuration to measure chirality in samples that promises higher spectral information that present methods. Finally, we present a new approach to nanoscale IR imaging that provides greater fidelity and speed at unprecedented levels of signal to noise ratio. Finally, we show how emerging machine learning approaches can further augment these advances. For each instrumentation advance, examples of use cases will be presented.

Recently developed mid-infrared photothermal (MIP) microscopy (for a Review, Science Advances, 2021, 7: eabg1559) not only overcomes the diffraction limit in direct IR imaging, but also circumvents the limitations in AFM-IR. In MIP microscopy, a visible beam probes the thermal effects induced by an intensity-modulated infrared beam. The MIP signals are measured in scanner manner or in wide-field manner. Video rate MIP imaging of living cells and tissues has been reached through a single pulse digitization approach. In this presentation, I will discuss the principle, instrumentation, and applications of MIP microscopy.

Current broadband hyperspectral imaging systems typically require 2 or more camera types (e.g. Si, InGaAs) to achieve high sensitivity in the wavelength range of 400 – 1700 nm. Given the tissue contrast available in this range, creating a compact real-time imaging system that could be deployed in the clinic would be advantageous. Here, we used a state-of-the-art camera based on the Sony SenSWIR image sensor that displays high sensitivity across the VIS-NIR-SWIR to create a broadband hyperspectral imaging system. After camera calibration, we acquired broadband spectra from patient specimens, demonstrating the ability to collect high quality spectra from biological tissues.

We developed a multimodal non-linear optical super resolution imaging platform for studying metabolic changes during aging and diseases. This platform integrates deuterium probed Stimulated Raman scattering (DO-SRS), multiphoton fluorescence (MPF), and second harmonic generation (SHG) into a super resolution multimolecular microscopy.

In the current report, we present a dual-mode approach for assessment of twisted light localization in turbid tissue-like scattering media. A unified open-access Monte Carlo (MC) model based on the tracking of transformations of electric filed vector is working in synergy with 3D geometrical exploration techniques for efficient identification of nonlinear structures produced by twisted light and subsequent tracking of their evolutions in turbid media. We report detection of the light’s twist for up to 6 ODs with <98% confidence intervals/threshold. Results are presented in comparison with exact theoretical solutions and experimental data obtained during laboratory studies. The models are accelerated by parallel programming on graphics cards and offer a responsive online interface.

In this study, we developed a prototype interstitial all-optical needle photoacoustic (PA) sensing probe for clinical translation. The performance of the PA needle probe was examined with intact human prostates ex vivo in a simulated transrectal ultrasound (US) guided transperineal prostate biopsy procedure. PA spectrum analysis shows statistical difference between the measurements acquired in benign and cancerous regions (n=49, p<0.05). Multivariate analysis using all quantitative measurements can obtain an accuracy close to 90%.

Optogenetic control of neuronal activity requires efficient light energy transmission through head tissues and bone. Here we evaluate the efficiency of ultrashort pulsed and continuous-wave light transmittance through the mouse scalp, skull, and brain tissues in near-IR optical windows. The outcomes of the experiments and computer modelling show that the brain cortex tissues can be exposed to 10-12% of the original laser irradiation. This finding together with the latest discovery of non-linear phytochrome conversion [S.Sokolovski et al. 2021] prove the possibility of phytochrome optogenetics in living animals and may be applied in the future for non-invasive photo-controlling of neural cells.

Extracellular vesicles (EVs) are small, sub-micron membrane-bound particles that function in cell-to-cell communication and have potential to be used in diagnostics, therapy, and biological investigations. However, common characterization methods for EVs lack functional information and rely on “bulk” metrics that lack single-EV resolution. Recent work has applied label-free multimodal nonlinear optical microscopy for characterization of EVs via NAD(P)H and FAD autofluorescence. Here, we characterize EVs isolated from urine and serum from human breast cancer surgery patients and breast reduction surgery patients who have no history of breast cancer to examine altered cancer-related metabolic signatures in cancer-associated EVs.

Real-time assessment of tissue morphology and function is a pressing clinical need. We present a low cost OCT probe based on combined position sensor feedback, as well as a data processing algorithm that enables real-time display of tissue morphology and birefringence. The preliminary evaluation of this instrument on various biological specimens has demonstrated its capability for real-time diagnosis. Its preclinical assessment in vivo on animal models of cancer was performed at MD Anderson Cancer Center. Reliable assessment of tissue morphology and birefringence has been successfully demonstrated.

Laser speckle contrast imaging (LSCI) is an imaging technique using a speckle pattern randomly produced on the image sensor based on the high coherency light source. LSCI is widely used for biological and clinical diagnoses such as blood flow measurement due to the advantages of real-time imaging, simplicity, and non-invasive. And due to the absorbance characteristics of hemoglobin, if two or more wavelengths in a specific near-infrared region are used, the change in concentration of each oxide, and reduced hemoglobin can be measured. In this study, by combining these two methods, using these systems, blood perfusion and hemodynamic responses in the body were measured in a large area.

We present a multiphoton excitation based hyperdimensional imaging microscope (HDIM). We demonstrate how this system may be used for metabolic readouts, how to evaluate samples with a very low photon budget (<50 photons/pixel), and how to combine such systems with real-time visualizations. We demonstrate how this HDIM approach can help us with metabolic interpretations in two use cases: cancer cells using NAD(P)H and in plant cells using chlorophyll fluorescence.

Eosinophilic esophagitis (EoE) is a clinicopathological condition requiring frequent upper endoscopy (EGD) with several biopsies and time-consuming histopathological diagnosis. We acquired in vivo fingerprint and high wavenumber Raman spectra from the esophageal mucosa of children undergoing EGD and assessed the efficacy of this non-obtrusive real-time approach for determining EoE activity. Spectral bands related to lipids (e.g., 1078, 1301, 1440, 2855cm-1), proteins (e.g., 935, 1003, 1342, 2931cm-1), and water (3075-3650cm-1) were found to differentiate between active, inactive, and non-EoE patients. The results from this study indicate that RS is a promising method for point-of-care assessment of EoE.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder characterized by synovial inflammation and pannus formation leading to destruction of local articular structure, bone erosion and functional disabilities. Common research models of inflammatory arthritis in rodents e.g. collagen antibody-induced arthritis (CAIA) in mice are associated with pain, discomfort, and distress. Here we demonstrate in vivo assessment of CAIA mice using a transflection Raman setup. Mice with induced arthritis and controls were clinically and spectroscopically assessed for 14 days. Raman measurements of tibiotarsal joint bone density correlated well with volumetric bone mineral density (vBMD) in mice exhibiting clinical symptoms of arthritis.

We present a virtual staining framework that can rapidly stain defocused autofluorescence images of label-free tissue, matching the performance of standard virtual staining models that use in-focus unlabeled images. We trained and blindly tested this deep learning-based framework using human lung tissue. Using coarsely-focused autofluorescence images acquired with 4× fewer focus points and 2× lower focusing precision, we achieved equivalent performance to the standard virtual staining that used finely-focused autofluorescence input images. We achieved a ~32% decrease in the total image acquisition time needed for virtual staining of a label-free whole-slide image, alongside a ~89% decrease in the autofocusing time.

Plasmonic metasurface for surface-enhanced infrared absorption (SEIRA) spectroscopy is integrated with multi-well cell culture chambers for application as a cellular assay based on Fourier transform infrared (FTIR) spectroscopy. Live cells are grown directly on the metasurface, and the enhanced infrared absorption through metasurface plasmonic resonance is probed in reflection mode using a custom-built FTIR-coupled inverted infrared microscope. The application of the proposed device as a cellular assay is demonstrated through the measurement of cellular adhesion with different surface coatings, as well as cellular response to the stimulation of the protease-activated receptors (PARs).

Diagnostic genomic profiling constitutes one of the major challenges to cure brain tumors. The deployment of such analyses depends on the quality of the surgical specimen sent for histopathological examination and further molecular studies. The aim of our study was to assess the potential added value of Stimulated Raman Histology (SRH) for the assessment of freshly excised central nervous system samples. We showed that SRH enabled a near-instant microscopic examination of various central nervous system samples without any tissue processing such as labelling, freezing nor sectioning. Following SRH imaging, we demonstrated that the samples could be readily recovered and reintroduced into a conventional pathology workflow including immunohistochemistry and genomic profiling to establish a definitive diagnosis.

Sickle Cell Disease (SCD) is an inherited hemoglobin disorder associated with multiple organ damage caused by continuous inflammation and systemic vasculopathy. There is a need for comprehensive technologies that can characterize vascular hemodynamics at the bedside. Near-infrared spectroscopy (NIRS) techniques show promise as a non-invasive diagnostic tool for monitoring tissue hemodynamics. Subjects with SCD were recruited to undergo longitudinal optical measurements while participating in a long-term efficacy study of Mitapivat, a pyruvate kinase activator under clinical development. We demonstrate the sensitivity of multiple modalities to hemodynamic changes in patients that achieved a < 1g/dL increase in hemoglobin level.