In this presentation, we will show how to use photodynamic action to reverse bacterial resistance to antibiotics. Using different types of resistant bacteria, the use of a low dose of light and a photosensitizer allows for reversing the resistance making the bacteria and its future generations again susceptible to antibiotics. In addition to works in this area, we will show several applications of photodynamic action in the control of infections such as Pneumonia, and pharyngotonsillitis, as well as avoiding infection during the process of mechanical ventilation.

We present a novel, cost-effective, versatile, and practical fluorescence imaging modality, termed Fluorescence Frequency-Response Imaging (F-FRI), capable of imaging the frequency response of the time-resolved fluorescence emission excited at multiple wavelengths and measured at multiple spectral emission bands. Rigorous validation experiments have demonstrated the capabilities of F-FRI to accurately measure fluorescence frequency-response of fluorophores with lifetimes in the range of ~0.4-12 ns. This novel F-FRI imaging modality has been successfully translated for label-free metabolic endoscopic imaging of epithelial oral mucosa, and for label-free biochemical wide-field imaging of resected skin tumors during Mohs surgery.

Cancer remains one of the leading causes of death worldwide despite advances in diagnostic and treatment approaches. Current methods of detection and diagnosis remain inaccessible or expensive in nature; therefore, the development of non-invasive strategies towards early-stage cancer detection are important to allow for early intervention, treatment, and access. Liquid biopsies have emerged as a non-invasive source to improve routine cancer monitoring, however cancer biomarker abundance is low, leading to limitations in detection and accuracy. The recent discovery of neoplastic circulating hybrid cells (CHCs) in peripheral patient blood provide the potential to improve detection sensitivity of blood-based assays using novel molecular-targeting contrast agents specific to both circulating tumor cells (CTCs) and CHCs. Additionally, these contrast agents can be detected using diffuse in vivo flow cytometry (DiFC), enabling non-invasive enumeration of cancer cell burden. Herein, the development and validation of near infrared (NIR) molecularly-targeted contrast agents with specificity towards epithelial biomarkers expressed on CHC and CTCs is discussed.

Fluorescence-guided surgery (FGS) has the potential to significantly enhance patient outcomes by enabling precise real-time visualization of vital nerve structures during surgical procedures. However, a clinically approved nerve-specific contrast agent does not exist. To address this need, we adopted a medicinal chemistry approach to design and develop novel near-infrared (NIR) nerve-binding small molecule fluorophore libraries. Our first-in-class NIR nerve-binding small molecule fluorophores represent a significant advancement in the field. By enabling precise nerve visualization in real-time during surgery, these contrast agents have the potential to revolutionize nerve-sparing procedures and ultimately improve patient outcomes.

In vivo imaging of a nerve injury and longitudinal monitoring of peripheral nerve processes in preclinical models can shed light on many biological process within the nerve. Optical imaging with its high spatial and temporal resolution is highly promising, however high scattering from the skin and other adjacent organs such as muscle limit the application to only in vitro or ex vivo studies. To address these limiting issues, we developed a minimally invasive in vivo model that enables continuous imaging of a peripheral nerve with a high, single axon resolution. Our approach uses a flexible skin-imbedded transparent optical window with the nerve surgically repositioned above the muscle layer. This modality allows longitudinal single-axonal resolution microscopy of the nerve with virtually any optical reporter that can be used in living animals.

Fluorescence microscopy relies on efficient emission of molecules excited by an incident laser light. However, their emission is often intrinsically limited by a low fluorescence quantum yield which results in low contrast images. In our recent efforts, we explored the fluorescence life-time imaging contrast for those low efficient fluorophores to find out that those molecules exhibit a rather intense emission over a relatively short time. This short fluorescence time was found to be dependent of the local structure and local environment providing a novel biomarker for biological imaging.

Unlocking the quantum potential of nitrogen-vacancy (NV) centers in diamonds has led to innovative advancements in sensing applications. By coupling NV centers with plasmonic nanostructures, ultrasensitive biolabels are envisioned. To this end, we exploit DNA self-assembly to create hybrid plasmonic nanodiamonds, featuring a closed nanocavity encapsulating a single nanodiamond. Correlated spectroscopy reveals enhanced brightness and emission rates, crucial for quantum sensing. This synergy between plasmonics and diamond fluorescence augments photon emission intensity and accelerates emission rates, enabling improved temporal resolution. These nanodiamonds hold promise as stable single-photon sources and versatile platforms for probing quantum effects in biology. This talk highlights the development, properties, and potential applications of these nanodiamonds, bridging quantum sensing and biomedicine, and fostering transformative changes in biophotonics.

The essential role of ferritin in iron homeostasis makes its study highly important for fundamental biochemistry research and clinical analysis of iron status, while the magnetic properties of its core mean that it holds interest for understanding magnetism in nanoscale condensed matter systems. Here we employ an emerging magnetic sensing technique using nitrogen-vacancy fluorescence defects in single crystal diamond and nanodiamonds, to quantify the magnetic properties of the iron core of ferritin as it is loaded. We observe anomalous magnetic behavior that can be explained using a theoretical model detailing a morphological change to the iron core occurring at relatively low iron loads. This model provides an L^(0.35±0.06) scaling of the uncompensated Fe spins, in agreement with previous theoretical predictions. The low detection limit (ferritin 2% loaded at a concentration of 7.5 ± 0.4 μg/mL) also makes this a promising method for precision applications where low analyte concentrations are unavoidable, such as in biological research or even clinical analysis.

Superfluorescence (SF) is a unique quantum mechanical behavior arising from the self-organization between emitters, thus forming a cooperatively coupled assembly. In contrast to isotropic spontaneous emission or normal fluorescence, SF produces a short but intense burst of light, which makes it ideal for a wide variety of applications in biophotonics, electronics, and optical computing. Due to the prerequisite of cooperative emitter coupling, SF has been conventionally observed under cryogenic conditions in limited systems, such as atomic gases, and a few bulk material systems. Here we show lanthanide-doped upconversion nanoparticles (UCNPs) as a medium to achieve anti-Stokes shift SF at room temperature.

Fluorescence imaging has emerged as a valuable tool for clinical angiographic and cardiovascular imaging, allowing for visualization and quantification of biological processes. Among the range of fluorescence imaging windows, near-infrared (NIR) imaging has shown great promise as a non-invasive modality for angiographic and cardiovascular imaging. To overcome limitations associated with indocyanine green dye (ICG), we developed a biocompatible DNA-based platform for conjugation with ICG dyes and targeting moieties. The primary objective of this pilot study is to evaluate the efficacy of the DNA-ICG platform for contrast-enhanced NIR-II (>1250 nm) fluorescence imaging in a mouse model. Throughout the experiment, various organs were observed, including the heart, liver, spleen, caecum, and intestines. Notably, vascular structures in the tail, spinal column, and head remained visible for hours after the administration of the contrast agent. The DNA-ICG platform holds promise as an effective imaging tool for angiographic and cardiovascular studies.

The results on design, synthesis, characterization and biomedical applications of core-thermoresponsive shell polymeric nanoparticles (CTS-PNPs) loaded with imaging and therapeutic agents will be presented. In CTS-PNPs, the polystyrene core (~30 nm) is coated with the shell of poly-N-isopropylacrylamide (PNIPAM) or co-polymer of NIPAM and acrylamide (AA). The shell “shrinks” in water at the temperatures higher than lower critical solution temperature, which can be tuned through co-polymerization of NIPAM with AA, and affects photophysics of the embedded fluorophores. This phenomenon has been employed by us in fluorescence imaging and photodynamic therapy of cancer using CTS-PNPs loaded with fluorescent dyes and photosensitizers.

Diabetic retinopathy (DR) is a debilitating organ manifestation of diabetes. Currently, there is no technology that allows detection of early molecular changes in the eyes of individuals with diabetes. The clinical diagnosis of DR is based on visualizations of structural damages that are irreversible. Absent of early diagnosis and intervention, vision tends to rapidly decline. Over the past two decades we developed a novel approach for non-invasive detection of molecules in the retinal microvessels. A hurdle to translation remained generation of biodegradable nanoprobes that are sufficiently bright for in vivo detection. An adhesive fluorescent nanoprobe with high brightness was developed using biodegradable materials. Upon systemic injection, the nanoprobes adhered in the retinal microvessels of diabetic mice and were visualized as bright spots in live retinal microscopy. Our results establish the translational potential of these newly generated nanoprobes and open new possibilities for early diagnosis of DR with high specificity and quantitative accuracy.

In vivo tumor imaging and delineating tumor margins serves a critical role in early detection and treatment of cancer. Together with our industrial collaborators, InnoSense LLC (Torrance, CA), we previously have developed ThermoDots™, temperature-responsive micelles that encapsulate an FDA approved near infrared (NIR) imaging agent, indocyanine green (ICG). ThermoDots enhances the signal and enable high resolution fluorescence imaging capability. In this work, they are conjugated with PSMA antibodies for prostate cancer imaging.

Solid tumors face the challenge of hypoxia, resulting from an imbalance between oxygen demands and supply. Hypoxia leads to resistance to conventional cancer therapies like radiation and chemotherapy, including photodynamic therapy (PDT) that relies on oxygen radicals. To address this, we developed perfluorocarbon nanodroplets for co-delivering oxygen and a photosensitizer. In vitro and in vivo studies validated oxygen release and enhanced tumor oxygenation. Histological analysis confirmed reduced hypoxic regions in nanodroplet-treated tumors. PDT using the nanodroplets demonstrated superior efficacy compared to a liposomal formulation. Overall, oxygen-loaded nanodroplets guided by photoacoustic imaging offer a promising approach for hypoxic tumor treatment.

We investigated the photophysical property of Yellow Cameleon 3.60 (YC3.60), a fluorescent calcium-ion (Ca2+) indicator based on Förster resonance energy transfer (FRET), under cryogenic conditions. By measuring the fluorescence intensity ratio of the donor and accepter at various Ca2+ concentrations under room and cryogenic temperatures, we confirmed that YC3.60 exhibits a Ca2+-dependent FRET efficiency. Although slight differences were observed in the fluorescence lifetime and spectral shape at the cryogenic temperature, which can affect the FRET efficiency, our measurement suggested that YC3.60 can be employed for quantitative Ca2+ measurement and imaging under cryogenic conditions with improved photostability and quantum yield.

Here we have validated a commercially available dye, CJ215 from ProImaging for enhanced cancer delineation. The dye (which does not require antibody or peptide conjugation) was found to be effective for both NIR and SWIR fluorescence imaging methods in preclinical breast, fibrosarcoma and prostate tumor models. The dye was effective for screening and resection and achieved some of the best-in-class tumor to organ ratios, e.g., tumor to muscle of 88.1, to liver of 17.9, to lungs of 12.9 and to kidney of 5 highlighting its renal clearance method. We briefly summarize the proposed mechanism of uptake and highlight the potential for clinical translation.

Skin disease is primarily diagnosed visually, but this subjective approach can lead to misdiagnosis, particularly for darkly-pigmented patients where increased melanin leads to more subtle disease appearance. Inflammation is characterized by shifts in tissue fluid, and the short-wave infrared (SWIR 900-1700 nm) regime, where water absorbs strongly and melanin absorbs weakly, may therefore be a pigment-insensitive modality for assessing skin inflammation. We built a multispectral SWIR imaging system and tested its ability to detect tissue fluid after intradermal saline injection in 24 healthy subjects with diverse pigmentation. Saline injection regions had 20-50 times more contrast than unaffected skin in SWIR images compared to visible photography, regardless of the degree of pigmentation. SWIR multispectral imaging may offer a new window into assessing inflammatory skin diseases in a pigmentation-independent manner.

We will present a new method to reduce the photobleaching of fluorescent proteins and the associated phototoxicity. Our method exploits a photophysical process known as reverse intersystem crossing, which we induce by near-infrared co-illumination during fluorophore excitation. This dual illumination method typically reduces photobleaching effects 4-fold, can be easily implemented on commercial microscopes and is effective in eukaryotic and prokaryotic cells with a wide range of fluorescent proteins.

Stem cell-derived organoids which are miniature self-organized three-dimensional tissue cultures could be a limitless source of functional tissue for transplantations in many organs and also have the potential to significantly improve the drug discovery process. One common feature to all organoids is that they are generated from either pluripotent stem cells (PSCs) or adult stem cells through replicating human development or organ regeneration in vitro. A key challenge with developing any organoid model is monitoring the maturation process or the response of organoids to changes in environment. Here, we will show that a combination of chromatin-sensitive coherent confocal light absorption and scattering spectroscopic (C-CLASS) microscopy and Raman microspectroscopy, which is sensitive to the vibrational modes of molecules and enables direct classification of a broad range of biomolecules with spectral unmixing, provides an efficient way of functional analysis of organoids and 3D cell cultures without the need to sacrifice a sample.

Molecular analysis of tissue samples plays an important role in understanding basic functionality of organisms and detecting abnormality. Acquiring comprehensive information on heterogeneous and complex biological systems with unique chemical specificity and high spatial resolution provides a powerful tool for early-stage disease detection and monitoring. However, MALDI mass spectrometry imaging of whole tissue sections at high spatial resolution (≤10 μm pixel size) requires extensive acquisition time. Here, Bruker’s proposed end-to-end workflow for quantum cascade laser (QCL) based infrared (IR) guided MALDI imaging was employed to obtain deep molecular information on various tissue samples. MALDI imaging is more sensitive and provides deeper molecular information than IR based imaging, but QCL-IR based imaging is much faster. QCL-IR imaging is therefore used as a screening tool for disease detection, where if disease is detected, then the more time-consuming MALDI imaging can target only the region of the tissue section in question. Examples presented include a proof-of-concept mouse brain sample and a practical multistage pancreatic cancer tissue sample. This approach facilitates region of interest (ROI) selection and significantly increases sample throughput – thereby enabling large-scale MALDI imaging studies in cancer research.

The combination of bright, tunable semiconductor quantum dots (QDs) emitting in the shortwave infrared (SWIR, 1000 – 1600 nm) and InGaAs camera-equipped preclinical imagers opens the door to non-invasive, multiplexed, deep tissue imaging in live mice. We demonstrate the simultaneous imaging of three distinct QDs to highlight different regions of the mouse anatomy (lymphatic system, vasculature, liver, spleen, lungs, cerebrospinal fluid, gastrointestinal tract) through different routes of delivery. Video framerate imaging enables dynamic observation of anatomical processes in exquisite detail. By changing the surface chemistry of the different colored nanoparticles, the impact of nanoparticle surface coatings on circulation half-life can be visualized and the final biodistribution determined quantitatively. This deep tissue imaging with quantitative validation is being applied to paired agent imaging of tumor biomarkers and nanomedicine drug delivery investigations.

Label-free spectroscopic detection of single viruses provides component analysis of virus strains. Current methods suffer from low throughput and weak signal contrast of individual virions. Here, we report a widefield interferometric defocus-enhanced mid-infrared photothermal (WIDE-MIP) microscopy for high-throughput fingerprinting of single viruses. WIDE-MIP not only reveals the contents of viral proteins and nucleic acids in single DNA vaccinia viruses and RNA vesicular stomatitis viruses, but also uncovers an enriched β sheet components in DNA varicella-zoster virus proteins. Different nucleic acids signatures of thymine and uracil residue vibrations are also obtained to differentiate DNA and RNA viruses.

This work proposes developing a water-soluble-chlorophyll-binding protein (WSCP) from Chenopodium album plant species (CaWSCP) as a contrast agent for cancer targeting with photoacoustic (PA) imaging. CaWSCP exhibits red-shifted absorption, good solubility, and photoconvertible properties that can generate enhanced contrast images while integrated SpyTag and SpyCatcher system enables active targeting of cancer cells. In vitro and in vivo experiments demonstrate the non-cytotoxic nature of WSCP and validate the efficacy of the SpyTag-SpyCatcher system for cancer cell targeting. Moreover, in vivo imaging confirm the contrast-enhancing capability of CaWSCP, positioning it as a promising candidate for PA imaging in cancer diagnostics.

pH has been shown to affect the phenotype of immune and leukemic cells. Using multi-photon microscopy with a ratiometric pH probe, SNARF-1, we previously developed a technique to map absolute interstitial pH in vivo at single-cell resolution. The methodology revealed a heterogenous pH distribution in the bone marrow microenvironment, where acidic regions were found in active bone remodeling sites that can support healthy and leukemic cells surrounding a macrophage subset. To elucidate how interstitial pH modulates hematopoietic function, we first established protocols to preserve RNA quality in histology slides and co-register them to 3D in vivo stacks. We then performed initial transcriptomic analysis using GeoMx Digital Spatial Profiling under image guidance which enables preservation of the pH information from the microenvironment.

This conference presentation was prepared for SPIE BiOS, 2024.

Quantitative imaging biomarkers are essential to advancing precision medicine within ophthalmology, providing objective insights into structural changes, disease progression, and treatment response. However, the path to their qualification and widespread adoption faces a significant hurdle: interoperability among imaging devices. In this presentation, we explore the role of industry standards and calibration phantoms in addressing the challenge of quantitative interoperability. We will review Maxwellian optics of the eye and implications for calibrating fundus cameras, OCT, and Adaptive Optics enabled retinal imaging systems. We explore how uniform and transparent calibrations can empower structural biomarker discovery and drive progress in ophthalmic clinical trials and personalized care.

International efforts to standardize emerging biomedical imaging approaches, such as photoacoustic imaging (PAI), require stable physical phantoms to enable routine quality control and robust performance evaluation across devices. Addressing this necessity, the International Photoacoustic Standardization Consortium (IPASC) has undertaken a consensus-finding exercise to establish recommendations for the properties of tissue-mimicking phantom materials and their characterization in the field of PAI. The manufacturing reproducibility of a stable copolymer-in-oil tissue-mimicking material fulfilling these recommendations was tested in an international multi-center study involving n=18 different partner sites. Here, the progress made toward these standardization efforts is outlined, highlighting prospects, challenges, and future trajectories.

This conference presentation was prepared for SPIE BiOS, 2024.