This talk will highlight a genetic engineering technique to obtain a series of albumin fragments and recombinant proteins containing one or multiple domains that form covalent bonds with chloro-containing cyanine dyes. These albumin variants protect the inserted dyes and remarkably enhance their brightness. The albumin variants can also be genetically edited to develop size-tunable complexes with precisely tailored pharmacokinetics. The proteins can also be conjugated to biofunctional molecules without impacting the complexed dyes. This combination of albumin mutants and clinically-used cyanine dyes can help widen the clinical application prospects of NIR I fluorophores to be visualized in the NIR II window.

Our current study using deuterium oxide probed stimulated Raman Scattering (DO-SRS) imaging provides new insights in brain lipid metabolism in female and male Drosophila individuals during aging processes. We found that the 3D volumetric distribution of newly synthesized lipids is heterogeneous, aged male and female flies have different metabolic activities in brain LDs, the glial LDs remain predominantly TAGs during aging, lipogenic diets improve brain functions by promoting lipid biosynthesis, and the IIS pathway mediates diets-regulated brain lipid metabolism.

The shortwave infrared (SWIR) shows a significantly higher level of contrast between different types of tissues compared to the Vis-NIR. This contrast can be further enhanced by using hyperspectral imaging (HSI). Unlike conventional imaging methods, each HSI pixel contains a full high-resolution spectral signal. Complex computational approaches are required to analyze HSI-measured optical alterations. Many of these approaches including machine learning and deep learning algorithms have been developed in the field of geospatial imaging. We adapted and optimized algorithms used in remote sensing to build an image processing platform IDCube that processes biomedical data and identifies tissues abnormalities in SWIR.

The advantages of shortwave infrared (SWIR, 900-1700 nm) imaging over NIR (650-900 nm) include reduced tissue optical aberrations, increased depth penetration along with improved resolution and contrast. Preclinical SWIR imaging has focused on system development and novel fluorophore development with a goal of targeted tumor resection for clinical translation. Clinical SWIR imaging has focused on indocyanine green (ICG) and the enhanced permeability and retention effect for resection with significant advantages. In this work we preclinically validate the pH low insertion peptide (pHLIP) conjugated to ICG (pHLIP ICG, currently in clinical trials) as a suitable cancer targeted fluorophore for cancer resection.

New technologies are needed for the identification of therapeutic vulnerabilities in cancers and neurodegenerative diseases. Autophagy is a cellular process with important functions that drive many such diseases. We developed a near-infrared optical nanosensor that measures autophagy-mediated changes in intralysosomal pH in cells and in vivo. The nanosensors, based on quantum defects on carbon nanotubes, are taken up by the lysosomes, where emission shifts in response to local pH. Using hyperspectral imaging, the sensors facilitated spatial, dynamic, and quantitative mapping of subtle changes in pH within tumor models. Monitoring the intratumorally-injected nanosensors enabled dynamic tracking of autophagy upon pharmacologic perturbation.

Silica nanoparticle optical probe properties are ultimately determined by the modified TEOS physical and chemical properties. This presentation will discuss the virtually unlimited possibilities of changing surface and internal properties of silica nanoparticles made of modified TEOS molecules. The resulting probe behavior is only limited by the chemical properties of modified TEOS and the proximity of the modified TEOS molecules in the nanoparticles which is determined by the concentration of the modified TEOS and the size of the nanoparticle. Silica nanoparticles are one of the few nanodevices that allow such a large variability. Any known optically active molecules that can covalently be attached to modified TEOS can be used to design these probes. Typical applications of these particles are for immunochemistry, flow cytometry, CE, forensic applications and other optical probe applications will be presented.

Absolute oxygen sensors based on quenching of phosphorescence have been the subject of numerous studies for the monitoring of biological environments. Here, we exploited self-assembly based fabrication of porous polymer films to encapsulate the phosphorescent dye onto four different types of porous films. Specifically, evaporation-based phase separation and the breath figure technique were used to induce porosity. The oxygen sensing properties were evaluated via a standardized calibration procedure with an optoelectronic setup in both transmission and reflection based configurations. We have also undertaken a thorough characterisation of the film porosities using an optical microscope, a scanning electron microscope and microCT imaging. Despite the use of common polymers, the fabrication techniques employed led to the significant enhancement of oxygen sensing properties and elucidated the relation between porous film morphologies and sensing performance.

Tumor-targeting contrast agents promise indispensable value for therapeutic monitoring, image guided therapy, early stage detection, and tumor growth surveillance. Many contrast agents, however, are limited by millimeter-ranged penetration depths, ionizing radiation sources, full-body scan imaging modalities, or a short half-life. Photoacoustic ultrasound offers a chairside and real-time platform for tumor imaging using both endogenous and exogenous sources for optical contrast. In this work, we formulated micelle-inspired nanoparticles via noncovalent intermolecular forces with (1) robust photoacoustic signal and (2) specificity to the tumor microenvironment. The particles are formed by tagging cyclic iRGD peptides with aspartic acid residues and linking them with methylene blue molecules; electrostatic attraction between the negatively charged residues of the peptide and the positively charged dyes enable their controlled assembly into nanoparticles that are stable in 150 mM saline solution on the order of days. The nanoparticles possessed a hydrodynamic diameter of 180 nm based on optimized peptide to dye molar ratios. The tagged iRGD peptides demonstrated targeted uptake in SKOV-3, MCF-7, and A549 cells measured via alpha v beta 3 competitive inhibition. On the other hand, the formulated nanoparticles possessed a photoacoustic peak excitation wavelength of 725 nm, but activatable photodynamic toxicity at 660 nm. The particles induced 9.31, 14.64, and 14.15-fold increase of efficient reactive oxygen species (ROS) generation in cancerous A549, MCF-7, and SKOV-3 cells, respectively, compared to noncancerous HEK 293-T cells. Future work will investigate the half-life and early stage detection capabilities of these nanoparticles in murine cancer models.

The talk will present our recent results on development of multifunctional biocompatible nanoplatforms for cancer theranostics. The three nanoplatforms targeting tumors include polymeric nanoparticles (PNPs), nanoliposomes and protein nanocrystals; they can be equipped with NIR luminescence, photoacoustic (PA) and computed tomography (CT) imaging contrasts, along with PDT/PTT and chemotherapy modalities, which can be selectively activated by tumor microenvironment (TME). The talk will also demonstrate application of PNPs for visible or NIR fluorescence labeling of the systemically injected mesenchymal stem cells, which manifest tumor tropism in metastatic cancer animal model, allowing for fluorescence imaging based detection of primary tumor and metastases.

Perfluorocarbon nanodroplets (PFCs) are a new class of liquid contrast agents that have been explored for ultrasound and photoacoustic imaging applications by the biomedical imaging community. These contrast agents can undergo a reversible or irreversible liquid-to-gas phase transitions upon an external excitation by a light or an ultrasound stimulus. Here, we evaluate the influence of apparently minute changes to the coating of PFCs and of dye-embedding strategies (i.e., for light activation) on the activation efficiency of the phase transition and the corresponding signal generation by PFCs. We discuss implications of these findings for further development of PFC contrast agents.

Cells use molecules to exchange information. A very prominent example is neurotransmitter signaling between neurons or paracrine signaling between blood cells. However, it is up to date difficult to visualize it with high spatial and temporal resolution. We have developed near infrared (NIR) fluorescent sensors based on single-walled carbon nanotubes (SWCNTs). They are chemically modified to render them selective for signaling molecules (dopamine, serotonin, ROS) using novel chemical strategies. These sensors change their fluorescence in the presence of the target molecule and detect down to single molecules. Fast parallel imaging of many of those sensors provides a NIR image (>900 nm) of the signaling molecules around single cells. This technique was used to image dopamine release from neurons as well as serotonin and ROS release from human blood platelets. These results illustrate that tailored SWCNTs are powerful tools for quantitative chemical imaging.

Fluorescent gold nanoclusters (AuNCs) with emission in near infrared (NIR) range have drawn wide attention due to its bright fluorescence, good biocompatibility and photostability. In this work, AuNCs with emission at 800 nm were synthesized through a simple glutathione reduction method through heating. Cyclic RGD peptide will be conjugated through thiol-PEG-amine as targeting ligand. The AuNCs are then used for two photon fluorescent imaging, due to the wide stokes shift. In vivo NIR fluorescent study showed the accumulation of the AuNCs in tumor areas. Moreover, the AuNCs also showed brain uptake, which indicates possible applications in brain imaging.

Profiling the heterogeneous landscape of cell types and biomolecules is rapidly being adopted to address current imperative research questions. Precision medicine seeks advancements in molecular spatial profiling techniques with highly multiplexed imaging capabilities and sub-cellular resolution, which remains an extremely complex task. Surface-enhanced Raman spectroscopy (SERS) imaging offers new promise through the utilization of nanoparticle-based contrast agents that exhibit narrow spectral features and molecular specificity. Herein, we report the first demonstration of simultaneously multiplexing 26 different nanoparticles in a single imaging pixel with subcellular resolution.

We performed synthesis and testing of Fab’-based NIR fluorescent probes with the goal of achieving sufficiently high tumor/background (muscle) ratios at the earlier time points after intravenous injection in comparison to the full-size IgG or larger fragments. Conjugation of NIRDye 800CW to Fab’ resulted in partially quenched imaging probes. Fluorescence intensity imaging of orthotopic xenografts in both EGFR-attenuated and wild-type triple negative breast cancer tumors using Fab’-800CW allowed earlier detection of EGFR expression in vivo.

Recent progress in the translation of antibody-drug conjugates (ADCs) has validated the potential of ligand-targeted drug delivery strategies. However, the clinical application of these strategies has encountered significant, often unanticipated, toxicity. Critically, many of these toxicities do not result from monoclonal antibody (mAb) binding to its cognate target, but rather from deleterious effects of the hydrophobic small molecule/linker combination on in vivo targeting of the mAb. Novel experimental approaches are needed to assess targeting early in the design, synthesis, and testing process. We hypothesize that in vivo optical imaging is uniquely poised to assess the role of payloads and linkers on ADC properties. This is because optical probes are small molecules of similar molecular weight and physical properties to drug payloads. We first set out to address the role of payload properties. By developing synthetic methods that enable the rapid synthesis of chemically varied heptamethine cyanines, we have assembled and quantitatively compared the targeting of a series of substituted variants. These efforts suggest that highly polar, and specifically zwitterionic, substituents dramatically improve the in vivo properties of mAb conjugates. To examine the role of ADC linkers, conventional always-ON probes are not suitable to study the site and extent of bond cleavage. To address this, we have created a new class of fluorogenic probes in the near-infrared (NIR) range that result from modification of heptamethine norcyanines with stimuli-responsive carbamate linkers. These norcyanine carbamates (CyBams) exhibit exceptional turn-ON ratios and can be activated by a range of enzymatic and chemical triggers. By optimizing the cellular uptake and retention of these probes, we have created mAb-targeted variants that allow us to quantitatively study linker chemistry in animal models. Overall, our goal is to develop and ultimately apply an “imaging-first” workflow for the design and testing of well-tolerated targeted drug delivery agents.

Optical microscopy together with novel imaging probes offers us powerful tools for studying the brain in action and neurovascular coupling in animal models at unprecedented spatio-temporal resolution. In this talk, I will introduce methods that we developed and used for characterizing oxygenation and blood flow changes at rest and in response to functional activation in awake mice across cortical regions and laminar layers, during healthy and diseased conditions. Our results showed that stimulus-induced changes in intravascular partial pressure of O2 (pO2) were conserved across cortical layers, suggesting a tightly controlled neurovascular response to provide adequate O2 supply across cortical depth. I will also discuss the extension of our approach for quantifying oxygenation and blood flow measurements, in two different organs: the eye and the gut. We demonstrated the first examples of quantification of absolute pO2 in the individual capillaries at all three layers of the inner retinal capillary plexus noninvasively through the pupil and in the large intestinal wall of mice by using two-photon microscopy. These developments are opening many possibilities to investigate the concurrent development of microvascular dysfunction in the retina and brain cortex, as well as the progression of intestinal diseases and the role of the gut microbiota.

Intracellular drug target validation and target engagement quantification have proven to be challenging, and all drugs have some degree of non-specific accumulation due to variable drug affinity, biodistribution, pharmacokinetics, and metabolism. A dynamic ratiometric imaging approach for estimating the concentration of cell surface receptors, termed Paired Agent Imaging, could have a revitalizing effect on quantitative intracellular protein receptor imaging, offering a potential means for quantifying intracellular drug target availability. We have developed a dynamic, fluorescence-based, three-compartment model termed intracellular paired-agent imaging that utilizes fluorophore labeled small molecule therapeutics as imaging agents to measure drug target availability in live cells and tissues.

Increased permeability in the blood nerve barrier is found after peripheral nerve injury(1) and annexin A-2 has been shown to translocate to Schwann cell surface after injury(2). We hypothesize that this change in permeability can facilitate intraneaural transit of LS-301 to identify nerve injury. We subjected Lewis rats to a mild or severe unilateral sciatic nerve crush injury and performed NIR imaging after administration of Cypate-3 dye and LS-301. Fluorescence within the ROI of the mildly injured nerve and severely injured nerve was significantly increased compared to their contralateral control at 5 hrs after injury. It appears that intravenous administration of near-infrared fluorophores results in detectable changes in nerve fluorescence compared to baseline.

There is still great need in better characterizing new targeted therapies in vivo, especially prior to clinical translation. In this regard, preclinical molecular imaging is a central tool in the targeted drug development pipeline. However, there is still a lack of imaging platforms that can enable to monitor target engagement, a key in vitro/in vivo parameter to assess the probability of success for a lead compound. Here, we will report on our methodologies to quantify target engagement via lifetime FRET in live subjects. These will include our recent instrumental developments, including large SPAD arrays and structured light strategies, as well as, software developments, including AI based data processing pipelines for 2D/3D imaging and unmixing.

Neuromodulators such as acetylcholine are chemicals made in the brain that can transform animal behavior. Neuromodulator dynamics are essential for their functions. Optical sensors have transformed the study of neuromodulators because they capture their dynamics with high spatial and temporal resolution. However, fluorescence intensity-based sensors are restricted to measure acute changes within one animal over a short period because intensity varies with sensor expression level and excitation light fluctuation. In contrast, fluorescence lifetime is impervious to sensor expression level or excitation light power, allowing comparison between individuals and across long periods. Here, we discover fluorescence lifetime response in multiple intensity-based neuromodulator sensors, and show that fluorescence lifetime predicts animal behavior states accurately despite varying excitation power or changes in sensor expression level across weeks and animals.