This PDF file contains the front matter associated with SPIE Proceedings Volume 12862, including the Title Page, Copyright information, Table of Contents, and Conference Committee information.

Mechanoluminescent nanotransducers (MLNTs) are colloidal materials that emit light upon mechanical excitation (e.g., by ultrasound activation). Encapsulation of MLNTs into erythrocyte-derived particles may provide a method to increase their circulation time. Herein, we have investigated three methods based on passive diffusion, sonoporation, and electroporation for loading of MLNTs. Confocal fluorescence imaging of the particles suggests that under the current protocols employed in this study, sonoporation and electroporation yielded better loading of the MLNTs into erythrocyte-derived particles.

Understanding the nature and role of communication between distinct cellular populations in the brain will require simultaneous measurement and control of activity within those populations during behavior. Current optogenetic tools, present limitations such as spectral incompatibility with sensors and modest efficacy. To address these issues, we engineered novel red-shifted high potent cation-selective channelrhodopsin, rsChRmine, and K⁺-selective channelrhodopsins (KCRs), KALIs, with enhanced K⁺ selectivity based on our cryo-EM structures. By integrating these new opsins with multiple genetically-encoded Ca²⁺ indicators, we can selectively control specific neural circuits while simultaneously observing the responses of other elements within the same network. We applied this method to the mPFC of freely-behaving mice, quantifying dynamic information transmission between excitatory and inhibitory populations. Together, this work lays the foundation for new kinds of investigation into brain function and dysfunction.

Hydrogels and hydrogel-based materials, thanks to their biocompatibility and biodegradability, are widely used as a supporting matrix for embedding various kinds of luminescent probes for biological sensing applications. Here we describe a family of phosphorescent hydrogels, termed Oxygels, which were designed specifically for local sensing of oxygen by means of Cherenkov-Excited Luminescence Imaging (CELI) in and around tumors during application of radiation therapy. Previously, our group has developed soluble phosphorescent probes, known as Oxyphors, and demonstrated their performance in CELI of oxygen. Oxyphors comprise phosphorescent metalloporphyrins encapsulated inside hydrophobic dendrimers, whose periphery is modified with polyethyleneglycol (PEG) residues. The PEG layer creates a hydrophilic jacket around the dendrimer, precluding interactions of the probe with biomacromolecules. As a result, Oxyphors retain stable calibration parameters, enabling quantitative imaging of oxygen in in vivo. However, locally delivered Oxyphors rapidly diffuse away from the injection sites and spread throughout the body, posing challenges to local oxygen quantification as well as raising concerns in terms of regulatory (FDA) approval. To this end, hydrogel-supported phosphorescent sensors implanted into tissue should allow for continuous local monitoring of oxygen during RT, aiding optimization of treatment protocols and facilitating the development of new types of RT treatment.

We have developed a novel stimulated Raman scattering (SRS) imaging platform to visualize various biomolecules at a resolution beyond the traditional diffraction limit of optical microscopy. Applying a super-resolution deconvolution algorithm, Adam based Pointillism Deconvolution (A-PoD), to hyperspectral SRS images, we can measure nanoscopic distributions of these molecules in cells. In this study, we showcase the application of A-PoD for SRS images of lipids, protein, unsaturated lipid, and saturated lipids in rat olfactory bulbs under hypoxia. The analysis result shows distinct distributions of unsaturated lipid in glomeruli of olfactory bulbs in hypoxia and control rats. This approach shows the capability of revealing nanoscopic molecular composition and metabolic activities. When applied in a super-resolution context with new workflow, this approach holds significant promise for advancing early detection and understanding of diseases.

Collagen and elastin are prominent components in both normal and abnormal tissues, and their presence and distribution have great significance for fibrosis- and cancer-related processes. Collagen and elastin quantification in the context of fibrosis, often associated with irreparable organ injury, can predict the disease severity and patient prognosis. In the context of cancer, specific spatial collagen signatures are known to influence tumor microenvironments while identification of elastin is important in the context of treatment of metastatic cancers. Traditional methods to quantify collagen and elastin vary in accuracy, cost, and ease of use. Using DUET microscopy on H&E slides, high-resolution collagen and elastin mapping is possible without added staining steps or expensive optical instrumentation. We demonstrate this approach in chronic kidney disease (CKD), coronary artery disease (CAD), and for identifying vascular elastin in colon cancers.

All-solid-state time-resolved cameras based on CMOS single-photon avalanche diode (SPAD) arrays have made very significant advances since the fabrication of the first CMOS SPADs back in 2003. These natively digital sensors are now available in large formats and with timing resolutions of a few tens of picoseconds, when operated in time-correlated single-photon counting (TCPSC) mode, or nanoseconds in gated implementations. They are capable of virtually noiseless read-out at very high speed, up to a hundred kfps. Designers have explored a host of architectures over the years, ranging from reconfigurable linear arrays relying entirely on FPGA-based processing to kpx TCSPC systems with in-built timestamping and histogramming and reduced histogramming, all the way to Mpx cameras featuring built-in gates. State-of-the-art 3D-stacked sensors are also emerging, based both on fully-silicon and hybrid-technology solutions, with very high potential at the cost of demanding design and fabrication cycles. We will discuss the related challenges in terms of sensing and timing performance, data read-out and processing, before addressing several time-resolved applications of interest to the biomedical community, including shot-noise limited fluorescence lifetime imaging of endogenous and exogenous fluorophores, compressive Raman spectroscopy, and diffuse correlation spectroscopy. Thanks to unprecedented sensitivity, noise, and speed performance at sensor and system level, these applications have a considerable potential towards diagnostic and clinical uses, operating in real time. An outlook on chip-level hyperspectral and/or polarization enhancements will complete the review.

The development of fluorescent proteins emitting in the near infrared (NIR) range (i.e., 650 nm-950 nm) has improved our capabilities for lifetime multiplexing and fluorescence imaging in vivo. Wavelengths in the NIR window experience reduced scattering and increased penetration depth through living tissue. Additionally, autofluorescence of cells and tissues is less prevalent in the NIR range, further improving signal to noise ratio. We performed fluorescence lifetime imaging (FLI) on breast cancer (AU565) and ovarian cancer (SKOV3) cell lines expressing the NIR fluorescent proteins (FPs), miRFP680 and emiRFP670. Confocal microscopy with time-correlated single-photon counting (TCSPC) reveals unique fluorescence decays for these NIR FPs, allowing for lifetime-based multiplexing on a single channel. Despite similar emission spectra, we were able to unmix fluorescence signals from a co-culture of SKOV3 expressing emiRFP670 and AU565 expressing miRFP680 based upon their unique fluorescence decays. We then generated 3D liquid overlay tumor spheroids using SKOV3 expressing emiRFP670 or miRFP680 for lifetime imaging via mesoscopic fluorescence molecular tomography (MFMT). 2D lifetime values and images acquired from MFMT corroborated our findings. Future investigation includes 3D light sheet mesoscopic imaging of tumor spheroids, as well as imaging of in vivo tumor xenografts expressing NIR-FPs. The long wavelengths and unique fluorescence lifetimes of emiRFP670 and miRFP680 make them ideal for multiplexed imaging, as well as for defining tumor volumes in vivo, while also leveraging the benefits of NIR imaging.

Fluorescence lifetime analysis (FLA) has become essential for non-invasive, real-time analysis of a variegate realm of compounds and materials in scientific and industrial fields. Here we present a compact and versatile device designed to apply FLA to the screening of healthcare nanoformulations which contain a luminescent active principle. In the case study here: Doxil FLA enables to decode the supramolecular organization and stability of the luminescent active principle within the nanoformulation in a non-invasive, rapid (seconds), and cost-effective manner. Contrary to currently used methods (e.g. TEM, SEM, CryoEM, HPLC), this device performs the analysis in the natural solvent with no need for sample chemical manipulation or labelling. This advancement holds promise for enhancing research and quality control in pharmaceutical industries. Also, we envision in the near future application of this technology to evaluate drug entry into cells and to monitor the change of drug supramolecular organization upon contact with biological environments, such as biological fluids, cells, tissues.

Water-soluble chlorin–dextran conjugates with a range of chlorin / dextran loading have been prepared to investigate the change in brightness as a function of loading. A polydisperse amino-dextran (~110,000 Da) was employed that consists of ~600 dextran units and ~24 amino-dextran units (3.8% degree of substitution). A synthetic chlorin is substituted with a single-junction water-solubilization group (comprised of three short, monodisperse PEG groups attached at the 2,4,6- positions of an aryl group) at the 10-position, a bioconjugatable phenylpropanoic acid group at the 15-position, and a gemdimethyl group in the reduced ring characteristic of the chlorin chromophore. The synthetic chlorin employed herein is a bioinspired analogue of chlorophyll a, which itself is Nature’s preeminent advanced functional dye. Joining of the chlorin to the dextran was achieved by amide bond formation upon N-hydroxysuccinimidyl activation of the chlorin. Loading numbers of 2.1, 2.7, 6.8, and 7.5 chlorins per dextran were obtained. The chlorin–dextran conjugates in water showed absorption and fluorescence spectra essentially identical to those of the monomeric chlorin. Among the chlorin–dextran conjugates, the brightness (fluorescence quantum yield per dextran molecule) reached ~5-times that of the corresponding chlorin monomer. Taken together, the results show the utility of combining hydrophilic fluorophores with a hydrophilic scaffold for photosciences applications in aqueous solution, yet also highlight the need for improved scaffolds (e.g., structural regularity, functional group diversity, conformational rigidity, monodispersity, water-solubility, built-in chromophores for ratiometric analyses) for development of materials with higher performance.

Absorption spectroscopy is a simple yet robust method for qualitative and quantitative analysis. The technique is most commonly applied for molecules in solution that absorb in the ultraviolet (UV), visible (Vis), and/or near-infrared (NIR) spectral region. The presence of the particles of size comparable to or greater than the wavelength of light can cause scattering. The contribution from light scattering can overwhelm intrinsic absorption especially in the shorter wavelength, UV regions, which impedes accurate analysis. The problem of light scattering is common in absorption spectroscopy. To compensate for the alterations caused by light scattering, various correction methods have been applied over the years. The chief correction methods are based on Rayleigh or Mie light-scattering theory. Displayed here are >40 spectra drawn from spectroscopy and photochemistry journals published over 7 decades (since the 1950s) that illustrate the lightscattering problem. The displays include >25 spectra beset by light scattering and 15 examples wherein spectral corrections have been applied. The present survey of the scope of the light-scattering problem in absorption spectroscopy (200–1000 nm) serves as a prelude to the development of a systematic calculational module for spectral correction.

Liposomal J-Aggregates of Indocyanine Green (L-JA) offer significant advantages over conventionally used monomeric indocyanine green (IcG) for photoacoustic imaging. When compared to IcG, which is often hindered by low circulation time and poor photostability, L-JA are characterized by longer circulation times, vastly improved photostability, elevated absorption at longer wavelengths, and increased photoacoustic signal generation. However, the documented methods for production of L-JA varies widely, which can lead to significant batch variability. We developed an approach to form IcG J-aggregates (IcG-JA) directly in liposomes efficiently and reproducibly at elevated temperatures. Aggregating within fully formed liposomes ensures particle uniformity and allows for the control of J-Aggregate size within the final particle. This technique provides a more robust and facile approach to producing these viable agents.