Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi-pass acquisition.

The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins, and can destroy chemically-sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi-pass flow cytometry approach to address this long-standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single-cell resolution maintained. Chemically-fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular fluorescent proteins and methanol-sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cell analysis without the constraints of conventional one-time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno-oncology, stem cell research, and cell biology.

High-Frequency Optical Coherence Elastography for Gingival Tissue Characterization: Variability in Stiffness and Response to Physiological Conditions.

Accurate measurement of gingiva's biomechanical properties in vivo has been an active field of research but remained an unmet challenge. Currently, there are no noninvasive tools that can accurately quantify tensile and shear moduli, which govern gingival health, with sufficiently high accuracy. This study presents the application of high-frequency optical coherence elastography (OCE) for characterizing gingival tissue in both porcine models and human subjects. Dynamic mechanical analysis, histology studies, and strain analysis are performed to support the OCE result. Our findings demonstrate substantial differences in tissue stiffness between supra-dental and inter-dental gingiva, validated by dynamic mechanical analysis and OCE. We confirmed the viscoelastic, nearly linear, and transverse-isotropic properties of gingiva in situ, establishing the reliability of OCE measurements. Further, we investigated the effects of tissue hydration, collagen degradation, and dehydration on gingival stiffness. These conditions showed a decrease and increase in stiffness, respectively. While preliminary, our study suggests OCE's potential in periodontal diagnosis and oral tissue engineering, offering real-time, millimeter-scale resolution assessments of tissue stiffness, crucial for clinical applications and biomaterial optimization in reconstructive surgeries.

High-dimensional multi-pass flow cytometry via spectrally encoded cellular barcoding.

Advances in immunology, immuno-oncology, drug discovery and vaccine development demand improvements in the capabilities of flow cytometry to allow it to measure more protein markers per cell at multiple timepoints. However, the size of panels of fluorophore markers is limited by overlaps in fluorescence-emission spectra, and flow cytometers typically perform cell measurements at one timepoint. Here we describe multi-pass high-dimensional flow cytometry, a method leveraging cellular barcoding via microparticles emitting near-infrared laser light to track and repeatedly measure each cell using more markers and fewer colours. By using live human peripheral blood mononuclear cells, we show that the method enables the time-resolved characterization of the same cells before and after stimulation, their analysis via a 10-marker panel with minimal compensation for spectral spillover and their deep immunophenotyping via a 32-marker panel, where the same cells are analysed in 3 back-to-back cycles with 10-13 markers per cycle, reducing overall spillover and simplifying marker-panel design. Cellular barcoding in flow cytometry extends the utility of the technique for high-dimensional multi-pass single-cell analyses.

Simultaneous tensile and shear measurement of the human cornea in vivo using S0- and A0-wave optical coherence elastography.

Understanding corneal stiffness is valuable for improving refractive surgery, detecting corneal abnormalities, and assessing intraocular pressure. However, accurately measuring the elastic properties, specifically the tensile and shear moduli that govern mechanical deformation, has been challenging. To tackle this issue, we have developed guided-wave optical coherence elastography that can simultaneously excite and analyze symmetric (S0) and anti-symmetric (A0) elastic waves in the cornea at around 10 kHz frequencies, enabling us to extract tensile and shear properties from measured wave dispersion curves. We verified the technique using elastomer phantoms and ex vivo porcine corneas and investigated the dependence on intraocular pressure using acoustoelastic theory that incorporates corneal tension and a nonlinear constitutive tissue model. In a pilot study involving six healthy human subjects aged 31 to 62, we measured shear moduli (Gzx) of 94±20 kPa (mean±standard deviation) and tensile moduli (Exx) of 4.0±1.1 MPa at central corneas. Our preliminary analysis of age-dependence revealed contrasting trends: -8.3±4.5 kPa/decade for shear and 0.30±0.21 MPa/decade for tensile modulus. This OCE technique has the potential to become a highly useful clinical tool for the quantitative biomechanical assessment of the cornea. STATEMENT OF SIGNIFICANCE: This article reports an innovative elastography technique using two guided elastic waves, demonstrating the measurement of both tensile and shear moduli in human cornea in vivo with unprecedented precision. This technique paves the way for comprehensive investigations into corneal mechanics and holds clinical significance in various aspects of corneal health and disease management.

Aggregation-Induced Stimulated Emission of 100% Dye Microspheres.

Dyes with aggregation-induced emission (AIE) properties have gained interests due to their bright luminescence in solid-state aggregates. While fluorescence from AIE dyes have been widely exploited, relatively little is known about aggregation-induced stimulated emission. Here, we investigated stimulated emission of tetraphenylethene (TPE)-based organoboron AIE dyes, TPEQBN, in thin films and in microcavity lasers. Using femtosecond pump-probe spectroscopy, gain coefficients up to 230 cm-1 at 500 nm were measured. Using rate equations, we analyzed concentration- and pump-dependent gain dynamics as well as laser build up dynamics. During laser oscillation, radiative stimulated emission allows high instantaneous quantum yield greater than 90% to be achieved. We fabricated solid-state microspheres made of 100% AIE dyes via microfluidic emulsion and solvent evaporation method. Coupled with high gain and high refractive index of 1.76, microspheres as small as 2 µm in diameter showed lasing by nanosecond pumping with a threshold of ~10 pJ µm-2. Polymer coated, but not bare, microspheres were internalized by live cells and generated narrowband cavity mode emission from within the cytoplasm. Our work shows the potential of AIE dyes as laser materials.

Facile layer-by-layer fabrication of semiconductor microdisk laser particles.

Semiconductor-based laser particles (LPs) with exceptionally narrowband spectral emission have been used in biological systems for cell tagging purposes. Fabrication of these LPs typically requires highly specialized lithography and etching equipment, and is typically done in a cleanroom environment, hindering the broad adoption of this exciting new technology. Here, using only easily accessible laboratory equipment, we demonstrate a simple layer-by-layer fabrication strategy that overcomes this obstacle. We start from an indium phosphide (InP) substrate with multiple epitaxial indium gallium arsenide phosphide (InGaAsP) layers which are sequentially processed to yield LPs of various compositions and spectral properties. The LPs isolated from each layer are characterized, exhibiting excellent optical properties with lasing emission full width at half maximum as narrow as < 0.3 nm and typical thresholds of approximately 6 pJ upon excitation using a 3 ns pulse duration 1064 nm pump laser. The high quality of these particles renders them suitable for large-scale biological experiments including those requiring spectral multiplexing.

In vivo Optical Coherence Elastography Unveils Spatial Variation of Human Corneal Stiffness.

OBJECTIVE: The mechanical properties of corneal tissues play a crucial role in determining corneal shape and have significant implications in vision care. This study aimed to address the challenge of obtaining accurate in vivo data for the human cornea. METHODS: We have developed a high-frequency optical coherence elastography (OCE) technique using shear-like antisymmetric (A0)-mode Lamb waves at frequencies above 10 kHz. RESULTS: By incorporating an anisotropic, nonlinear constitutive model and utilizing the acoustoelastic theory, we gained quantitative insights into the influence of corneal tension on wave speeds and elastic moduli. Our study revealed significant spatial variations in the shear modulus of the corneal stroma on healthy subjects for the first time. Over an age span from 21 to 34 (N = 6), the central corneas exhibited a mean shear modulus of 87 kPa, while the corneal periphery showed a significant decrease to 44 kPa. The central cornea's shear modulus decreases with age with a slope of -19 +/- 8 kPa per decade, whereas the periphery showed non-significant age dependence. The limbus demonstrated an increased shear modulus exceeding 100 kPa. We obtained wave displacement profiles that are consistent with highly anisotropic corneal tissues. CONCLUSION: Our approach enabled precise measurement of corneal tissue elastic moduli in situ with high precision (< 7%) and high spatial resolution (< 1 mm). Our results revealed significant stiffness variation from the central to peripheral corneas. SIGNIFICANCE: The high-frequency OCE technique holds promise for biomechanical evaluation in clinical settings, providing valuable information for refractive surgeries, degenerative disorder diagnoses, and intraocular pressure assessments.

Poster Session: Optical coherence elastography for In situ measurement of stiffness increase in posterior sclera after crosslinking.

During eye growth, scleral development critically determine eye size and thus the refractive status of the eye. Scleral remodeling in myopia includes scleral thinning, loss of scleral tissue, and weakening of the mechanical properties. Therefore, an intervention aiming at stiffening scleral tissues (crosslinking, SCXL) may provide a way to prevent or treat myopia. The development of SCXL requires tools to evaluate the effects of crosslinking on the mechanical properties of tissues, particularly in sclera where the mechanical properties are more spatially heterogeneous than in the cornea, anisotropic, and varying locally from the anterior to posterior regions. Here, we apply the high-frequency OCE technique to measure the heterogeneous mechanical properties of posterior scleral tissues and, evaluate the changes in shear moduli after SCXL. As a model system, we use ex vivo in porcine eyes and riboflavin-assisted UV crosslinking. From measured elastic wave speeds (6-16kHz), the average out-of-plane shear modulus was 0.71±0.12MPa (n=20) for normal scleras. After treatment, the shear modulus increased to 1.50±0.39MPa. This 2-fold change was consistent with the increase of static Young's modulus from 5.5±.1 to 9.3±1.9MPa after crosslinking, using conventional uniaxial extensometry. OCE revealed regional stiffness differences across the temporal, nasal, and deeper posterior sclera, demonstrating its potential as a noninvasive tool to test the effect of scleral crosslinking.

Simultaneous tensile and shear measurement of the human cornea in vivo using S0- and A0-wave optical coherence elastography.

Understanding corneal stiffness is valuable for improving refractive surgery, detecting corneal abnormalities, and assessing intraocular pressure. However, accurately measuring the elastic properties, particularly the tensile and shear moduli that govern mechanical deformation, has been challenging. To tackle this issue, we have developed guided-wave optical coherence elastography that can simultaneously excite and analyze symmetric (S0) and anti-symmetric (A0) elastic waves in the cornea at frequencies around 10 kHz and allows us to extract tensile and shear properties from measured wave dispersion curves. By applying acoustoelastic theory that incorporates corneal tension and a nonlinear constitutive tissue model, we verified the technique using elastomer phantoms and ex vivo porcine corneas and investigated the dependence on intraocular pressure. For two healthy human subjects, we measured a mean tensile modulus of 3.6 MPa and a mean shear modulus of 76 kPa in vivo with estimated errors of < 4%. This technique shows promise for the quantitative biomechanical assessment of the cornea in a clinical setting.

Ultra-wideband optical coherence elastography from acoustic to ultrasonic frequencies.

Visualizing viscoelastic waves in materials and tissues through noninvasive imaging is valuable for analyzing their mechanical properties and detecting internal anomalies. However, traditional elastography techniques have been limited by a maximum wave frequency below 1-10 kHz, which hampers temporal and spatial resolution. Here, we introduce an optical coherence elastography technique that overcomes the limitation by extending the frequency range to MHz. Our system can measure the stiffness of hard materials including bones and extract viscoelastic shear moduli for polymers and hydrogels in conventionally inaccessible ranges between 100 Hz and 1 MHz. The dispersion of Rayleigh surface waves across the ultrawide band allowed us to profile depth-dependent shear modulus in cartilages ex vivo and human skin in vivo with sub-mm anatomical resolution. This technique holds immense potential as a noninvasive measurement tool for material sciences, tissue engineering, and medical diagnostics.

Ultrasmall InGa(As)P Dielectric and Plasmonic Nanolasers.

Nanolasers have great potential for both on-chip light sources and optical barcoding particles. We demonstrate ultrasmall InGaP and InGaAsP disk lasers with diameters down to 360 nm (198 nm in height) in the red spectral range. Optically pumped, room-temperature, single-mode lasing was achieved from both disk-on-pillar and isolated particles. When isolated disks were placed on gold, plasmon polariton lasing was obtained with Purcell-enhanced stimulated emission. UV lithography and plasma ashing enabled wafer-scale fabrication of nanodisks with an intended random size variation. Silica-coated nanodisk particles generated stable subnanometer spectra from within biological cells across an 80 nm bandwidth from 635 to 715 nm.

Pulsed stimulated Brillouin microscopy.

Stimulated Brillouin scattering is an emerging technique for probing the mechanical properties of biological samples. However, the nonlinear process requires high optical intensities to generate sufficient signal-to-noise ratio (SNR). Here, we show that the SNR of stimulated Brillouin scattering can exceed that of spontaneous Brillouin scattering with the same average power levels suitable for biological samples. We verify the theoretical prediction by developing a novel scheme using low duty cycle, nanosecond pulses for the pump and probe. A shot noise-limited SNR over 1000 was measured with a total average power of 10 mW for 2 ms or 50 mW for 200 µs integration on water samples. High-resolution maps of Brillouin frequency shift, linewidth, and gain amplitude from cells in vitro are obtained with a spectral acquisition time of 20 ms. Our results demonstrate the superior SNR of pulsed stimulated Brillouin over spontaneous Brillouin microscopy.

Excess pancreatic elastase alters acinar-ß cell communication by impairing the mechano-signaling and the PAR2 pathways.

Type 1 (T1D) or type 2 diabetes (T2D) are caused by a deficit of functional insulin-producing ß cells. Thus, the identification of ß cell trophic agents could allow the development of therapeutic strategies to counteract diabetes. The discovery of SerpinB1, an elastase inhibitor that promotes human ß cell growth, prompted us to hypothesize that pancreatic elastase (PE) regulates ß cell viability. Here, we report that PE is up-regulated in acinar cells and in islets from T2D patients, and negatively impacts ß cell viability. Using high-throughput screening assays, we identified telaprevir as a potent PE inhibitor that can increase human and rodent ß cell viability in vitro and in vivo and improve glucose tolerance in insulin-resistant mice. Phospho-antibody microarrays and single-cell RNA sequencing analysis identified PAR2 and mechano-signaling pathways as potential mediators of PE. Taken together, our work highlights PE as a potential regulator of acinar-ß cell crosstalk that acts to limit ß cell viability, leading to T2D.

Single-cell transcriptomics of a dynamic cell behavior in murine airways.

Despite advances in high-dimensional cellular analysis, the molecular profiling of dynamic behaviors of cells in their native environment remains a major challenge. We present a method that allows us to couple the physiological behaviors of cells in an intact murine tissue to deep molecular profiling of individual cells. This method enabled us to establish a novel molecular signature for a striking migratory cellular behavior following injury in murine airways.

All-natural-molecule, bioluminescent photodynamic therapy results in complete tumor regression and prevents metastasis.

Self-luminescent photodynamic therapy (PDT) has gained attention owing to its potential to enable effective phototherapy without the bottleneck of shallow light penetration into tissues. However, the biosafety concerns and low cytotoxic effect of self-luminescent reagents in vivo have been problems. Here, we demonstrate efficacious bioluminescence (BL)-PDT by using bioluminescence resonance energy transfer (BRET) conjugates of a clinically approved photosensitizer, Chlorin e6, and a luciferase, Renilla reniformis; both derived from biocompatible, natural molecules. With over 80% biophoton utilization efficiency and membrane-fusion liposome-assisted intracellular delivery, these conjugates produce effective, targeted cancer cell killing. Specifically, in an orthotopic mouse model of 4T1 triple-negative breast cancer, BL-PDT showed strong therapeutic effects on large primary tumors and a neoadjuvant outcome in invasive tumors. Furthermore, BL-PDT resulted in complete tumor remission and prevention of metastasis for early-stage tumors. Our results demonstrate the promise of molecularly-activated, clinically viable, depth-unlimited phototherapy.

Planted Graphene Quantum Dots for Targeted, Enhanced Tumor Imaging and Long-Term Visualization of Local Pharmacokinetics.

While photoluminescent graphene quantum dots (GQDs) have long been considered very suitable for bioimaging owing to their protein-like size, superhigh photostability and in vivo long-term biosafety, their unique and crucial bioimaging applications in vivo remain unreachable. Herein, planted GQDs are presented as an excellent tool for in vivo fluorescent, sustainable and multimodality tumor bioimaging in various scenarios. The GQDs are in situ planted in the poly(ethylene glycol) (PEG) layer of PEGylated nanoparticles via a bottom-up molecular approach to obtain the NPs-GQDs-PEG nanocomposite. The planted GQDs show more than four times prolonged blood circulation and 7-8 times increased tumor accumulation than typical GQDs in vivo. After accessible specificity modification, the multifunctional NPs-GQDs-PEG provides targeted, multimodal molecular imaging for various tumor models in vitro or in vivo. Moreover, the highly photostable GQDs enable long-term, real-time visualization of the local pharmacokinetics of NPs in vivo. Planting GQDs in PEGylated nanomedicine offers a new strategy for broad in vivo biomedical applications of GQDs.

An ultra-small bispecific protein augments tumor penetration and treatment for pancreatic cancer.

PURPOSE: The once highly anticipated antibody-based pathway-targeted therapies have not achieved promising outcomes for deadly pancreatic ductal adenocarcinoma (PDAC), mainly due to drugs' low intrinsic anticancer activity and poor penetration across the dense physiological barrier. This study aims to develop an ultra-small-sized, EGFR/VEGF bispecific therapeutic protein to largely penetrate deep tumor tissue and effectively inhibit PDAC tumor growth in vivo. METHODS: The bispecific protein, Bi-fp50, was constructed by a typical synthetic biology method and labeled with fluorescent dyes for in vitro and in vivo imaging. Physicochemical properties, protein dual-binding affinity, and specificity of the Bi-fp50 were evaluated in several PDAC cell lines. In vitro quantitatively and qualitatively anticancer activity of Bi-fp50 was assessed by live/dead staining, MTT assay, and flow cytometry. In vivo pharmacokinetic and biodistribution were evaluated using blood biopsy samples and near-infrared fluorescence imaging. In vivo real-time tracking of Bi-fp50 in the local tumor was conducted by fibered confocal fluorescence microscopy. The subcutaneous PDAC tumor model was used to assess the in vivo antitumor effect of Bi-fp50. RESULTS: Bi-fp50 with an ultra-small size of 50 kDa (5 ~ 6 nm) showed an excellent binding ability to VEGF and EGFR simultaneously and had enhanced, accumulated binding capability for Bxpc3 PDAC cells compared with anti-VEGF scFv and anti-EGFR scFv alone. Additionally, bi-fp50 significantly inhibited the proliferation and growth of Bxpc3 and Aspc1 PDAC cells even under a relatively low concentration (0.3 µM). It showed synergistically enhanced therapeutic effects relative to two individual scFv and Bi-fp50x control in vitro. The half-life of blood clearance of Bi-fp50 was 4.33 ± 0.23 h. After intravenous injection, Bi-fp50 gradually penetrated the deep tumor, widely distributed throughout the whole tissue, and primarily enriched in the tumor with nearly twice the accumulation than scFv2 in the orthotopic PDAC tumor model. Furthermore, the Bi-fp50 protein could induce broad apoptosis in the whole tumor and significantly inhibited tumor growth 3 weeks after injection in vivo without other noticeable side effects. CONCLUSION: The proof-of-concept study demonstrated that the ultra-small-sized, bispecific protein Bi-fp50 could be a potential tumor suppressor and an efficient, safe theranostic tool for treating PDAC tumors.

Precise photoelectrochemical tuning of semiconductor microdisk lasers.

Micro- and nano-disk lasers have emerged as promising optical sources and probes for on-chip and free-space applications. However, the randomness in disk diameter introduced by standard nanofabrication makes it challenging to obtain deterministic wavelengths. To address this, we developed a photoelectrochemical (PEC) etching-based technique that enables us to precisely tune the lasing wavelength with sub-nanometer accuracy. We examined the PEC mechanism and compound semiconductor etching rate in diluted sulfuric acid solution. Using this technique, we produced microlasers on a chip and isolated particles with distinct lasing wavelengths. Our results demonstrate that this scalable technique can be used to produce groups of lasers with precise emission wavelengths for various nanophotonic and biomedical applications.