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.

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.

Label-free histological imaging of tissues using Brillouin light scattering contrast.

Brillouin light scattering offers a unique label-free approach to measure biomechanical properties non-invasively. While this technique is used in biomechanical analysis of cells and tissues, its potential for visualizing structural features of tissues based on the biomechanical contrast has not been much exploited. Here, we present high-resolution Brillouin microscopy images of four basic tissue types: muscular, connective, epithelial, and nervous tissues. The Brillouin contrast distinguishes between muscle fiber cells and endomysium in skeletal muscle and reveals chondrocytes along with spatially varying stiffness of the extracellular matrix in articular cartilage. The hydration-sensitive contrast can visualize the stratum corneum, epidermis, and dermis in the skin epithelium. In brain tissues, the Brillouin images show the mechanical heterogeneity across the cortex and deeper regions. This work demonstrates the versatility of using the Brillouin shift as histological contrast for examining intact tissue substructures via longitudinal modulus without the need for laborious tissue processing steps.

Multiplexed laser particles for spatially resolved single-cell analysis.

Biomolecular analysis at the single-cell level is increasingly important in the study of cellular heterogeneity and its consequences, particularly in organismic development and complex diseases such as cancer. Single-cell molecular analyses have led to the identification of new cell types1 and the discovery of novel targets for diagnosis and therapy2. While these analyses are performed predominantly on dissociated single cells, emerging techniques seek understanding of cellular state, cellular function and cell-cell interactions within the native tissue environment by combining optical microscopy and single-cell molecular analyses. These techniques include in situ multiplexed imaging of fluorescently labeled proteins and nucleotides, as well as low-throughput ex vivo methods in which specific cells are isolated for downstream molecular analyses. However, these methods are limited in either the number and type of molecular species they can identify or the number of cells that can be analyzed. High-throughput methods are needed for comprehensive profiling of many cells (>1000) to detect rare cell types, discriminate relevant biomarkers from intrinsic population noise, and reduce the time and cost of measurement. Many established, high-throughput single-cell analyses are not directly applicable because they require tissue dissociation, leading to a loss of spatial information3. No current methods exist that can seamlessly connect spatial mapping to single-cell techniques. In this Perspective, we review current methods for spatially resolved single-cell analysis and discuss the prospect of novel multiplexed imaging probes, called laser particles, which allow individual cells to be tagged in tissue and analyzed subsequently using high-throughput, comprehensive single-cell techniques.

Selective Equatorial Sclera Crosslinking in the Orbit Using a Metal-Coated Polymer Waveguide.

Purpose: Photochemical crosslinking of the sclera is an emerging technique that may prevent excessive eye elongation in pathologic myopia by stiffening the scleral tissue. To overcome the challenge of uniform light delivery in an anatomically restricted space, we previously introduced the use of flexible polymer waveguides. We presently demonstrate advanced waveguides that are optimized to deliver light selectively to equatorial sclera in the intact orbit. Methods: Our waveguides consist of a polydimethylsiloxane cladding and a polyurethane core, coupled to an optical fiber. A reflective silver coating deposited on the top and side surfaces of the waveguide prevents light leakage to nontarget, periorbital tissue. Postmortem rabbits were used to test the feasibility of in situ equatorial sclera crosslinking. Tensometry measurements were performed on ex vivo rabbit eyes to confirm a biomechanical stiffening effect. Results: Metal-coated waveguides enabled efficient light delivery to the entire circumference of the equatorial sclera with minimal light leakage to the periorbital tissues. Blue light was delivered to the intact orbit with a coefficient of variation in intensity of 22%, resulting in a 45 ± 11% bleaching of riboflavin fluorescence. A 2-fold increase in the Young's modulus at 5% strain (increase of 92% P < 0.05, at 25 J/cm2) was achieved for ex vivo crosslinked eyes. Conclusions: Flexible polymer waveguides with reflective, biocompatible surfaces are useful for sclera crosslinking to achieve targeted light delivery. We anticipate that our demonstrated procedure will be applicable to sclera crosslinking in live animal models and, potentially, humans in vivo.

Wavelength-encoded laser particles for massively multiplexed cell tagging.

Large-scale single-cell analyses have become increasingly important given the role of cellular heterogeneity in complex biological systems. However, no current techniques enable optical imaging of uniquely-tagged individual cells. Fluorescence-based approaches can only distinguish a small number of distinct cells or cell groups at a time because of spectral crosstalk between conventional fluorophores. Here we investigate large-scale cell tracking using intracellular laser particles as imaging probes that emit coherent laser light with a characteristic wavelength. Made of silica-coated semiconductor microcavities, these laser particles have single-mode emission over a broad range from 1170 to 1580 nm with sub-nm linewidths, enabling massive spectral multiplexing. We explore the stability and biocompatibility of these probes in vitro and their utility for wavelength-multiplexed cell tagging and imaging. We demonstrate real-time tracking of thousands of individual cells in a 3D tumour model over several days showing different behavioural phenotypes.

Millisecond cellular labelling in situ with two-photon photoconversion.

In situ labeling of cells within living biological tissues using photoconversion has provided valuable information on cellular physiology in their natural environments. However, current photoconvertible probes typically require seconds to minutes of light exposure, limiting their uses in rapid biological processes such as intracellular diffusion and circulating cells. Here, we report that two-photon photoconversion of cyanine-based dyes offers unprecedentedly rapid photoconversion down to millisecond timescales per cell. We demonstrate potential biological applications including measuring intracellular diffusion kinetics in a spinal nerve, labeling of rapidly flowing cells in a microfluidic channel, and photoconversion of a circulating cell in vivo.

Effects of Corneal Hydration on Brillouin Microscopy In Vivo.

Purpose: To investigate how corneal hydration affects the Brillouin frequency of corneal stroma. Methods: From a simple analytical model considering the volume fraction of water in corneal stroma, we derived the dependence of Brillouin frequency on hydration and hydration-induced corneal thickness variation. The Brillouin frequencies of fresh ex vivo porcine corneas were measured as their hydration was varied in dextran solution and water. Healthy volunteers (8 eyes) were scanned in vivo repeatedly over the course of 9 hours, and the diurnal variations of Brillouin frequency and central corneal thickness (CCT) were measured. Results: The measured dependence of Brillouin frequency on hydration, both ex vivo and in vivo, agreed well with the theoretical prediction. The Brillouin frequencies of human corneas scanned immediately after waking were on average ∼25 MHz lower than their daytime average values. For stabilized corneas, the typical variation of Brillouin frequency was ± 7.2 MHz. With respect to CCT increase or swelling, the Brillouin frequency decreased with a slope of -1.06 MHz/µm in vivo. Conclusions: The ex vivo and in vivo data agree with our theoretical model and support that the effect of corneal hydration on Brillouin frequency comes predominantly from the dependence of the tissue compressibility on the water. Corneal hydration correlates negatively with the Brillouin frequency. During daytime activities, the influence of physiological hydration changes in human corneas is < ± 10 MHz. The sensitivity to hydration may potentially be useful in detecting abnormal hydration change in patients with endothelial disorders.

Light in diagnosis, therapy and surgery.

Light and optical techniques have made profound impacts on modern medicine, with numerous lasers and optical devices being currently used in clinical practice to assess health and treat disease. Recent advances in biomedical optics have enabled increasingly sophisticated technologies - in particular those that integrate photonics with nanotechnology, biomaterials and genetic engineering. In this Review, we revisit the fundamentals of light-matter interactions, describe the applications of light in imaging, diagnosis, therapy and surgery, overview their clinical use, and discuss the promise of emerging light-based technologies.

Flexible Optical Waveguides for Uniform Periscleral Cross-Linking.

Purpose: Scleral cross-linking (SXL) with a photosensitizer and light is a potential strategy to mechanically reinforce the sclera and prevent progressive axial elongation responsible for severe myopia. Current approaches for light delivery to the sclera are cumbersome, do not provide uniform illumination, and only treat a limited area of sclera. To overcome these challenges, we developed flexible optical waveguides optimized for efficient, homogeneous light delivery. Methods: Waveguides were fabricated from polydimethylsiloxane elastomer. Blue light (445 nm) is coupled into the waveguide with an input fiber. Light delivery efficiency from the waveguide to scleral tissue was measured and fit to a theoretical model. SXL was performed on fresh porcine eyes stained with 0.5% riboflavin, using irradiances of 0, 25, and 50 mW/cm2 around the entire equator of the eye. Stiffness of scleral strips was characterized with tensiometry. Results: Light delivery with a waveguide of tapered thickness (1.4-0.5 mm) enhanced the uniformity of light delivery, compared to a flat waveguide, achieving a coefficient of variation of less than 10%. At 8% strain, sclera cross-linked with the waveguides at 50 mW/cm2 for 30 minutes had a Young's modulus of 10.7 ± 1.0 MPa, compared to 5.9 ± 0.5 MPa for no irradiation, with no difference in stiffness between proximally and distally treated halves. The stiffness of waveguide-irradiated samples did not differ from direct irradiation at the same irradiance. Conclusions: We developed flexible waveguides for periscleral cross-linking. We demonstrated efficient and uniform stiffening of a 5-mm-wide equatorial band of scleral tissue.

Optical lens-microneedle array for percutaneous light delivery.

The limited penetration depth of light in skin tissues is a practical bottleneck in dermatologic applications of light-induced therapies, including anti-microbial blue light therapy and photodynamic skin cancer therapy. Here, we demonstrate a novel device, termed optical microneedle array (OMNA), for percutaneous light delivery. A prototype device with a 11 by 11 array of needles at a spacing of 1 mm and a length of 1.6 mm was fabricated by press-molding poly-(lactic acid) (PLA) polymers. The device also incorporates a matched microlens array that focuses the light through the needle tips at specific points to achieve an optimal intensity profile in the tissue. In experiments done with bovine tissues, the OMNA enabled us to deliver a total of 7.5% of the input photons at a wavelength of 491 nm, compared to only 0.85% without the device. This 9-fold enhancement of light delivery was close to the prediction of 10.8 dB by ray-tracing simulation and is expected to increase the effective treatment depth of anti-microbial blue light therapy significantly from 1.3 to 2.5 mm in human skin.

Noninvasive Transdermal Vaccination Using Hyaluronan Nanocarriers and Laser Adjuvant.

Vaccines are commonly administered by injection using needles. Although transdermal microneedles are less-invasive promising alternatives, needle-free topical vaccination without involving physical damage to the natural skin barrier is still sought after as it can further reduce needle-induced anxiety and simply administration. However, this long-standing goal has been elusive since the intact skin is impermeable to most macromolecules. Here, we show an efficient, non-invasive transdermal vaccination in mice by employing two key innovations: first, the use of hyaluronan (HA) as vaccine carriers and, second, non-ablative laser adjuvants. Conjugates of a model vaccine ovalbumin (OVA) and HA-HA-OVA conjugates-induced more effective maturation of dendritic cells in vitro, compared to OVA or HA alone, through synergistic HA receptor-mediated effects. Following topical administration in the back skin, HA-OVA conjugates penetrated into the epidermis and dermis in murine and porcine skins up to 30% of the total applied quantity, as revealed by intravital microscopy and quantitative fluorescence assay. Topical administration of HA-OVA conjugates significantly elevated both anti-OVA IgG antibody levels in serum and IgA antibody levels in bronchioalveolar lavage, with peak levels at 4 weeks, while OVA alone had a negligible effect. An OVA challenge at week 8 elicited strong immune-recall humoral responses. With pre-treatment of the skin using non-ablative fractional laser beams (1410 nm wavelength, 10 ms pulse duration, 0.2 mJ/pulse) as laser adjuvant, strong immunization was achieved with much reduced doses of HA-OVA (1 mg/kg OVA). Our results demonstrate the potential of the non-invasive patch-type transdermal vaccination platform.

Two-photon excited photoconversion of cyanine-based dyes.

The advent of phototransformable fluorescent proteins has led to significant advances in optical imaging, including the unambiguous tracking of cells over large spatiotemporal scales. However, these proteins typically require activating light in the UV-blue spectrum, which limits their in vivo applicability due to poor light penetration and associated phototoxicity on cells and tissue. We report that cyanine-based, organic dyes can be efficiently photoconverted by nonlinear excitation at the near infrared (NIR) window. Photoconversion likely involves singlet-oxygen mediated photochemical cleavage, yielding blue-shifted fluorescent products. Using SYTO62, a biocompatible and cell-permeable dye, we demonstrate photoconversion in a variety of cell lines, including depth-resolved labeling of cells in 3D culture. Two-photon photoconversion of cyanine-based dyes offer several advantages over existing photoconvertible proteins, including use of minimally toxic NIR light, labeling without need for genetic intervention, rapid kinetics, remote subsurface targeting, and long persistence of photoconverted signal. These findings are expected to be useful for applications involving rapid labeling of cells deep in tissue.

Shear Brillouin light scattering microscope.

Brillouin spectroscopy has been used to characterize shear acoustic phonons in materials. However, conventional instruments had slow acquisition times over 10 min per 1 mW of input optical power, and they required two objective lenses to form a 90° scattering geometry necessary for polarization coupling by shear phonons. Here, we demonstrate a confocal Brillouin microscope capable of detecting both shear and longitudinal phonons with improved speeds and with a single objective lens. Brillouin scattering spectra were measured from polycarbonate, fused quartz, and borosilicate in 1-10 s at an optical power level of 10 mW. The elastic constants, phonon mean free path and the ratio of the Pockels coefficients were determined at microscopic resolution.

Photonic hydrogel sensors.

Analyte-sensitive hydrogels that incorporate optical structures have emerged as sensing platforms for point-of-care diagnostics. The optical properties of the hydrogel sensors can be rationally designed and fabricated through self-assembly, microfabrication or laser writing. The advantages of photonic hydrogel sensors over conventional assay formats include label-free, quantitative, reusable, and continuous measurement capability that can be integrated with equipment-free text or image display. This Review explains the operation principles of photonic hydrogel sensors, presents syntheses of stimuli-responsive polymers, and provides an overview of qualitative and quantitative readout technologies. Applications in clinical samples are discussed, and potential future directions are identified.

Selective two-photon collagen crosslinking in situ measured by Brillouin microscopy.

Two-photon polymerization has enabled precise microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, and cellular scaffolds. We present two-photon collagen crosslinking (2P-CXL) of intact corneal tissue using riboflavin and femtosecond laser irradiation. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Measurement of local changes in longitudinal mechanical moduli with confocal Brillouin microscopy enabled the visualization of the cross-linked pattern without perturbation of the surrounding non-irradiated regions. 2P-CXL induced stiffening was comparable to that achieved with conventional one-photon CXL. Our results demonstrate the ability to selectively stiffen biological tissue in situ at high resolution with broad implications in ophthalmology, laser surgery, and tissue engineering.

In vivo fluorescence microscopy: lessons from observing cell behavior in their native environment.

Microscopic imaging techniques to visualize cellular behaviors in their natural environment play a pivotal role in biomedical research. Here, we review how recent technical advances in intravital microscopy have enabled unprecedented access to cellular physiology in various organs of mice in normal and diseased states.

Label-free probe of HIV-1 TAT peptide binding to mimetic membranes.

The transacting activator of transduction (TAT) protein plays a key role in the progression of AIDS. Studies have shown that a +8 charged sequence of amino acids in the protein, called the TAT peptide, enables the TAT protein to penetrate cell membranes. To probe mechanisms of binding and translocation of the TAT peptide into the cell, investigators have used phospholipid liposomes as cell membrane mimics. We have used the method of surface potential sensitive second harmonic generation (SHG), which is a label-free and interface-selective method, to study the binding of TAT to anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1'-rac-glycerol (POPG) and neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. It is the SHG sensitivity to the electrostatic field generated by a charged interface that enabled us to obtain the interfacial electrostatic potential. SHG together with the Poisson-Boltzmann equation yielded the dependence of the surface potential on the density of adsorbed TAT. We obtained the dissociation constants Kd for TAT binding to POPC and POPG liposomes and the maximum number of TATs that can bind to a given liposome surface. For POPC Kd was found to be 7.5 ± 2 µM, and for POPG Kd was 29.0 ± 4.0 µM. As TAT was added to the liposome solution the POPC surface potential changed from 0 mV to +37 mV, and for POPG it changed from -57 mV to -37 mV. A numerical calculation of Kd, which included all terms obtained from application of the Poisson-Boltzmann equation to the TAT liposome SHG data, was shown to be in good agreement with an approximated solution.

Ultrasound-mediated microbubble enhancement of radiation therapy studied using three-dimensional high-frequency power Doppler ultrasound.

Tumor responses to high-dose (>8 Gy) radiation therapy are tightly connected to endothelial cell death. In the study described here, we investigated whether ultrasound-activated microbubbles can locally enhance tumor response to radiation treatments of 2 and 8 Gy by mechanically perturbing the endothelial lining of tumors. We evaluated vascular changes resulting from combined microbubble and radiation treatments using high-frequency 3-D power Doppler ultrasound in a breast cancer xenograft model. We compared treatment effects and monitored vasculature damage 3 hours, 24 hours and 7 days after treatment delivery. Mice treated with 2 Gy radiation and ultrasound-activated microbubbles exhibited a decrease in vascular index to 48 ± 10% at 24 hours, whereas vascular indices of mice treated with 2 Gy radiation alone or microbubbles alone were relatively unchanged at 95 ± 14% and 78 ± 14%, respectively. These results suggest that ultrasound-activated microbubbles enhance the effects of 2 Gy radiation through a synergistic mechanism, resulting in alterations of tumor blood flow. This novel therapy may potentiate lower radiation doses to preferentially target endothelial cells, thus reducing effects on neighboring normal tissue and increasing the efficacy of cancer treatments.