High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging.

Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .

Diamond Raman laser and Yb fiber amplifier for in vivo multiphoton fluorescence microscopy.

Here we introduce a fiber amplifier and a diamond Raman laser that output high powers (6.5 W, 1.3 W) at valuable wavelengths (1060 nm, 1250 nm) for two-photon excitation of red-shifted fluorophores. These custom excitation sources are both simple to construct and cost-efficient in comparison to similar custom and commercial alternatives. Furthermore, they operate at a repetition rate (80 MHz) that allows fast image acquisition using resonant scanners. With our system we demonstrate compatibility with fast resonant scanning, the ability to acquire neuronal images, and the capability to image vasculature at deep locations (>1 mm) within the mouse cerebral cortex.

Three-Dimensional Two-Color Dual-Particle Tracking Microscope for Monitoring DNA Conformational Changes and Nanoparticle Landings on Live Cells.

Here, we present a three-dimensional two-color dual-particle tracking (3D-2C-DPT) technique that can simultaneously localize two spectrally distinct targets in three dimensions with a time resolution down to 5 ms. The dual-targets can be tracked with separation distances from 33 to 250 nm with tracking precisions of ∼15 nm (for static targets) and ∼35 nm (for freely diffusing targets). Since each target is individually localized, a wealth of data can be extracted, such as the relative 3D position, the 2D rotation, and the separation distance between the two targets. Using this technique, we turn a double-stranded DNA (dsDNA)-linked dumbbell-like dimer into a nanoscopic optical ruler to quantify the bending dynamics of nicked or gapped dsDNA molecules in free solution by manipulating the design of dsDNA linkers (1-nick, 3-nt, 6-nt, or 9-nt single-strand gap), and the results show the increase of kon (linear to bent) from 3.2 to 10.7 s-1. The 3D-2C-DPT is then applied to observe translational and rotational motions of the landing of an antibody-conjugated nanoparticle on the plasma membrane of living cells, revealing the reduction of rotations possibly due to interactions with membrane receptors. This study demonstrates that this 3D-2C-DPT technique is a new tool to shed light on the conformational changes of biomolecules and the intermolecular interactions on plasma membrane.

Spatial EGFR Dynamics and Metastatic Phenotypes Modulated by Upregulated EphB2 and Src Pathways in Advanced Prostate Cancer.

Advanced prostate cancer is a very heterogeneous disease reflecting in diverse regulations of oncogenic signaling pathways. Aberrant spatial dynamics of epidermal growth factor receptor (EGFR) promote their dimerization and clustering, leading to constitutive activation in oncogenesis. The EphB2 and Src signaling pathways are associated with the reorganization of the cytoskeleton leading to malignancy, but their roles in regulating EGFR dynamics and activation are scarcely reported. Using single-particle tracking techniques, we found that highly phosphorylated EGFR in the advanced prostate cancer cell line, PC3, was associated with higher EGFR diffusivity, as compared with LNCaP and less aggressive DU145. The increased EGFR activation and biophysical dynamics were consistent with high proliferation, migration, and invasion. After performing single-cell RNA-seq on prostate cancer cell lines and circulating tumor cells from patients, we identified that upregulated gene expression in the EphB2 and Src pathways are associated with advanced malignancy. After dasatinib treatment or siRNA knockdowns of EphB2 or Src, the PC3 cells exhibited significantly lower EGFR dynamics, cell motility, and invasion. Partial inhibitory effects were also found in DU145 cells. The upregulation of parts of the EphB2 and Src pathways also predicts poor prognosis in the prostate cancer patient cohort of The Cancer Genome Atlas. Our results provide evidence that overexpression of the EphB2 and Src signaling pathways regulate EGFR dynamics and cellular aggressiveness in some advanced prostate cancer cells.

Assessing metastatic potential of breast cancer cells based on EGFR dynamics.

Derailed transmembrane receptor trafficking could be a hallmark of tumorigenesis and increased tumor invasiveness, but receptor dynamics have not been used to differentiate metastatic cancer cells from less invasive ones. Using single-particle tracking techniques, we  developed a phenotyping asssay named Transmembrane Receptor Dynamics (TReD), studied the dynamics of epidermal growth factor receptor (EGFR) in seven breast epithelial cell lines and developed a phenotyping assay named Transmembrane Receptor Dynamics (TReD). Here we show a clear evidence that increased EGFR diffusivity and enlarged EGFR confinement size in the plasma membrane (PM) are correlated with the enhanced metastatic potential in these cell lines. By comparing the TReD results with the gene expression profiles, we found a clear negative correlation between the EGFR diffusivities and the breast cancer luminal differentiation scores (r = -0.75). Upon the induction of epithelial-mesenchymal transition (EMT), EGFR diffusivity significantly increased for the non-tumorigenic MCF10A (99%) and the non-invasive MCF7 (56%) cells, but not for the highly metastatic MDA-MB-231 cell. We believe that the reorganization of actin filaments during EMT modified the PM structures, causing the receptor dynamics to change. TReD can thus serve as a new biophysical marker to probe the metastatic potential of cancer cells and even to monitor the transition of metastasis.

Polymer dots enable deep in vivo multiphoton fluorescence imaging of microvasculature.

Deep in vivo imaging of vasculature requires small, bright, and photostable fluorophores suitable for multiphoton microscopy (MPM). Although semiconducting polymer dots (pdots) are an emerging class of highly fluorescent contrast agents with favorable advantages for the next generation of in vivo imaging, their use for deep MPM has never before been demonstrated. Herein, we characterize the multiphoton properties of three pdot variants and perform deep in vivo MPM imaging of cortical rodent microvasculature. We find pdot brightness exceeds conventional fluorophores, including quantum dots, and their broad multiphoton absorption spectrum permits imaging at wavelengths better-suited for biological imaging and confers compatibility with a range of longer excitation wavelengths. This results in substantial improvements in signal-to-background ratio (>3.5-fold) and greater cortical imaging depths (z = 1,300 µm). Ultimately, pdots are a versatile tool for MPM due to their extraordinary brightness and broad absorption, enabling interrogation of deep structures in vivo.

Eradication of Triple-Negative Breast Cancer Cells by Targeting Glycosylated PD-L1.

Protein glycosylation provides proteomic diversity in regulating protein localization, stability, and activity; it remains largely unknown whether the sugar moiety contributes to immunosuppression. In the study of immune receptor glycosylation, we showed that EGF induces programmed death ligand 1 (PD-L1) and receptor programmed cell death protein 1 (PD-1) interaction, requiring ß-1,3-N-acetylglucosaminyl transferase (B3GNT3) expression in triple-negative breast cancer. Downregulation of B3GNT3 enhances cytotoxic T cell-mediated anti-tumor immunity. A monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) blocks PD-L1/PD-1 interaction and promotes PD-L1 internalization and degradation. In addition to immune reactivation, drug-conjugated gPD-L1 antibody induces a potent cell-killing effect as well as a bystander-killing effect on adjacent cancer cells lacking PD-L1 expression without any detectable toxicity. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy.

Controlling Plasmon-Enhanced Fluorescence via Intersystem Crossing in Photoswitchable Molecules.

By harnessing photoswitchable intersystem crossing (ISC) in spiropyran (SP) molecules, active control of plasmon-enhanced fluorescence in the hybrid systems of SP molecules and plasmonic nanostructures is achieved. Specifically, SP-derived merocyanine (MC) molecules formed by photochemical ring-opening reaction display efficient ISC due to their zwitterionic character. In contrast, ISC in quinoidal MC molecules formed by thermal ring-opening reaction is negligible. The high ISC rate can improve fluorescence quantum yield of the plasmon-modified spontaneous emission, only when the plasmonic electromagnetic field enhancement is sufficiently high. Along this line, extensive photomodulation of fluorescence is demonstrated by switching the ISC in MC molecules at Au nanoparticle aggregates, where strongly enhanced plasmonic hot spots exist. The ISC-mediated plasmon-enhanced fluorescence represents a new approach toward controlling the spontaneous emission of fluorophores near plasmonic nanostructures, which expands the applications of active molecular plasmonics in information processing, biosensing, and bioimaging.

In vivo multiphoton imaging of a diverse array of fluorophores to investigate deep neurovascular structure.

We perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier laser source tunable between λ=1,100 and 1,400 nm. By combining the high repetition rate (511 kHz) and high pulse energy (400 nJ) of our amplifier laser system, we demonstrate imaging of vasculature labeled with Texas Red and Indocyanine Green, and neurons expressing tdTomato and yellow fluorescent protein. We measure the blood flow speed of a single capillary at a depth of 1.2 mm, and image vasculature to a depth of 1.53 mm with fine axial steps (5 µm) and reasonable acquisition times. The high image quality enabled analysis of vascular morphology at depths to 1.45 mm.

High-Resolution Bubble Printing of Quantum Dots.

Semiconductor quantum dots (QDs) feature excellent properties, such as high quantum efficiency, tunable emission frequency, and good fluorescence stability. Incorporation of QDs into new devices relies upon high-resolution and high-throughput patterning techniques. Herein, we report a new printing technique known as bubble printing (BP), which exploits a light-generated microbubble at the interface of colloidal QD solution and a substrate to directly write QDs into arbitrary patterns. With the uniform plasmonic hot spot distribution for high bubble stability and the optimum light-scanning parameters, we have achieved full-color QD printing with submicron resolution (650 nm), high throughput (scanning rate of ∼10-2 m/s), and high adhesion of the QDs to the substrates. The printing parameters can be optimized to further control the fluorescence properties of the patterned QDs, such as emission wavelength and lifetime. The patterning of QDs on flexible substrates further demonstrates the wide applicability of this new technique. Thus, BP technique addresses the barrier of achieving a widely applicable, high-throughput and user-friendly patterning technique in the submicrometer regime, along with simultaneous fluorescence modification capability.

Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser.

Two-color multiphoton microscopy through wavelength mixing of synchronized lasers has been shown to increase the spectral window of excitable fluorophores without the need for wavelength tuning. However, most currently available dual output laser sources rely on the costly and complicated optical parametric generation approach. In this report, we detail a relatively simple and low cost diamond Raman laser pumped by a ytterbium fiber amplifier emitting at 1055 nm, which generates a first Stokes emission centered at 1240 nm with a pulse width of 100 fs. The two excitation wavelengths of 1055 and 1240 nm, along with the effective two-color excitation wavelength of 1140 nm, provide an almost complete coverage of fluorophores excitable within the range of 1000-1300 nm. When compared with 1055 nm excitation, two-color excitation at 1140 nm offers a 90% increase in signal for many far-red emitting fluorescent proteins (for example, tdKatushka2). We demonstrate multicolor imaging of tdKa-tushka2 and Hoechst 33342 via simultaneous two-color two-photon, and two-color three-photon microscopy in engineered 3D multicellular spheroids. We further discuss potential benefits and applications for two-color three-photon excitation. In addition, we show that this laser system is capable of in vivo imaging in mouse cortex to nearly 1 mm in depth with two-color excitation.

Patterning and fluorescence tuning of quantum dots with haptic-interfaced bubble printing.

Semiconductor quantum dots (QDs) are attractive for a wide range of applications such as displays, light-emitting devices, and sensors due to their properties such as tunable fluorescence wavelength, high brightness, and narrow bandwidth. Most of the applications require precise patterning of QDs with targeted properties on solid-state substrates. Herein, we have developed a haptic-interfaced bubble printing (HIBP) technique to enable high-resolution (510 nm) high-throughput (>104 µm s-1) patterning of QDs with strong emission tunability and to significantly enhance the accessibility of the technique via a smartphone device. The scalability and versatility of the HIBP are demonstrated in our arbitrary patterning of QDs on plasmonic substrates. A detailed study of plasmonic and photothermal interactions is performed via programmed stage movements to realize tunability of the emission wavelength and lifetime. Finally, the influence of the hand movement on the properties of the printed QDs in terms of emission wavelength tuning from yellow to blue is established. This work provides a single-step macroscale platform to manipulate nanoscale properties at high resolution and high throughput.

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking.

Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of intracellular processes using single-particle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups-immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis.

Single-Molecule Tracking and Its Application in Biomolecular Binding Detection.

In the past two decades significant advances have been made in single-molecule detection, which enables the direct observation of single biomolecules at work in real time and under physiological conditions. In particular, the development of single-molecule tracking (SMT) microscopy allows us to monitor the motion paths of individual biomolecules in living systems, unveiling the localization dynamics and transport modalities of the biomolecules that support the development of life. Beyond the capabilities of traditional camera-based tracking techniques, state-of-the-art SMT microscopies developed in recent years can record fluorescence lifetime while tracking a single molecule in the 3D space. This multiparameter detection capability can open the door to a wide range of investigations at the cellular or tissue level, including identification of molecular interaction hotspots and characterization of association/dissociation kinetics between molecules. In this review, we discuss various SMT techniques developed to date, with an emphasis on our recent development of the next generation 3D tracking system that not only achieves ultrahigh spatiotemporal resolution but also provides sufficient working depth suitable for live animal imaging. We also discuss the challenges that current SMT techniques are facing and the potential strategies to tackle those challenges.

Deep in vivo two-photon microscopy with a low cost custom built mode-locked 1060 nm fiber laser.

Here we demonstrate that a mode-locked ytterbium fiber laser for two-photon fluorescence microscopy can be built for $13,000. The laser emits at a wavelength of 1060 nm with a usable average power of 1 W at a repetition rate of 40 MHz and a compressed pulse width of 81 fs at the sample. The laser is used to obtain deep in vivo two-color images of layer-V pyramidal neurons expressing YFP and vasculature labelled with Texas Red at depths up to 900 µm. The sub-1 µm features of dendritic spines can be resolved at a 200 µm depth.

Bubble-Pen Lithography.

Current lithography techniques, which employ photon, electron, or ion beams to induce chemical or physical reactions for micro/nano-fabrication, have remained challenging in patterning chemically synthesized colloidal particles, which are emerging as building blocks for functional devices. Herein, we develop a new technique - bubble-pen lithography (BPL) - to pattern colloidal particles on substrates using optically controlled microbubbles. Briefly, a single laser beam generates a microbubble at the interface of colloidal suspension and a plasmonic substrate via plasmon-enhanced photothermal effects. The microbubble captures and immobilizes the colloidal particles on the substrate through coordinated actions of Marangoni convection, surface tension, gas pressure, and substrate adhesion. Through directing the laser beam to move the microbubble, we create arbitrary single-particle patterns and particle assemblies with different resolutions and architectures. Furthermore, we have applied BPL to pattern CdSe/ZnS quantum dots on plasmonic substrates and polystyrene (PS) microparticles on two-dimensional (2D) atomic-layer materials. With the low-power operation, arbitrary patterning and applicability to general colloidal particles, BPL will find a wide range of applications in microelectronics, nanophotonics, and nanomedicine.

Molecular-Fluorescence Enhancement via Blue-Shifted Plasmon-Induced Resonance Energy Transfer.

We report molecular-fluorescence enhancement via the blue-shifted plasmon-induced resonance energy transfer (PIRET) from single Au nanorods (AuNRs) to merocyanine (MC) dye molecules. The blue-shifted PIRET occurs when there is a proper spectral overlap between the scattering of AuNRs and the absorption of MC molecules. Along with the quenching of scattering from AuNRs, the blue-shifted PIRET enhances the fluorescence of nearby molecules. On the basis of the fluorescence enhancement, we conclude that AuNRs can be used as donors with clear advantages to excite the fluorescence of molecules as acceptors in AuNR-molecule hybrids. On the one hand, compared to conventional molecular donors in Förster resonance energy transfer (FRET), AuNRs have much larger absorption cross sections at the plasmon resonance frequencies. On the other hand, energy-transfer efficiency of PIRET decreases at a lower rate than that of FRET when the donor-acceptor distance is increased. Besides, the blue-shifted PIRET allows excitation with incident light of lower energy than the acceptor's absorption, which is difficult to achieve in FRET because of the Stokes shift. With the capability of enhancing molecular fluorescence with excitation light of low intensity and long wavelength, the blue-shifted PIRET will expand the applications of nanoparticle- molecule hybrids in biosensing and bioimaging by increasing signal-to-noise ratio and by reducing photodamage to biological cells and organelles at the targeted areas.

Deep and high-resolution three-dimensional tracking of single particles using nonlinear and multiplexed illumination.

Molecular trafficking within cells, tissues and engineered three-dimensional multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. However, current tracking methods are either confined to two dimensions or limited to an interrogation depth of ∼15 µm. Here we present a three-dimensional tracking method capable of quantifying rapid molecular transport dynamics in highly scattering environments at depths up to 200 µm. The system has a response time of 1 ms with a temporal resolution down to 50 µs in high signal-to-noise conditions, and a spatial localization precision as good as 35 nm. Built on spatiotemporally multiplexed two-photon excitation, this approach requires only one detector for three-dimensional particle tracking and allows for two-photon, multicolour imaging. Here we demonstrate three-dimensional tracking of epidermal growth factor receptor complexes at a depth of ∼100 µm in tumour spheroids.