Multiplexed and Millimeter-Scale Fluorescence Nanoscopy of Cells and Tissue Sections via Prism-Illumination and Microfluidics-Enhanced DNA-PAINT.

Fluorescence nanoscopy has become increasingly powerful for biomedical research, but it has historically afforded a small field-of-view (FOV) of around 50 µm × 50 µm at once and more recently up to ∼200 µm × 200 µm. Efforts to further increase the FOV in fluorescence nanoscopy have thus far relied on the use of fabricated waveguide substrates, adding cost and sample constraints to the applications. Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT (PRIME-PAINT) for multiplexed fluorescence nanoscopy across millimeter-scale FOVs. Built upon the well-established prism-type total internal reflection microscopy, PRIME-PAINT achieves robust single-molecule localization with up to ∼520 µm × 520 µm single FOVs and 25-40 nm lateral resolutions. Through stitching, nanoscopic imaging over mm2 sample areas can be completed in as little as 40 min per target. An on-stage microfluidics chamber facilitates probe exchange for multiplexing and enhances image quality, particularly for formalin-fixed paraffin-embedded (FFPE) tissue sections. We demonstrate the utility of PRIME-PAINT by analyzing ∼106 caveolae structures in ∼1,000 cells and imaging entire pancreatic cancer lesions from patient tissue biopsies. By imaging from nanometers to millimeters with multiplexity and broad sample compatibility, PRIME-PAINT will be useful for building multiscale, Google-Earth-like views of biological systems.

Ultrafast Background-Free Ultrasound Imaging Using Blinking Nanoparticles.

Localization-based ultrasound imaging methods that use microbubbles or nanodroplets offer high-resolution imaging with improved sensitivity and reduced background signal. However, these methods require long acquisition times (typically seconds to minutes), preventing their use for real-time imaging and, thus, limiting their clinical translational potential. Here, we present a new ultrafast localization method using blinking ultrasound-responsive nanoparticles (BNPs). When activated with high frame rate (1 kHz) plane wave ultrasound pulses with a mechanical index of 1.5, the BNPs incept growth of micrometer-sized bubbles, which in turn collapse and generate a blinking ultrasound signal. We showed that background-free ultrasound images could be obtained by localizing these blinking events using acquisition times as low as 11 ms. In addition, we demonstrated that BNPs enable in vivo background-free ultrasound imaging in mice. We envision that BNPs will facilitate the clinical translation of localization-based ultrasound imaging for more sensitive detection of cancer and other diseases.

Multispectral Localized Surface Plasmon Resonance (msLSPR) Reveals and Overcomes Spectral and Sensing Heterogeneities of Single Gold Nanoparticles.

Metal nanoparticles can be sensitive molecular sensors due to enhanced absorption and scattering of light near a localized surface plasmon resonance (LSPR). Variations in both intrinsic properties such as the geometry and extrinsic properties such as the environment can cause heterogeneity in nanoparticle LSPR and impact the overall sensing responses. To date, however, few studies have examined LSPR and sensing heterogeneities, due to technical challenges in obtaining the full LSPR spectra of individual nanoparticles in dynamic assays. Here, we report multispectral LSPR (msLSPR), a wide-field imaging technique for real-time spectral monitoring of light scattering from individual nanoparticles across the whole field of view (FOV) at ∼0.5 nm spectral and ∼100 ms temporal resolutions. Using msLSPR, we studied the spectral and sensing properties of gold nanoparticles commonly used in LSPR assays, including spheres, rods, and bipyramids. Complemented with electron microscopy imaging, msLSPR analysis revealed that all classes of gold nanoparticles exhibited variations in LSPR peak wavelengths that largely paralleled variations in morphology. Compared with the rods and spheres, gold nanobipyramids exhibited both more uniform and stronger sensing responses as long as the bipyramids are structurally intact. Simulations incorporating the experimental LSPR properties demonstrate the negative impact of spectral heterogeneity on the overall performance of conventional, intensity-based LSPR assays and the ability of msLSPR in overcoming both particle heterogeneity and measurement noise. These results highlight the importance of spectral heterogeneity in LSPR-based sensors and the potential advantage of performing LSPR assays in the spectral domain.

A Versatile Synthetic Pathway for Producing Mesostructured Plasmonic Nanostructures.

Highly branched gold (Au) nanostructures with sharp tips are considered excellent substrates for surface-enhanced Raman scattering (SERS)-based sensing technologies. Here, a simple synthetic route for producing Au or Au-Ag bimetallic mesostructures with multiple sharpened tips in the presence of carbon quantum dots (CQDs) is presented. The morphologies of these mesostructured plasmonic nanoparticles (MSPNs) can be controlled by adjusting the concentration of CQDs, reaction temperatures, and seed particles. The optimal molar ratio for [HAuCl4 ]/[CQDs] is found to be ≈25. At this molar ratio, the diameters of MSPNs can be tuned from 80 to 200 nm by changing the reaction temperature from 25 to 80 °C. In addition, it is found that hierarchical MSPNs consisting of multiple Au nanocrystals can be formed over the entire seed particle surface. Finally, the SERS activity of these MSPNs is examined through the detection of rhodamine 6G and methylene blue. Of the different mesostructures, the bimetallic MSPNs have the highest sensitivity with the ability to detect 10-7  m of rhodamine 6G and 10-6  m of methylene blue. The properties of these MSPN particles, made using a novel synthetic process, make them excellent candidates for SERS-based chemical sensing applications.

Plasma cell-free RNA profiling distinguishes cancers from pre-malignant conditions in solid and hematologic malignancies.

Cell-free RNA (cfRNA) in plasma reflects phenotypic alterations of both localized sites of cancer and the systemic host response. Here we report that cfRNA sequencing enables the discovery of messenger RNA (mRNA) biomarkers in plasma with the tissue of origin-specific to cancer types and precancerous conditions in both solid and hematologic malignancies. To explore the diagnostic potential of total cfRNA from blood, we sequenced plasma samples of eight hepatocellular carcinoma (HCC) and ten multiple myeloma (MM) patients, 12 patients of their respective precancerous conditions, and 20 non-cancer (NC) donors. We identified distinct gene sets and built classification models using Random Forest and linear discriminant analysis algorithms that could distinguish cancer patients from premalignant conditions and NC individuals with high accuracy. Plasma cfRNA biomarkers of HCC are liver-specific genes and biomarkers of MM are highly expressed in the bone marrow compared to other tissues and are related to cell cycle processes. The cfRNA level of these biomarkers displayed a gradual transition from noncancerous states through precancerous conditions and cancer. Sequencing data were cross-validated by quantitative reverse transcription PCR and cfRNA biomarkers were validated in an independent sample set (20 HCC, 9 MM, and 10 NC) with AUC greater than 0.86. cfRNA results observed in precancerous conditions require further validation. This work demonstrates a proof of principle for using mRNA transcripts in plasma with a small panel of genes to distinguish between cancers, noncancerous states, and precancerous conditions.

Irreversible alteration of extracellular vesicle and cell-free messenger RNA profiles in human plasma associated with blood processing and storage.

The discovery and utility of clinically relevant circulating biomarkers depend on standardized methods that minimize preanalytical errors. Despite growing interest in studying extracellular vesicles (EVs) and cell-free messenger RNA (cf-mRNA) as potential biomarkers, how blood processing and freeze/thaw impacts the profiles of these analytes in plasma was not thoroughly understood. We utilized flow cytometric analysis to examine the effect of differential centrifugation and a freeze/thaw cycle on EV profiles. Utilizing flow cytometry postacquisition analysis software (FCMpass) to calibrate light scattering and fluorescence, we revealed how differential centrifugation and post-freeze/thaw processing removes and retains EV subpopulations. Additionally, cf-mRNA levels measured by RT-qPCR profiles from a panel of housekeeping, platelet, and tissue-specific genes were preferentially affected by differential centrifugation and post-freeze/thaw processing. Critically, freezing plasma containing residual platelets yielded irreversible ex vivo generation of EV subpopulations and cf-mRNA transcripts, which were not removable by additional processing after freeze/thaw. Our findings suggest the importance of minimizing confounding variation attributed to plasma processing and platelet contamination.

Subcellular Targeted Nanohoop for One- and Two-Photon Live Cell Imaging.

Fluorophores are powerful tools for interrogating biological systems. Carbon nanotubes (CNTs) have long been attractive materials for biological imaging due to their near-infrared excitation and bright, tunable optical properties. The difficulty in synthesizing and functionalizing these materials with precision, however, has hampered progress in this area. Carbon nanohoops, which are macrocyclic CNT substructures, are carbon nanostructures that possess ideal photophysical characteristics of nanomaterials, while maintaining the precise synthesis of small molecules. However, much work remains to advance the nanohoop class of fluorophores as biological imaging agents. Herein, we report an intracellular targeted nanohoop. This fluorescent nanostructure is noncytotoxic at concentrations up to 50 µM, and cellular uptake investigations indicate internalization through endocytic pathways. Additionally, we employ this nanohoop for two-photon fluorescence imaging, demonstrating a high two-photon absorption cross-section (65 GM) and photostability comparable to a commercial probe. This work further motivates continued investigations into carbon nanohoop photophysics and their biological imaging applications.

A Caenorhabditis elegans behavioral assay distinguishes early stage prostate cancer patient urine from controls.

Current methods for non-invasive prostate cancer (PrCa) detection have a high false-positive rate and often result in unnecessary biopsies. Previous work has suggested that urinary volatile organic compound (VOC) biomarkers may be able to distinguish PrCa cases from benign disease. The behavior of the nematode Caenorhabditis elegans has been proposed as a tool to take advantage of these potential VOC profiles. To test the ability of C. elegans Bristol N2 to distinguish PrCa cases from controls, we performed chemotaxis assays using human urine samples collected from men screened for PrCa. Behavioral response of nematodes towards diluted urine from PrCa cases was compared to response to samples from cancer-free controls. Overall, we observed a significant attraction of young adult-stage C. elegans nematodes to 1:100 diluted urine from confirmed PrCa cases and repulsion of C. elegans to urine from controls. When C. elegans chemotaxis index was considered alongside prostate-specific antigen levels for distinguishing cancer from cancer-free controls, the accuracy of patient classification was 81%. We also observed behavioral attraction of C. elegans to two previously reported VOCs to be increased in PrCa patient urine. We conclude nematode behavior distinguishes PrCa case urine from controls in a dilution-dependent manner.

Gas-Stabilizing Sub-100 nm Mesoporous Silica Nanoparticles for Ultrasound Theranostics.

Recent studies have demonstrated that gas-stabilizing particles can generate cavitating micron-sized bubbles when exposed to ultrasound, offering excellent application potential, including ultrasound imaging, drug delivery, and tumor ablation. However, the majority of the reported gas-stabilizing particles are relatively large (>200 nm), and smaller particles require high acoustic pressures to promote cavitation. Here, this paper reports the preparation of sub-100 nm gas-stabilizing nanoparticles (GSNs) that can initiate cavitation at low acoustic intensities, which can be delivered using a conventional medical ultrasound imaging system. The highly echogenic GSNs (F127-hMSN) were prepared by carefully engineering the surfaces of ∼50 nm mesoporous silica nanoparticles. It was demonstrated that the F127-hMSNs could be continuously imaged with ultrasound in buffer or biological solutions or agarose phantoms for up to 20 min. Also, the F127-hMSN can be stored in phosphate-buffered saline for at least a month with no loss in ultrasound responsiveness. The particles significantly degraded when diluted in simulated body fluids, indicating possible biodegradation of the F127-hMSNs in vivo. Furthermore, at ultrasound imaging conditions, F127-hMSNs did not cause detectable cell death, supporting the potential safety of these particles. Finally, strong cavitation activity generation by the F127-hMSNs under high-intensity focused ultrasound insonation was demonstrated and applied to effectively ablate cancer cells.

Author Correction: Fast and multiplexed superresolution imaging with DNA-PAINT-ERS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fast and multiplexed superresolution imaging with DNA-PAINT-ERS.

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. To address this issue, we propose the additions of ethylene carbonate (EC) to the imaging buffer, sequence repeats to the docking strand, and a spacer between the docking strand and the affinity agent. Collectively termed DNA-PAINT-ERS (E = EC, R = Repeating sequence, and S = Spacer), these strategies can be easily integrated into current DNA-PAINT workflows for both accelerated imaging speed and improved image quality through optimized DNA hybridization kinetics and efficiency. We demonstrate the general applicability of DNA-PAINT-ERS for fast, multiplexed superresolution imaging using previously validated oligonucleotide constructs with slight modifications.

Scanning fiber microdisplay: design, implementation, and comparison to MEMS mirror-based scanning displays.

In this study, we propose a compact, lightweight scanning fiber microdisplay towards virtual and augmented reality applications. Our design that is tailored as a head-worn-display simply consists of a four-quadrant piezoelectric tube actuator through which a fiber optics cable is extended and actuated, and a reflective (or semi-reflective) ellipsoidal surface that relays the moving tip of the fiber onto the viewer's retina. The proposed display, offers significant advantages in terms of architectural simplicity, form-factor, fabrication complexity and cost over other fiber scanner and MEMS mirror counterparts towards practical realization. We demonstrate the display of various patterns with ∼VGA resolution and further provide analytical formulas for mechanical and optical constraints to compare the performance of the proposed scanning fiber microdisplay with that of MEMS mirror-based microdisplays. Also we discuss the road steps towards improving the performance of the proposed scanning fiber microdisplay to high-definition video formats (such as HD1440), which is beyond what has been achieved by MEMS mirror based laser scanning displays.

Coagulation measurement from whole blood using vibrating optical fiber in a disposable cartridge.

In clinics, blood coagulation time measurements are performed using mechanical measurements with blood plasma. Such measurements are challenging to do in a lab-on-a-chip (LoC) system using a small volume of whole blood. Existing LoC systems use indirect measurement principles employing optical or electrochemical methods. We developed an LoC system using mechanical measurements with a small volume of whole blood without requiring sample preparation. The measurement is performed in a microfluidic channel where two fibers are placed inline with a small gap in between. The first fiber operates near its mechanical resonance using remote magnetic actuation and immersed in the sample. The second fiber is a pick-up fiber acting as an optical sensor. The microfluidic channel is engineered innovatively such that the blood does not block the gap between the vibrating fiber and the pick-up fiber, resulting in high signal-to-noise ratio optical output. The control plasma test results matched well with the plasma manufacturer's datasheet. Activated-partial-thromboplastin-time tests were successfully performed also with human whole blood samples, and the method is proven to be effective. Simplicity of the cartridge design and cost of readily available materials enable a low-cost point-of-care device for blood coagulation measurements.

Efficiency of integrated waveguide probes for the detection of light backscattered from weakly scattering media.

A semianalytical model for light collection by integrated waveguide probes is developed by extending previous models used to describe fiber probes. The efficiency of waveguide probes is compared to that of different types of fiber probes for different thicknesses of a weakly scattering sample. The simulation results show that integrated probes have a collection efficiency that is higher than that of small-core fiber probes, and, in the particular case of thin samples, also exceeds the collection efficiency of large-core highly multimode fiber probes. An integrated waveguide probe with one excitation and eight collector waveguides is fabricated and applied to excite and collect luminescence from a ruby rod. The experimental results are in good agreement with the simulation and validate the semianalytical model.