Semi-automated approaches for interrogating spatial heterogeneity of tissue samples.

Tissues are spatially orchestrated ecosystems composed of heterogeneous cell populations and non-cellular elements. Tissue components' interactions shape the biological processes that govern homeostasis and disease, thus comprehensive insights into tissues' composition are crucial for understanding their biology. Recently, advancements in the spatial biology field enabled the in-depth analyses of tissue architecture at single-cell resolution, while preserving the structural context. The increasing number of biomarkers analyzed, together with whole tissue imaging, generate datasets approaching several hundreds of gigabytes in size, which are rich sources of valuable knowledge but require investments in infrastructure and resources for extracting quantitative information. The analysis of multiplex whole-tissue images requires extensive training and experience in data analysis. Here, we showcase how a set of open-source tools can allow semi-automated image data extraction to study the spatial composition of tissues with a focus on tumor microenvironment (TME). With the use of Lunaphore COMET platform, we interrogated lung cancer specimens where we examined the expression of 20 biomarkers. Subsequently, the tissue composition was interrogated using an in-house optimized nuclei detection algorithm followed by a newly developed image artifact exclusion approach. Thereafter, the data was processed using several publicly available tools, highlighting the compatibility of COMET-derived data with currently available image analysis frameworks. In summary, we showcased an innovative semi-automated workflow that highlights the ease of adoption of multiplex imaging to explore TME composition at single-cell resolution using a simple slide in, data out approach. Our workflow is easily transferrable to various cohorts of specimens to provide a toolset for spatial cellular dissection of the tissue composition.

Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics.

Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 h, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings.

Engineering Optically Active Defects in Hexagonal Boron Nitride Using Focused Ion Beam and Water.

Hexagonal boron nitride (hBN) has emerged as a promising material platform for nanophotonics and quantum sensing, hosting optically active defects with exceptional properties such as high brightness and large spectral tuning. However, precise control over deterministic spatial positioning of emitters in hBN remained elusive for a long time, limiting their proper correlative characterization and applications in hybrid devices. Recently, focused ion beam (FIB) systems proved to be useful to engineer several types of spatially defined emitters with various structural and photophysical properties. Here we systematically explore the physical processes leading to the creation of optically active defects in hBN using FIB and find that beam-substrate interaction plays a key role in the formation of defects. These findings are confirmed using transmission electron microscopy, which reveals local mechanical deterioration of the hBN layers and local amorphization of ion beam irradiated hBN. Additionally, we show that, upon exposure to water, amorphized hBN undergoes a structural and optical transition between two defect types with distinctive emission properties. Moreover, using super-resolution optical microscopy combined with atomic force microscopy, we pinpoint the exact location of emitters within the defect sites, confirming the role of defected edges as primary sources of fluorescent emission. This lays the foundation for FIB-assisted engineering of optically active defects in hBN with high spatial and spectral control for applications ranging from integrated photonics, to nanoscale sensing, and to nanofluidics.

Time-Resolved Scanning Ion Conductance Microscopy for Three-Dimensional Tracking of Nanoscale Cell Surface Dynamics.

Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy, EM, AFM). However, time-resolved imaging techniques tend to either have a lower resolution, are limited to small areas, or cause damage to the cells, thereby preventing long-term time-lapse studies. Scanning probe microscopy methods such as atomic force microscopy (AFM) combine high-resolution imaging with the ability to image living cells in physiological conditions. The mechanical contact between the tip and the sample, however, deforms the cell surface, disturbs the native state, and prohibits long-term time-lapse imaging. Here, we develop a scanning ion conductance microscope (SICM) for high-speed and long-term nanoscale imaging of eukaryotic cells. By utilizing advances in nanopositioning, nanopore fabrication, microelectronics, and controls engineering, we developed a microscopy method that can resolve spatiotemporally diverse three-dimensional (3D) processes on the cell membrane at sub-5-nm axial resolution. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of subsecond to days, imaging diverse processes ranging from endocytosis, micropinocytosis, and mitosis to bacterial infection and cell differentiation in cancer cells. This technique enables a detailed look at membrane events and may offer insights into cell-cell interactions for infection, immunology, and cancer research.

Bio-orthogonal Red and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-resolution Microscopy.

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazine-dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine-dye conjugates with minimal distance between tetrazine and the fluorophore. Two synthetic routes give access to a series of cell-permeable and -impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as the dominant excited-state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multicolor and super-resolution STED, and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size.

Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy.

High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization. AFM routinely resolves atomic steps, however on soft biological samples, the forces between the tip and the sample deform the fragile membrane, thereby distorting the otherwise high axial resolution of the technique. Here we present scanning ion-conductance microscopy (SICM) as an alternative approach for topographical imaging of soft biological samples, preserving high axial resolution on cells. SICM is complemented with live-cell compatible super-resolution optical fluctuation imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. Finally, we employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffraction-resolution.

High-Throughput Nanocapillary Filling Enabled by Microwave Radiation for Scanning Ion Conductance Microscopy Imaging.

Solid-state nanopores provide a highly sensitive tool for single-molecule sensing and probing nanofluidic effects in solutions. Glass nanopipettes are a cheap and robust type of solid-state nanopore produced from pulling glass capillaries with opening orifice diameters down to below tens of nanometers. Sub-50 nm nanocapillaries allow an unprecedented resolution for translocating single molecules or for scanning ion conductance microscopy imaging. Due to the small opening orifice diameters, such nanocapillaries are difficult to fill with solutions, compromising their advantages of low cost, availability, and experimental simplicity. We present a simple and cheap method to reliably fill nanocapillaries down to sub-10 nm diameters by microwave radiation heating. Using a large statistic of filled nanocapillaries, we determine the filling efficiency and physical principle of the filling process using sub-50 nm quartz nanocapillaries. Finally, we have used multiple nanocapillaries filled by our method for high-resolution scanning ion conductance microscopy imaging.

Scanning Probe-Directed Assembly and Rapid Chemical Writing Using Nanoscopic Flow of Phospholipids.

Nanofluidic systems offer a huge potential for discovery of new molecular transport and chemical phenomena that can be employed for future technologies. Herein, we report on the transport behavior of surface-reactive compounds in a nanometer-scale flow of phospholipids from a scanning probe. We have investigated microscopic deposit formation on polycrystalline gold by lithographic printing and writing of 1,2-dioleoyl-sn-glycero-3-phosphocholine and eicosanethiol mixtures, with the latter compound being a model case for self-assembled monolayers (SAMs). By analyzing the ink transport rates, we found that the transfer of thiols was fully controlled by the fluid lipid matrix allowing to achieve a certain jetting regime, i.e., transport rates previously not reported in dip-pen nanolithography (DPN) studies on surface-reactive, SAM-forming molecules. Such a transport behavior deviated significantly from the so-called molecular diffusion models, and it was most obvious at the high writing speeds, close to 100 µm s-1. Moreover, the combined data from imaging ellipsometry, scanning electron microscopy, atomic force microscopy (AFM), and spectroscopy revealed a rapid and efficient ink phase separation occurring in the AFM tip-gold contact zone. The force curve analysis indicated formation of a mixed ink meniscus behaving as a self-organizing liquid. Based on our data, it has to be considered as one of the co-acting mechanisms driving the surface reactions and self-assembly under such highly nonequilibrium, crowded environment conditions. The results of the present study significantly extend the capabilities of DPN using standard AFM instrumentation: in the writing regime, the patterning speed was already comparable to that achievable by using electron beam systems. We demonstrate that lipid flow-controlled chemical patterning process is directly applicable for rapid prototyping of solid-state devices having mesoscopic features as well as for biomolecular architectures.

Facile Production of Hexagonal Boron Nitride Nanoparticles by Cryogenic Exfoliation.

Fluorescent nanoparticles with optically robust luminescence are imperative to applications in imaging and labeling. Here we demonstrate that hexagonal boron nitride (hBN) nanoparticles can be reliably produced using a scalable cryogenic exfoliation technique with sizes below 10 nm. The particles exhibit bright fluorescence generated by color centers that act as atomic-size quantum emitters. We analyze their optical properties, including emission wavelength, photon-statistics, and photodynamics, and show that they are suitable for far-field super-resolution fluorescence nanoscopy. Our results provide a foundation for exploration of hBN nanoparticles as candidates for bioimaging, labeling, as well as biomarkers that are suitable for quantum sensing.

Fixed DNA Molecule Arrays for High-Throughput Single DNA-Protein Interaction Studies.

The DNA Curtains assay is a recently developed experimental platform for protein-DNA interaction studies at the single-molecule level that is based on anchoring and alignment of DNA fragments. The DNA Curtains so far have been made by using chromium barriers and fluid lipid bilayer membranes, which makes such a specialized assay technically challenging and relatively unstable. Herein, we report on an alternative strategy for DNA arraying for analysis of individual DNA-protein interactions. It relies on stable DNA tethering onto nanopatterned protein templates via high affinity molecular recognition. We describe fabrication of streptavidin templates (line features as narrow as 200 nm) onto modified glass coverslips by combining surface chemistry, atomic force microscopy (AFM), and soft lithography techniques with affinity-driven assembly. We have employed such chips for arraying single- and double-tethered DNA strands, and we characterized the obtained molecular architecture: we evaluated the structural characteristics and specific versus nonspecific binding of fluorescence-labeled DNA using AFM and total internal reflection fluorescence microscopy. We demonstrate the feasibility of our DNA molecule arrays for short single-tethered as well as for lambda single- and double-tethered DNA. The latter type of arrays proved very suitable for localization of single DNA-protein interactions employing restriction endonucleases. The presented molecular architecture and facile method of fabrication of our nanoscale platform does not require clean room equipment, and it offers advanced functional studies of DNA machineries and the development of future nanodevices.

Wide-Field Spectral Super-Resolution Mapping of Optically Active Defects in Hexagonal Boron Nitride.

Point defects can have significant impact on the mechanical, electronic, and optical properties of materials. The development of robust, multidimensional, high-throughput, and large-scale characterization techniques of defects is thus crucial for the establishment of integrated nanophotonic technologies and material growth optimization. Here, we demonstrate the potential of wide-field spectral single-molecule localization microscopy (SMLM) for the determination of ensemble spectral properties as well as the characterization of spatial, spectral, and temporal dynamics of single defects in chemical vapor deposition (CVD)-grown and irradiated exfoliated hexagonal boron-nitride materials. We characterize the heterogeneous spectral response of our samples and identify at least two types of defects in CVD-grown materials, while irradiated exfoliated flakes show predominantly only one type of defects. We analyze the blinking kinetics and spectral emission for each type of defects and discuss their implications with respect to the observed spectral heterogeneity of our samples. Our study shows the potential of wide-field spectral SMLM techniques in material science and paves the way toward the quantitative multidimensional mapping of defect properties.

Fluorescent Nanodiamonds as Versatile Intracellular Temperature Sensors.

Temperature is a widely known phenomenon, which plays an extremely important role in biological systems. Its behavior on the macro-scale has been quite well investigated and understood, thanks to the availability of reliable and precise thermometers such as thermocouples and infrared cameras. However, temperature measurements on the subcellular scale present an ongoing challenge due to the absence of universal nanoscale temperature sensors. Recent work on fluorescent nanodiamonds has revealed their unique ability to measure temperature with high spatial and temporal resolution, of particular importance in the intracellular environment. This review summarizes recent progress in the field and highlights the future directions for intracellular temperature sensing using fluorescent nanodiamonds.