Nanoscopic Imaging Research Articles

Achieving low-power single-wavelength-pair nanoscopy with NIR-II continuous-wave laser for multi-chromatic probes

Stimulated emission depletion (STED) microscopy is a powerful diffraction-unlimited technique for fluorescence imaging. Despite its rapid evolution, STED fundamentally suffers from high-intensity light illumination, sophisticated probe-defined laser schemes, and limited photon budget of the probes. Here, we demonstrate a versatile strategy, stimulated-emission induced excitation depletion (STExD), to deplete the emission of multi-chromatic probes using a single pair of low-power, near-infrared (NIR), continuous-wave (CW) lasers with fixed wavelengths. With the effect of cascade amplified depletion in lanthanide upconversion systems, we achieve emission inhibition for a wide range of emitters (e.g., Nd3+, Yb3+, Er3+, Ho3+, Pr3+, Eu3+, Tm3+, Gd3+, and Tb3+) by manipulating their common sensitizer, i.e., Nd3+ ions, using a 1064-nm laser. With NaYF4:Nd nanoparticles, we demonstrate an ultrahigh depletion efficiency of 99.3 ± 0.3% for the 450 nm emission with a low saturation intensity of 23.8 ± 0.4 kW cm−2. We further demonstrate nanoscopic imaging with a series of multi-chromatic nanoprobes with a lateral resolution down to 34 nm, two-color STExD imaging, and subcellular imaging of the immunolabelled actin filaments. The strategy expounded here promotes single wavelength-pair nanoscopy for multi-chromatic probes and for multi-color imaging under low-intensity-level NIR-II CW laser depletion.

En route to nanoscopic quantum optical imaging: counting emitters with photon-number-resolving detectors.

The fundamental understanding of biological pathways requires minimally invasive nanoscopic optical resolution imaging. Many approaches to high-resolution imaging rely on localization of single emitters, such as fluorescent molecules or quantum dots. Additionally, the exact determination of the number of such emitters in an imaging volume is essential for a number of applications; however, in standard intensity-based microscopy it is not possible to determine the number of individual emitters within a diffraction limited spot without initial knowledge of system parameters. Here we explore how quantum measurements of the emitted photons using photon number resolving detectors can be used to address this challenging task. In the proposed new approach, the problem of counting emitters reduces to the task of determining differences between the emitted photon distribution and the Poisson limit. We show that quantum measurements of the number of photons emitted from an ensemble of emitters enable the determination of both the number of emitters and the probability of emission. This method can be applied for any type of single-photon emitters. The scaling laws of this new approach are presented by the Cramer-Rao Lower Bounds, and this technique has great potential in quantum optical imaging with nanoscopic resolution.

Single-exposure X-ray phase imaging microscopy with a grating interferometer.

The advent of hard X-ray free-electron lasers enables nanoscopic X-ray imaging with sub-picosecond temporal resolution. X-ray grating interferometry offers a phase-sensitive full-field imaging technique where the phase retrieval can be carried out from a single exposure alone. Thus, the method is attractive for imaging applications at X-ray free-electron lasers where intrinsic pulse-to-pulse fluctuations pose a major challenge. In this work, the single-exposure phase imaging capabilities of grating interferometry are characterized by an implementation at the I13-1 beamline of Diamond Light Source (Oxfordshire, UK). For comparison purposes, propagation-based phase contrast imaging was also performed at the same instrument. The characterization is carried out in terms of the quantitativeness and the contrast-to-noise ratio of the phase reconstructions as well as via the achievable spatial resolution. By using a statistical image reconstruction scheme, previous limitations of grating interferometry regarding the spatial resolution can be mitigated as well as the experimental applicability of the technique.

High Resolution of Plasmonic Resonance Scattering Imaging with Deep Learning.

The dark-field microscopy (DFM) imaging technology has the advantage of a high signal-to-noise ratio, and it is often used for real-time monitoring of plasmonic resonance scattering and biological imaging at the single-nanoparticle level. Due to the limitation of the optical diffraction limit, it is still a challenging task to accurately distinguish two or more nanoparticles whose distance is less than the diffraction limit. Here, we propose a computational strategy based on a deep learning framework (NanoNet), which will realize the effective segmentation of the scattered light spots in diffraction-limited DFM images and obtain high-resolution plasmonic light scattering imaging. A small data set of DFM and the corresponding scanning electron microscopy (SEM) image pairs are used to learn for obtaining a highly resolved semantic imaging model using NanoNet, and thus highly resolved DFM images matching the resolution of those acquired using SEM can be obtained. Our method has the ability to transform diffraction-limited DFM images to highly resolved ones without adding a complex optical system. As a proof of concept, a highly resolved DFM image of living cells through the NanoNet technique is successfully made, opening up a new avenue for high-resolution optical nanoscopic imaging.

Versatile Near‐Infrared Super‐Resolution Imaging of Amyloid Fibrils with the Fluorogenic Probe CRANAD‐2

AbstractCRANAD‐2 is a fluorogenic curcumin derivative used for near‐infrared detection and imaging in vivo of amyloid aggregates, which are involved in neurodegenerative diseases. We explore the performance of CRANAD‐2 in two super‐resolution imaging techniques, namely stimulated emission depletion (STED) and single‐molecule localization microscopy (SMLM), with markedly different fluorophore requirements. By conveniently adapting the concentration of CRANAD‐2, which transiently binds to amyloid fibrils, we show that it performs well in both techniques, achieving a resolution in the range of 45–55 nm. Correlation of SMLM with atomic force microscopy (AFM) validates the resolution of fine features in the reconstructed super‐resolved image. The good performance and versatility of CRANAD‐2 provides a powerful tool for near‐infrared nanoscopic imaging of amyloids in vitro and in vivo.

Surface-Engineered Gold Nanoclusters for Stimulated Emission Depletion and Correlated Light and Electron Microscopy Imaging.

Stimulated emission depletion (STED) nanoscopy is an emerging super-resolution imaging platform for the study of the cellular structure. Developing suitable fluorescent probes of small size, good photostability, and easy functionalization is still in demand. Herein, we introduce a new type of surface-engineered gold nanoclusters (Au NCs) that are ultrasmall (1.7 nm) and ultrabright (QY = 60%) for STED bioimaging. A rigid shell formed by l-arginine (l-Arg) and 6-aza-2-thiothymine (ATT) on the Au NC surface enables not only its strong fluorescence in aqueous solution but also its easy chemical modification for specific biomolecule labeling. Au NCs show remarkable performance as STED nanoprobes, including high depletion efficiency, good photobleaching resistance, and low saturation intensity. Super-resolution imaging has been achieved with these Au NCs, and targeted nanoscopic imaging of cellular tubulin has been demonstrated. Moreover, the circular structure of lysosomes in live cells has been revealed. As a Au NC is also an ideal probe for electron microscopy, dual imaging of Aβ42 aggregates with the single labeling probe of Au NCs has been realized in correlative light and electron microscopy (CLEM). This work reports, for the first time, the application of Au NCs as a novel probe in STED and CLEM imaging. With their excellent properties, Au NCs show promising potential for nanoscale bioimaging.

Dosimetry of heavy ion exposure to human cells using nanoscopic imaging of double strand break repair protein clusters

The human body is constantly exposed to ionizing radiation of different qualities. Especially the exposure to high-LET (linear energy transfer) particles increases due to new tumor therapy methods using e.g. carbon ions. Furthermore, upon radiation accidents, a mixture of radiation of different quality is adding up to human radiation exposure. Finally, long-term space missions such as the mission to mars pose great challenges to the dose assessment an astronaut was exposed to. Currently, DSB counting using γH2AX foci is used as an exact dosimetric measure for individuals. Due to the size of the γH2AX IRIF of ~ 0.6 µm, it is only possible to count DSB when they are separated by this distance. For high-LET particle exposure, the distance of the DSB is too small to be separated and the dose will be underestimated. In this study, we developed a method where it is possible to count DSB which are separated by a distance of ~ 140 nm. We counted the number of ionizing radiation-induced pDNA-PKcs (DNA-PKcs phosphorylated at T2609) foci (size = 140 nm ± 20 nm) in human HeLa cells using STED super-resolution microscopy that has an intrinsic resolution of 100 nm. Irradiation was performed at the ion microprobe SNAKE using high-LET 20 MeV lithium (LET = 116 keV/µm) and 27 MeV carbon ions (LET = 500 keV/µm). pDNA-PKcs foci label all DSB as proven by counterstaining with 53BP1 after low-LET γ-irradiation where separation of individual DSB is in most cases larger than the 53BP1 gross size of about 0.6 µm. Lithium ions produce (1.5 ± 0.1) IRIF/µm track length, for carbon ions (2.2 ± 0.2) IRIF/µm are counted. These values are enhanced by a factor of 2–3 compared to conventional foci counting of high-LET tracks. Comparison of the measurements to PARTRAC simulation data proof the consistency of results. We used these data to develop a measure for dosimetry of high-LET or mixed particle radiation exposure directly in the biological sample. We show that proper dosimetry for radiation up to a LET of 240 keV/µm is possible.

Photon counting of extreme ultraviolet high harmonics using a superconducting nanowire single-photon detector

Laser-driven light sources in the extreme ultraviolet range (EUV) enable nanoscopic imaging with unique label-free elemental contrast. However, to fully exploit the unique properties of these new sources, novel detection schemes need to be developed. Here, we show in a proof-of-concept experiment that superconducting nanowire single-photon detectors (SNSPD) can be utilized to enable photon counting of a laser-driven EUV source based on high harmonic generation (HHG). These detectors are dark-count free and accommodate very high count rates—a perfect match for high repetition rate HHG sources. In addition to the advantages of SNSPDs for classical imaging applications with laser-driven EUV sources, the ability to count single photons paves the way for very promising applications in quantum optics and quantum imaging with high energetic radiation like, e.g., quantum ghost imaging with nanoscale resolution.

Curved carbon photo-oxygenation catalysts for the suppression and nanoscopic imaging of β-amyloid peptides fibrillation

Curved carbon photo-oxygenation catalysts for the suppression and nanoscopic imaging of β-amyloid peptides fibrillation

A distyrylbenzene-based fluorescent probe with high photostability and large Stokes shift for STED nanoscopy imaging of cellular lipid droplets

Directly limited by the availability of suitable organic fluorescent probes, the stimulated emission depletion (STED) nanoscopy imaging of cellular lipid droplets (LDs) is a highly challenge task. Herein, a sulfone-embedded distyrylbenzene derivative SO2-DSB has been reported as a novel LDs fluorescent probe suitable for STED nanoscopy imaging. This fluorescent probe exhibiting high photostability and large Stokes shift could be efficiently depleted while be not excited by the extremely strong STED lasers of 592 nm or 660 nm. Consequently, STED nanoscopy imaging of LDs employed with this fluorescent probe has achieved high resolutions of 73 nm and 84 nm under 592 nm and 660 nm STED lasers, respectively. Notably, these resolutions are significantly higher than the common confocal images (resolutions of about 250 nm) and represent one of the leading results of the nanoscopy imaging of LDs so far, highlight the ability of this fluorescent probe in STED nanoscopy. In addition, the general ability of this fluorescent probe in confocal microscopy has also been demonstrated by 3D imaging and multicolor imaging.

STED Nanoscopy Imaging of Cellular Lipid Droplets Employing a Superior Organic Fluorescent Probe

Lipid droplets (LDs) are spherical organelles that participate in numerous biological processes. In order to visualize LDs on the nanoscale, nanoscopy fluorescence imaging is considered as the most attractive technique but is substantially limited by the characteristics of fluorescent probes. Thus, the development of a superior fluorescent probe that is capable of nanoscopy fluorescence imaging has attracted enormous attention. Herein, a benzodithiophene-tetraoxide-based molecule Lipi-BDTO has been developed that can easily undergo the stimulated emission depletion (STED) process and displays high photostability. These two characteristics of fluorescent probes finely satisfy the requirements of STED nanoscopy imaging. Indeed, applying the probe for STED imaging achieves a high resolution of 65 nm, belonging to one of the leading results of LDs fluorescence imaging. Furthermore, the high photostability of this fluorescent probe enables it to monitor the dynamics of LDs by time-lapse STED imaging as well as to visualize the three-dimensional (3D) spatial distribution of LDs by 3D STED imaging. Notably, the resolution of the 3D STED image represents one of the best LDs fluorescence imaging results so far. Besides STED nanoscopy imaging, the superior utility of this fluorescent probe has been also demonstrated in two-color 3D confocal imaging and four-color confocal imaging.

Nanoscopic Imaging of Nucleolar Stress Enabled by Protein-Mimicking Carbon Dots.

The nucleolus is a central hub for coordinating cellular stress responses during cancer development and treatment. Accurate identification of nucleolar stress response is crucially desired for nucleolus-based diagnostics and therapeutics but technically challenging due to the need to address the ultrastructural analysis. Here, we report a protein-like CD with the integration of fluorescent blinking domains and RNA-binding motifs, which offers the ability to perform enhanced super-resolution imaging of the nucleolar ultrastructure. This image allows extraction of multidimensional information from the nucleolus for accurate distinguishment of different cells from the same cell types. Furthermore, we demonstrate for the first time this CD-depicted nucleolar ultrastructure as a sensitive hallmark to identify and discriminate subtle responses to various stressors as well as to afford RNA-related information that has been inaccessible by conventional immunofluorescence methods. This protein-mimicking CD could become a broadly useful probe for nucleolar stress studies in cell diagnostics and therapeutics.

Analysing errors in single-molecule localisation microscopy.

In single molecule localisation microscopy (SMLM) a super-resolution image of the distribution of fluorophores in the sample is built up from the localised positions of many individual molecules. It has become widely used due to its experimental simplicity and the high resolution that can be achieved. However, the factors which limit resolution in a reconstructed image, and the artefacts which can be present, are completely different to those present in standard fluorescent microscopy techniques. Artefacts may be difficult for users to identify, particularly as they can cause images to appear falsely sharp, an effect called artificial sharpening. Here we discuss the different sources of error and bias in SMLM, and the methods available for avoiding or detecting them.

Nanoscopic subcellular imaging enabled by ion beam tomography

Multiplexed ion beam imaging (MIBI) has been previously used to profile multiple parameters in two dimensions in single cells within tissue slices. Here, a mathematical and technical framework for three-dimensional (3D) subcellular MIBI is presented. Ion-beam tomography (IBT) compiles ion beam images that are acquired iteratively across successive, multiple scans, and later assembled into a 3D format without loss of depth resolution. Algorithmic deconvolution, tailored for ion beams, is then applied to the transformed ion image series, yielding 4-fold enhanced ion beam data cubes. To further generate 3D sub-ion-beam-width precision visuals, isolated ion molecules are localized in the raw ion beam images, creating an approach coined as SILM, secondary ion beam localization microscopy, providing sub-25 nm accuracy in original ion images. Using deep learning, a parameter-free reconstruction method for ion beam tomograms with high accuracy is developed for low-density targets. In cultured cancer cells and tissues, IBT enables accessible visualization of 3D volumetric distributions of genomic regions, RNA transcripts, and protein factors with 5 nm axial resolution using isotope-enrichments and label-free elemental analyses. Multiparameter imaging of subcellular features at near macromolecular resolution is implemented by the IBT tools as a general biocomputation pipeline for imaging mass spectrometry.

Extracting Organelle Membrane Topology from Super-Resolution Microscopy Data

Constant remodeling of the endoplasmic reticulum (ER) is essential for maintaining its complex structure and function, but difficult to study due to the ER's convoluted topology: the ER exhibits a broad continuum of morphologies ranging from flat, sheet-like domains to highly curved parking-garage structures, tubules, and nanoscale holes. It can transition between these morphologies on a timescale of seconds to minutes. While many proteins that are key players in ER organization have been identified, how the ER maintains its shape is poorly understood. It is particularly unclear how curvature-associated proteins know where and when to migrate along the ER. Due to the structural heterogeneity in the ER, we believe that remodeling processes must be studied in the context of local membrane morphology. To enable this, we have developed super-resolution microscopy techniques that provide a detailed view of ER morphology and dynamics. Single-molecule localization microscopy (SMLM), in particular, provides 3D data at 20-nm resolution. Rather than directly imaging membrane surfaces, however, SMLM gives us the positions of proteins that localize to the ER membrane. Here, we present our tool to create and visualize biophysically-constrained representations of a membrane passing through an SMLM point cloud and its evolution in time. These algorithms are implemented in the PYthon Microscopy Environment (PYME), an open-source nanoscopy imaging and analysis suite (https://www.biorxiv.org/content/10.1101/2020.09.29.315671v1). We are able to quantify the curvature of the ER membrane, and examine the correlation of curvature-associated protein distributions and local nanoscale curvature, which we can use to understand possible mechanisms behind ER remodeling.

Disulfide-Reduction-Triggered Spontaneous Photoblinking Cy5 Probe for Nanoscopic Imaging of Mitochondrial Dynamics in Live Cells.

Mitochondria are highly dynamic organelles with interconnected tubule structures that are sensitive to environmental stress and light illumination. Super-resolution optical imaging of mitochondrial dynamics is of significance for understanding such biological events. Direct stochastic optical reconstruction microscopy has the advantages of a high spatial resolution, low phototoxicity in live-cell imaging, and the capacity to incorporate smart fluorescent probes. However, dSTORM imaging in live cells is challenging because of the requirement for an imaging buffer and a low temporal resolution. In this work, we achieved dSTORM imaging of mitochondrial dynamics in live cells with a disulfide-substituted Cy5 probe without using any toxic imaging buffer. Under the illumination of very low laser power, the probe exhibited spontaneous photoblinking triggered by disulfide-bond reduction in mitochondria of live cells. The obtained thiol attacked nearby carbon to form a six-membered ring and the reversible opening/closing of the ring produced spontaneous photoblinking behavior. With this new STORM strategy, we achieved observation of mitochondrial dynamics for more than 3 min, which provides a promising tool for further studies of mitochondria with an ultrafine structure.

DNA nanotechnology-empowered nanoscopic imaging of biomolecules.

Advances in bioimaging technologies have led to unprecedented findings of novel biological processes at the nanoscale. However, there remains an ever-lasting demand for the improvement of spatiotemporal resolution, multiplexity, and smart responsiveness of bioimaging in living systems. In recent decades, self-assembled DNA nanostructures with highly programmable shape, nanometer addressability, and structural responsiveness have shown great promise in developing nanoscale probes and labels for high-performance bioimaging. Here, we briefly review the recent progress in structural DNA nanotechnology and the development of DNA frameworks, and summarize the bioimaging strategies empowered by DNA nanotechnology. We highlight the advantages of DNA nanostructures in overcoming the bottlenecks in bioimaging and discuss the challenges and opportunities in this field.

Length-Dependent Distinct Cytotoxic Effect of Amyloid Fibrils beyond Optical Diffraction Limit Revealed by Nanoscopic Imaging.

Fibrillar species have been proposed to play an essential role in the cytotoxicity of amyloid peptide and the pathogenesis of neurodegenerative diseases. Discrimination of Aβ aggregates in situ at high spatial resolution is therefore significant for the development of a therapeutic method. In this work, we adopt a rhodamine-like structure as luminescent centers to fabricate carbonized fluorescent nanoparticles (i.e., carbon dots, RhoCDs) with tunable emission wavelengths from green to red and burst-like photoblinking property for localization-based nanoscopic imaging. These RhoCDs contain lipophilic cationic and carboxyl groups which can specifically bind with Aβ1-40 aggregates via electrostatic interaction and hydrogen bonding. According to the nanoscopic imaging in the Aβ1-40 fibrillation and disaggregation process, different types of Aβ1-40 aggregates beyond the optical diffraction limit have been disclosed. Additionally, length-dependent toxic effect of Aβ1-40 aggregates beyond the optical diffraction limit is unveiled. Short amyloid assemblies with length of 187 ± 3.9 nm in the early stage are more toxic than the elongated amyloid fibrils. Second, disassembly of long fibrils into short species by Gramicidin S (GS-2) peptide might enhance the cytotoxicity. These results lay the foundation to develop functional fluorophore for nanoscopic imaging and also provide deep insight into morphology-dependent cytotoxicity from amyloid peptides.

Far-field transient absorption nanoscopy with sub-50 nm optical super-resolution

Nanoscopic imaging or characterizing is the mainstay of the development of advanced materials. Despite great progress in electronic and atomic force microscopies, label-free and far-field characterization of materials with deep sub-wavelength spatial resolution has long been highly desired. Herein, we demonstrate far-field super-resolution transient absorption (TA) imaging of two-dimensional material with a spatial resolution of sub-50 nm. By introducing a donut-shaped blue saturation laser, we effectively suppress the TA transition driven by near-infrared (NIR) pump–probe photons, and push the NIR-TA microscopy to sub-diffraction-limited resolution. Specifically, we demonstrate that our method can image the individual nano-grains in graphene with lateral resolution down to 36 nm. Further, we perform super-resolution TA imaging of nano-wrinkles in monolayer graphene, and the measured results are very consistent with the characterization by an atomic force microscope. This direct far-field optical nanoscopy holds great promise to achieve sub-20 nm spatial resolution and a few tens of femtoseconds temporal resolution upon further improvement and represents a paradigm shift in a broad range of hard and soft nanomaterial characterization.

Nanoscale optical assessment of photochemical changes of SU-8 photoresist induced by ultrashort near-IR optical excitation

Modern device manufacturing heavily relies on photolithographic techniques to fabricate three-dimensional (3D) nanostructures. Direct laser writing using multiphoton absorption is commonly employed. A key challenge in troubleshooting 3D structure fabrication is understanding why structures are destroyed and/or defects are created during development (collectively “fabrication errors”). In this report, we explore new approaches to avoid and assess fabrication errors. We demonstrate a novel way to expose photoresists by developing rows of SU-8 photoresist using intense (~ 10–40 TW/cm2) femtosecond pulses at much longer (1.7 µm) wavelength than conventionally used for multiphoton micromachining. We then apply atomic force microscopy-infrared absorption spectroscopy to provide chemically-specific nanoscopic imaging to distinguish photoinduced modifications of SU-8 photoresist. The focus of this report is on testing the feasibility of novel fabrication and detection methods. While no 3D structures were fabricated in this report, infrared femtosecond excitation can be combined with existing 3D patterning techniques to fabricate 3D structures. By inducing, detecting and imaging development at the nanoscale, the demonstrated new approach opens new doors for future 3D manufacturing and its quality assessment.