Super-resolution Imaging Technology Research Articles

Chromatin organization and behavior in HRAS-transformed mouse fibroblasts.

In higher eukaryotic cells, a string of nucleosomes, where long genomic DNA is wrapped around core histones, are rather irregularly folded into a number of condensed chromatin domains, which have been revealed by super-resolution imaging and Hi-C technologies. Inside these domains, nucleosomes fluctuate and locally behave like a liquid. The behavior of chromatin may be highly related to DNA transaction activities such as transcription and repair, which are often upregulated in cancer cells. To investigate chromatin behavior in cancer cells and compare those of cancer and non-cancer cells, we focused on oncogenic-HRAS (Gly12Val)-transformed mouse fibroblasts CIRAS-3 cells and their parental 10T1/2 cells. CIRAS-3 cells are tumorigenic and highly metastatic. First, we found that HRAS-induced transformation altered not only chromosome structure, but also nuclear morphology in the cell. Using single-nucleosome imaging/tracking in live cells, we demonstrated that nucleosomes are locally more constrained in CIRAS-3 cells than in 10T1/2 cells. Consistently, heterochromatin marked with H3K27me3 was upregulated in CIRAS-3 cells. Finally, Hi-C analysis showed enriched interactions of the B-B compartment in CIRAS-3 cells, which likely represents transcriptionally inactive chromatin. Increased heterochromatin may play an important role in cell migration, as they have been reported to increase during metastasis. Our study also suggests that single-nucleosome imaging provides new insights into how local chromatin is structured in living cells.

The Advances and Applications of Characterization Technique for Exosomes: From Dynamic Light Scattering to Super-Resolution Imaging Technology

Exosomes distributed by extracellular vesicles carry various information highly consistent with cells, becoming a new type of biomarker for tumor screening. However, although conventional characterization technologies can quantify size and morphology for exosomes, they are limited in related fields such as function tracing, protein quantification at unit point, and microstructural information. In this paper, firstly, different exosome characterization methods are systematically reviewed, such as dynamic light scattering, nanoparticle tracking analysis, flow cytometry, electron microscope, and emerging super-resolution imaging technologies. Then, advances in applications are described one by one. Last but not least, we compare the features of different technologies for exosomes and propose that super-resolution imaging technology can not only take into account the advantages of conventional characterization techniques but also provide accurate, real-time, and super-resolution quantitative analysis for exosomes. It provides a fine guide for exosome-related biomedical research, as well as application in liquid biopsy and analysis techniques.

Three-Dimensional Orientation and Localization Microscopy of Single Molecules Using Super-Resolution Imaging Technology (Invited)

Three-Dimensional Orientation and Localization Microscopy of Single Molecules Using Super-Resolution Imaging Technology (Invited)

Recent advances in super-resolution optical imaging based on aggregation-induced emission.

Super-resolution imaging has rapidly emerged as an optical microscopy technique, offering advantages of high optical resolution over the past two decades; achieving improved imaging resolution requires significant efforts in developing super-resolution imaging agents characterized by high brightness, high contrast and high sensitivity to fluorescence switching. Apart from technical requirements in optical systems and algorithms, super-resolution imaging relies on fluorescent dyes with special photophysical or photochemical properties. The concept of aggregation-induced emission (AIE) was proposed in 2001, coinciding with unprecedented advancements and innovations in super-resolution imaging technology. AIE probes offer many advantages, including high brightness in the aggregated state, low background signal, a larger Stokes shift, ultra-high photostability, and excellent biocompatibility, making them highly promising for applications in super-resolution imaging. In this review, we summarize the progress in implementation methods and provide insights into the mechanism of AIE-based super-resolution imaging, including fluorescence switching resulting from photochemically-converted aggregation-induced emission, electrostatically controlled aggregation-induced emission and specific binding-regulated aggregation-induced emission. Particularly, the aggregation-induced emission principle has been proposed to achieve spontaneous fluorescence switching, expanding the selection and application scenarios of super-resolution imaging probes. By combining the aggregation-induced emission principle and specific molecular design, we offer some comprehensive insights to facilitate the applications of AIEgens (AIE-active molecules) in super-resolution imaging.

No-wash fluorogenic labeling of proteins for reversible photoswitching in live cells.

Photoswitchable fluorescent molecules (PSFMs) are positioned as valuable tools for biomolecule localization tracking and super-resolution imaging technologies due to their unique ability to reversibly control fluorescence intensity upon light irradiation. Despite the high demand for PSFMs that are suitable for live-cell imaging, no general method has been reported that enables reversible fluorescence control on proteins of interest in living cells. Herein, we have established a platform to realize reversible fluorescence switching in living cells by adapting a protein labeling system. We have developed a new PSFM, named HTL-Trp-BODIPY-FF, which exhibits strong fluorogenicity upon recognition of Halo-tag protein and reversible fluorescence photoswitching in living cells. This is the first example of a PSFM that can be applicable to a general-purpose Halo-tag protein labeling system for no-wash live-cell imaging.

Spontaneously Blinking Rhodamine Dyes for Single-Molecule Localization Microscopy.

Single-molecule localization microscopy (SMLM) has found extensive applications in various fields of biology and chemistry. As a vital component of SMLM, fluorophores play an essential role in obtaining super-resolution fluorescence images. Recent research on spontaneously blinking fluorophores has greatly simplified the experimental setups and extended the imaging duration of SMLM. To support this crucial development, this review provides a comprehensive overview of the development of spontaneously blinking rhodamines from 2014 to 2023, as well as the key mechanistic aspects of intramolecular spirocyclization reactions. We hope that by offering insightful design guidelines, this review will contribute to accelerating the advancement of super-resolution imaging technologies.

Expanding NIR-II Lanthanide Toolboxes for Improved Biomedical Imaging and Detection

ConspectusOptical imaging with high spatiotemporal resolution has been developed as a vital technology to reveal biological mysteries. Researchers have made countless profound innovations to promote the investigations on cell biology, especially after the implementation of far-field optical super-resolution imaging technology. Intrinsically, in-depth understanding of physiological activities inevitably requires optical imaging in living organisms at tissue, cell, or even single molecule levels. However, such a requirement usually encounters a great bottleneck because traditional optical imaging has little ability to penetrate biological tissues. Recently, the newly emerged optical imaging technology based on the second near-infrared window (NIR-II, 1000∼1700 nm) has changed this predicament. With suppressed light-scattering and diminished tissue autofluorescence, NIR-II optical imaging can easily achieve micrometer scale resolution at sub-centimeter tissue depth. Therefore, researchers regard it as a new avenue for in vivo optical imaging research.The past decade has witnessed increased interest in the field of in vivo optical imaging in the NIR-II window, accompanied by the significant development of NIR-II luminescence probes including small organic molecules, single-walled carbon nanotubes (SWNTs), quantum dots (QDs), and lanthanide nanoprobes. Among these luminescent probes, lanthanide nanoprobes, including lanthanide nanoparticles and lanthanide complexes, have become one of the most promising contrast agents. Owing to their abundant emissions with a narrow-band, lanthanide nanoprobes are usually favored when performing in vivo multiplexing imaging. In addition, their inherently long luminescence lifetimes (>1 μs) offer them great opportunity to implement time-gated imaging that can efficiently avoid autofluorescence interference. Besides, the superior photostability of lanthanides probes satisfies the requirements of long-term in vivo imaging. Based on these advantages, researchers have carried out extensive exploration on optical properties and functionalities of lanthanide nanoprobes.In this Account, we highlight our contributions to developing the NIR-II lanthanide toolboxes for improved biomedical imaging and detection, with a special focus on the future development of lanthanide nanoprobes for biological applications. First, we summarize our efforts on optimizing the excitation and emission wavelengths of the NIR-II lanthanide nanoprobes and analyze how these wavelengths affect the spatial resolution and penetration depth of in vivo optical imaging. Then, we discuss the time domain relevant achievements that are realized by well-designed lanthanide nanoparticles and luminescence lifetime imaging system. After that, we present the attempts to achieve persistent luminescence lanthanide nanoparticles for excitation-free in vivo optical imaging. We also conclude the breakthroughs in the design and synthesis of lanthanide complexes as well as their unique biological imaging results. At the end, we emphasize the future challenges and developments of the NIR-II lanthanide nanoprobes for in vivo optical imaging. By taking our work as a guide, we hope that this Account will provide the rationale behind the development of the NIR-II lanthanide tools and reveal their future opportunities in bioapplications.

Quantifying Intracellular Nanoparticle Distributions with Three-Dimensional Super-Resolution Microscopy.

Super-resolution microscopy can transform our understanding of nanoparticle-cell interactions. Here, we established a super-resolution imaging technology to visualize nanoparticle distributions inside mammalian cells. The cells were exposed to metallic nanoparticles and then embedded within different swellable hydrogels to enable quantitative three-dimensional (3D) imaging approaching electron-microscopy-like resolution using a standard light microscope. By exploiting the nanoparticles' light scattering properties, we demonstrated quantitative label-free imaging of intracellular nanoparticles with ultrastructural context. We confirmed the compatibility of two expansion microscopy protocols, protein retention and pan-expansion microscopy, with nanoparticle uptake studies. We validated relative differences between nanoparticle cellular accumulation for various surface modifications using mass spectrometry and determined the intracellular nanoparticle spatial distribution in 3D for entire single cells. This super-resolution imaging platform technology may be broadly used to understand the nanoparticle intracellular fate in fundamental and applied studies to potentially inform the engineering of safer and more effective nanomedicines.

Photochemical Mechanisms of Fluorophores Employed in Single-Molecule Localization Microscopy.

Decoding cellular processes requires visualization of the spatial distribution and dynamic interactions of biomolecules. It is therefore not surprising that innovations in imaging technologies have facilitated advances in biomedical research. The advent of super-resolution imaging technologies has empowered biomedical researchers with the ability to answer long-standing questions about cellular processes at an entirely new level. Fluorescent probes greatly enhance the specificity and resolution of super-resolution imaging experiments. Here, we introduce key super-resolution imaging technologies, with a brief discussion on single-molecule localization microscopy (SMLM). We evaluate the chemistry and photochemical mechanisms of fluorescent probes employed in SMLM. This Review provides guidance on the identification and adoption of fluorescent probes in single molecule localization microscopy to inspire the design of next-generation fluorescent probes amenable to single-molecule imaging.

Reduction in required volume of imaging data and image reconstruction time for adaptive-illumination Fourier ptychographic microscopy.

Fourier ptychographic microscopy (FPM) is a promising super-resolution computational imaging technology. It stitches a series of low-resolution (LR) images in the Fourier domain by an iterative method. Thus, it obtains a large field of view and high-resolution quantitative phase images. Owing to its capability to perform high-spatial bandwidth product imaging, FPM is widely used in the reconstruction of conventional static samples. However, the influence of the FPM imaging mechanism limits its application in high-speed dynamic imaging. To solve this problem, an adaptive-illumination FPM scheme using regional energy estimation is proposed. Starting with several captured real LR images, the energy distribution of all LR images is estimated, and select the measurement images with large information to perform FPM reconstruction. Simulation and experimental results show that the method produces efficient imaging performance and reduces the required volume of data to more than 65% while ensuring the quality of FPM reconstruction.

Polymer Models of Chromatin Imaging Data in Single Cells

Recent super-resolution imaging technologies enable tracing chromatin conformation with nanometer-scale precision at the single-cell level. They revealed, for example, that human chromosomes fold into a complex three-dimensional structure within the cell nucleus that is essential to establish biological activities, such as the regulation of the genes. Yet, to decode from imaging data the molecular mechanisms that shape the structure of the genome, quantitative methods are required. In this review, we consider models of polymer physics of chromosome folding that we benchmark against multiplexed FISH data available in human loci in IMR90 fibroblast cells. By combining polymer theory, numerical simulations and machine learning strategies, the predictions of the models are validated at the single-cell level, showing that chromosome structure is controlled by the interplay of distinct physical processes, such as active loop-extrusion and thermodynamic phase-separation.

Hyperbolic metamaterial-assisted structured illumination microscopy using periodic sub-diffraction speckles

Structured illumination microscopy (SIM), as a wide-field, rapid, super-resolution imaging technology, is widely employed in the field of biology. In this work, we propose a hyperbolic metamaterial (HMM)-assisted super-resolution structured illumination microscopy technique. By utilizing the HMM, a sub-diffraction illumination pattern of bulk plasmon polariton (BPP) with pure and higher spatial frequency replaces the conventional laser interference fringes, thus the imaging resolution of BPPSIM could surpass that of conventional SIM and reach 65 nm for HMM with 8-layers periodic structure. Moreover, an HMM with 10-layers aperiodic structure designed with particle swarm optimization was obtained, and BPPSIM could bring the imaging resolution down to 60 nm (1/9 of the fluorescence wavelength), which is a 3.3-fold improvement compared with the diffraction-limited image. This BPPSIM would provide a super-resolution, wide field of view, and good bio-compatibility approach in biological imaging.

A novel rat model of cerebral small vessel disease and evaluation by super-resolution ultrasound imaging

Cerebral small vessel disease (CSVD), which causes cognitive, functional and emotional decline, is related to stroke events, and it is a major cause of Alzheimer's disease. In the social context of an aging population, the incidence of CSVD is on the rise yearly, and the exact pathogenesis is still controversial and remains unclear. Exploring the pathological mechanism of CSVD on the histological level using animal models is important for the investigation of new clinical diagnostic methods and treatment options. The existing surgical CSVD model preparation methods are difficult to operate and cannot control the injury location or degree. This study used ultrasound combined with microbubbles (MBs) to induce an easy-to-operate and non-invasive animal model of CSVD with controllable location and degree. The rat model was evaluated from the perspective of histology, ethology, and imageology, respectively. In addition, we utilized super-resolution ultrasound imaging (SR-US) technology to directly observe the microvessels of the model. The histological results showed that the modeling was successful in the preset position, and neurology deficits were observed in 62.5% of 8 rats. The SR-US results of one rat showed that compared with the non-sonication region, the number of cerebral small blood vessels discovered in the sonication area was reduced (43 vs 11), the blood flow speed decreased significantly (p 0.001), and blood flow volume decreased (144.7 vs 11.7 μL/s) because of vasoconstriction. This study provides a new modeling method with controllable damage location and degree for the study of CSVD, and SR-US is found to be an effective evaluation method, which can directly assess the hemodynamic changes of CSVD in vivo.

Boosting optical nonlinearities of graphene oxide films by laser direct writing

In this work, we study the boosted optical nonlinearities of graphene oxide (GO) films by laser direct writing with femtosecond laser (532 nm, 330fs) excitation. We firstly prepare the uniform GO films by vacuum filtration method, which are then partly reduced by commercial carbon dioxide laser machine. The morphology, structure and luminescence traits of both the pristine and reduced GO are further characterized by SEM, XRD, Raman spectra, XPS, PL and UV–Vis absorption spectrum. Moreover, their third-order optical nonlinearities (TONs) are measured by the femtosecond laser Z-scan technique. The experimental results show that the reduced GO films take on stronger TONs and the related degree of enhancement depends on the spot size of laser processing. In particular, when the processed diameter is around 400 μm, the optical nonlinear absorption and refraction coefficients of the reduced GO are −2.9 × 10−7 m/W and 2.5 × 10−15 m2/W, respectively, which are in turn 4 and 2 times higher than that of GO film. Our results provide powerful backing for the GO-based applications, such as ultra-fast optical devices, super-resolution imaging technology and photodetector.

The Influence of Optical Alignment Error on Compression Coding Superresolution Imaging.

Superresolution (SR) imaging technology based on compression coding has always been considered as the key to break through the geometric resolution of the detector. In addition to factors such as the reconstruction algorithm and mounting platform vibrations, the impact of inherent errors in the optical system itself on the reconstruction results of SR imaging is also obvious. To address this issue, a study on the design of the SR optical system and the influence of optical alignment errors on SR imaging was conducted. The design of the SR optical system based on digital micro-mirror device (DMD) for long-wave infrared wavelength was completed, and an athermal analysis of the system was carried out. The design results showed that the SR optical system has good imaging quality in the operating temperature range. The imaging model of the DMD SR imaging optical system is established according to the designed SR optical system. We investigated the influence of various alignment errors, including decenter, tilt, lens interval error and defocus, on the imaging properties of the SR optical system. Various random combinations of alignment errors were introduced into the optical system, respectively, and the SR reconstructed image quality of the imaging system was analyzed using the inverse sensitivity method to obtain the tolerance limits when the system was assembled. Finally, the effectiveness of the method to obtain the alignment tolerance limit of the compression coding SR imaging optical system was verified through a desktop demonstration experiment.

Virus morphology: Insights from super-resolution fluorescence microscopy.

As epitomised by the COVID-19 pandemic, diseases caused by viruses are one of the greatest health and economic burdens to human society. Viruses are ‘nanostructures’, and their small size (typically less than 200 nm in diameter) can make it challenging to obtain images of their morphology and structure. Recent advances in fluorescence microscopy have given rise to super-resolution techniques, which have enabled the structure of viruses to be visualised directly at a resolution in the order of 20 nm. This mini-review discusses how recent state-of-the-art super-resolution imaging technologies are providing new nanoscale insights into virus structure.

Progress in microspheric lens based super-resolution microscopic imaging technology with large field of view

Progress in microspheric lens based super-resolution microscopic imaging technology with large field of view

Recent progress on the reconstruction algorithms of structured illumination microscopy

Abstract: As an early component of modern super-resolution (SR) imaging technology, structured illumination microscopy (SIM) has been developed for nearly twenty years. With up to ~60 nm and 564 Hz frame rate, it has achieved an optimal combination of spatiotemporal resolution in live cells recently. Despite these advantages, SIM also suffers disadvantages, some of which originated from the intrinsic reconstruction process. Here we review recent technical advances in SIM, including SR reconstruction, performance evaluation, and its integration with other technologies to provide a practical guide for biologists.

Heme Induced Progressive Loss of Endothelial Protein C Receptor Promotes Development of Chronic Kidney Disease in Sickle Cell Disorders

Chronic kidney disease (CKD), widespread among the individuals with sickle cell disease (SCD), is a major contributor to early death in this patient population. The progressive deterioration of renal health in SCD is associated with chronic persistent intravascular hemolysis leading to anemia. We have previously reported that extracellular heme, released during acute intravascular hemolysis triggers clinically relevant acute kidney injury (AKI) in SCD mice (SS) (Blood (2020) 135 (13): 1044-1048). Although AKI is reversible, it is considered as a risk factor for CKD. The mechanistic approach elucidating the hemolysis driven pathogenesis of AKI-to-CKD transition in SCD is unknown. We found that CKD develops in the SS mice by progressive (1-10 months of age) increase in albuminuria (urinary albumin and creatinine ratio, uACR) and decrease in glomerular filtration rate (GFR) (n=5; p<0.001). Histopathology of the kidney showed age-dependent deterioration in renal peritubular vascular congestion in SS mice compared to that of the AA mice. Alongside, Evan's blue extravasation experiments showed that the SS mice are susceptible to vascular leakage that is correlated positively with age (Pearson r=0.98, p<0.001) and negatively with anemia (Pearson r= -0.46, p<0.05). We hypothesized that multiple acute hemolytic events may instigate persistent endothelial damage that ensues CKD development in SCD. To test this hypothesis, we intravenously infused vehicle or heme (14 μmoles/kg body weight; 5 times on alternate days) to 1-month old SS mice and monitored for 3 weeks following first heme injection. These mice developed severe albuminuria (n=5; p<0.01) with substantial loss of GFR (n=5; p<0.001), indicating heme induced CKD development. Next, we used ultrasound super-resolution (USR) imaging technology to determine renal microvascular changes in older SS mice (6-months) without heme challenge and in young SS mice (1-month) challenged with heme. Analysis of the USR data showed reduced renal blood volume (rBV) and substantial loss of vessel density in renal cortex as well as in corticomedullary areas of the older SS mice compared to the age-matched AA mice. Accordingly, multiple heme challenge reduced rBV and vessel density extensively in young SS mice comparable to the older SS mice without heme challenge. Since endothelial protein C receptor (EPCR) maintains vascular barrier integrity by activating protease activated receptor-1 (PAR1) signaling in the endothelium, we tested whether alterations in EPCR expression contribute to progressive endothelial damage in SS mice during CKD development. Using immunofluorescence microscopy, we determined that renal endothelium lacks expression of EPCR in older SS mice while younger SS mice retains EPCR cellular expression. In corroboration, infusion of heme in younger SS mice results in loss of renal endothelial EPCR. Shedding of EPCR from endothelium often results in a soluble form of EPCR (sEPCR). We found that SS mice had higher plasma levels of soluble EPCR (sEPCR) compared to their AA counterparts. While age dependent increase in plasma and urinary sEPCR were evident in SS mice (n=6; p<0.01), infusion of heme in younger SS mice results in significant increase in plasma sEPCR (n=6; p<0.01). In consistent with the mouse data, we discovered that the plasma sEPCR was significantly elevated in SCD patients compared to normal individuals (n=8-16; p<0.05). Moreover, the plasma sEPCR was significantly associated with the baseline albuminuria in a cohort of SCD patients (n=16; Pearson r=0.64; p<0.01). This study supports the conclusion that multiple hemolytic events may trigger CKD development in SCD by gradual loss of renal microvascular EPCR expression. Clinically, the sEPCR can be developed as risk factor for sickle CKD. Finally, our data suggest that restoration of EPCR function may protect SCD patients from CKD. DisclosuresNo relevant conflicts of interest to declare.

The Influence of Noise in the Super-Resolution Reconstruction of Structured Illumination Microscopy

As one of the mainstream super-resolution imaging technologies, structured illumination microscopy (SIM) is popular for its fast imaging speed and simple optical path structure. Spectrum separation is a key step in the reconstruction of super-resolution images. However, in the process of imaging, the unavoidable noise will seriously affect the accuracy of frequency spectrum separation. This paper carries out a simulation study on the influence of noise in the process of frequency spectrum separation. The results show that although noise can cause distortion of low-frequency information in frequency spectrum separation results, it has little influence on high-frequency information. Therefore, a super-resolution image reconstruction method is proposed to effectively suppress the influence of noise. Both simulation and experimental results are shown the method can suppress the influence of noise without losing the details of super-resolution.