Biological Microscopy Research Articles

Unmixing Biological Fluorescence Image Data with Sparse and Low-Rank Poisson Regression.

Multispectral biological fluorescence microscopy has enabled the identification of multiple targets in complex samples. The accuracy in the unmixing result degrades (1) as the number of fluorophores used in any experiment increases and (2) as the signal-to-noise ratio in the recorded images decreases. Further, the availability of prior knowledge regarding the expected spatial distributions of fluorophores in images of labeled cells provides an opportunity to improve the accuracy of fluorophore identification and abundance. We propose a regularized sparse and low-rank Poisson unmixing approach (SL-PRU) to deconvolve spectral images labeled with highly overlapping fluorophores which are recorded in low signal-to-noise regimes. Firstly, SL-PRU implements multi-penalty terms when pursuing sparseness and spatial correlation of the resulting abundances in small neighborhoods simultaneously. Secondly, SL-PRU makes use of Poisson regression for unmixing instead of least squares regression to better estimate photon abundance. Thirdly, we propose a method to tune the SL-PRU parameters involved in the unmixing procedure in the absence of knowledge of the ground truth abundance information in a recorded image. By validating on simulated and real-world images, we show that our proposed method leads to improved accuracy in unmixing fluorophores with highly overlapping spectra. The source code used for this paper was written in MATLAB and is available with the test data at https://github.com/WANGRUOGU/SL-PRU. Supplementary data are available at Bioinformatics online.

Inverse design of a near-infrared metalens with an extended depth of focus based on double-process genetic algorithm optimization.

Metalens with extended depth of focus (EDOF) can extend the mapping area of the image, which leads to novel applications in imaging and microscopy. Since there are still some disadvantages for existing EDOF metalenses based on forward design, such as asymmetric point spread function (PSF) and non-uniformly distributed focal spot, which impair the quality of images, we propose a double-process genetic algorithm (DPGA) optimization to inversely design the EDOF metalens for addressing these drawbacks. By separately adopting different mutation operators in successive two genetic algorithm (GA) processes, DPGA exhibits significant advantages in searching for the ideal solution in the whole parameter space. Here, the 1D and 2D EDOF metalenses operating at 980 nm are separately designed via this method, and both of them exhibit significant depth of focus (DOF) improvement to that of conventional focusing. Furthermore, a uniformly distributed focal spot can be maintained well, which can guarantee stable imaging quality along the longitudinal direction. The proposed EDOF metalenses have considerable potential applications in biological microscopy and imaging, and the scheme of DPGA can be promoted to the inverse design of other nanophotonics devices.

Broadband squeezed microwaves and amplification with a Josephson travelling-wave parametric amplifier

Squeezing of the electromagnetic vacuum is an essential metrological technique used to reduce quantum noise in applications spanning gravitational wave detection, biological microscopy, and quantum information science. In superconducting circuits, the resonator-based Josephson-junction parametric amplifiers conventionally used to generate squeezed microwaves are constrained by a narrow bandwidth and low dynamic range. In this work, we develop a dual-pump, broadband Josephson traveling-wave parametric amplifier that combines a phase-sensitive extinction ratio of 56 dB with single-mode squeezing on par with the best resonator-based squeezers. We also demonstrate two-mode squeezing at microwave frequencies with bandwidth in the gigahertz range that is almost two orders of magnitude wider than that of contemporary resonator-based squeezers. Our amplifier is capable of simultaneously creating entangled microwave photon pairs with large frequency separation, with potential applications including high-fidelity qubit readout, quantum illumination and teleportation.

High-speed atomic force microscopy for dynamic single molecule structural biology.

High-speed atomic force microscopy (HS-AFM) is a powerful technique that provides dynamic movies of biomolecules at work. We successfully used HS-AFM to take movies and determine dynamic parameters of membrane trafficking systems, transporters and channels. To break current temporal limitations to characterize molecular dynamics using HS-AFM, we developed HS-AFM line scanning (HS-AFM-LS) and HS-AFM height spectroscopy (HS-AFM-HS), methods whereby we scan the HS-AFM tip along a single scan line or keep it at a fixed position, respectively, and detect the motions of the molecules under the tip with sub-nanometer spatial and millisecond to microseconds temporal resolution to characterize single molecule kinetics. To break resolution limitations, we developed Localization AFM (LAFM) by applying localization image reconstruction algorithms to peak positions in high-speed AFM and conventional AFM data and increased the resolution beyond the limits set by the tip radius to reach quasi-atomic resolution on soft protein surfaces in native and dynamic conditions. Here, I will review these recent achievements and present novel data and methods to integrate and strengthen HS-AFM as a powerful tool in structural biology and molecular biophysics. We reason that the progress of AFM must comprise two axes: First, making AFM/HS-AFM a better tool. With the above-mentioned methods, we made significant progress in this axis over the last ∼5 years. Second, making AFM/HS-AFM data an integrated part of the structural biology / molecular biophysics toolbox. To this end, we are developing now methods and data analysis and interpretation methods that interface with cryo-EM and X-ray crystallography, where AFM/HS-AFM can contribute the urgently needed information about structure, dynamics and conformations under close-to-native conditions.

An Effective Approach for Automatic River Features Extraction Using High-Resolution UAV Imagery

The effects of climate change are causing an increase in the frequency and extent of natural disasters. Because of their morphological characteristics, rivers can cause major flooding events. Indeed, they can be subjected to variations in discharge in response to heavy rainfall and riverbank failures. Among the emerging methodologies that address the monitoring of river flooding, those that include the combination of Unmanned Aerial Vehicle (UAV) and photogrammetric techniques (i.e., Structure from Motion-SfM) ensure the high-frequency acquisition of high-resolution spatial data over wide areas and so the generation of orthomosaics, useful for automatic feature extraction. Trainable Weka Segmentation (TWS) is an automatic feature extraction open-source tool. It was developed to primarily fulfill supervised classification purposes of biological microscope images, but its usefulness has been demonstrated in several image pipelines. At the same time, there is a significant lack of published studies on the applicability of TWS with the identification of a universal and efficient combination of machine learning classifiers and segmentation approach, in particular with respect to classifying UAV images of riverine environments. In this perspective, we present a study comparing the accuracy of nine combinations, classifier plus image segmentation filter, using TWS, also with respect to human photo-interpretation, in order to identify an effective supervised approach for automatic river features extraction from UAV multi-temporal orthomosaics. The results, which are very close to human interpretation, indicate that the proposed approach could prove to be a valuable tool to support and improve the hydro-geomorphological and flooding hazard assessments in riverine environments.

Tissue mechanics modulate PCNP expression in oral squamous cell carcinomas with different differentiation.

PEST-containing nuclear protein (PCNP), a novel zinc finger protein, participates in cell cycle regulation. Previous studies have confirmed that PCNP plays a role in mediating cellular development and invasion in a variety of cancer types. However, the relationship between PCNP expression and the occurrence and development of oral squamous cell carcinoma (OSCC) requires further exploration. In this study, we used biological atomic force microscopy to examine the histomorphological and mechanical properties of OSCC to explore the relationship between PCNP expression and differentiation of OSCC. Seventy-seven OSCC samples with varying degrees of differentiation were selected for hematoxylin and eosin staining, immunohistochemistry, and cellular mechanical measurement. The expression of PCNP and the mechanical properties such as stiffness and roughness of the tissue interface in OSCC samples were investigated. The Kaplan-Meier survival curve was utilized to assess the relationship of PCNP expression with patient survival. The level of PCNP was significantly higher in well-differentiated OSCC than in moderately and poorly differentiated OSCC (P < 0.001). High expression of PCNP was specifically associated with higher tumor differentiation, lack of lymph node metastasis, and lower tumor node metastasis stage (all P < 0.05). Patients with high PCNP expression had a higher survival rate than those with low PCNP expression. The average variation of stiffness within a single tissue ranged from 347 kPa to 539 kPa. The mean surface roughness of highly, moderately, and poorly differentiated OSCC and paraneoplastic tissues were 795.53 ± 47.2 nm, 598.37 ± 45.76 nm, 410.16 ± 38.44 nm, and 1010.94 ± 119.07 nm, respectively. Pearson correlation coefficient demonstrated a positive correlation between PCNP expression and tissue stiffness of OSCC (R = 0.86, P < 0.001). The expression of PCNP was positively correlated with patient survival, tumor differentiation, and mechanical properties of tissue interfaces. PCNP is a potential biomarker for the early diagnosis and staging of OSCC. Furthermore, determination of the mechanical properties of the tissue interface could provide further useful information required for the detection and differentiation of OSCC.

Advanced fluorescence microscopy in respiratory virus cell biology.

Respiratory viruses are a major public health burden across all age groups around the globe, and are associated with high morbidity and mortality rates. They can be transmitted by multiple routes, including physical contact or droplets and aerosols, resulting in efficient spreading within the human population. Investigations of the cell biology of virus replication are thus of utmost importance to gain a better understanding of virus-induced pathogenicity and the development of antiviral countermeasures. Light and fluorescence microscopy techniques have revolutionized investigations of the cell biology of virus infection by allowing the study of the localization and dynamics of viral or cellular components directly in infected cells. Advanced microscopy including high- and super-resolution microscopy techniques available today can visualize biological processes at the single-virus and even single-molecule level, thus opening a unique view on virus infection. We will highlight how fluorescence microscopy has supported investigations on virus cell biology by focusing on three major respiratory viruses: respiratory syncytial virus (RSV), Influenza A virus (IAV) and SARS-CoV-2. We will review our current knowledge of virus replication and highlight how fluorescence microscopy has helped to improve our state of understanding. We will start by introducing major imaging and labeling modalities and conclude the chapter with a perspective discussion on remaining challenges and potential opportunities.

ICL Postimplantation Decentration and Tilt in Myopic Patients with Primary Iridociliary Cysts.

To observe the decentration and tilt of implantable collamer lens (ICL) as well as possible visual effects postimplantation in primary iridociliary cysts. The present investigation was a retrospective cohort study. All 48 patients (91 eyes) who underwent ICL surgery at the Center of Refraction Surgery of Tianjin Medical University Eye Hospital between July 2018 and May 2020 were split into two groups based on the absence or presence of primary iridociliary cysts established by ultrasonic biological microscopy (UBM) examination. Intraocular pressure (IOP), corrected distance visual acuity (CDVA), uncorrected distance visual acuity (UDVA), anterior chamber angle (ACA), anterior chamber volume (ACV), and anterior chamber depth (ACD) were recorded preoperatively and postoperatively at 1, 6, and 12 months. Additionally, we performed an analysis of the ICL vault, decentration, and tilt using a rotating Scheimpflug Oculus Pentacam camera system at 1, 6, and 12 months after surgery. No serious complications were observed. Significant postoperative improvement (P < 0.05) of UDVA was established in the two studied groups; however, we did not observe statistically significant intergroup differences (P > 0.05) throughout the entire research period. In each group, the preoperative ACA, ACV, and ACD were statistically significantly reduced (P < 0.05), but no such decrease was established between their postoperative values (P > 0.05). We observed no statistical differences between both groups with regard to their values of IOP, ACA, ACV, ACD, ICL vault, ICL decentration, and tilt at 1, 6, and 12 months after surgery. Similarly, no statistically significant within-group correlation (P > 0.05) of the decentration of ICL and the tilt and the CDVA values was established. No postimplantation effect of ICL with a central hole on vision was established in myopia patients with primary iridociliary cysts, within certain limits of ICL decentration and tilt values.

A supercritical relaxor phase boundary for ultrahigh electrostrictive properties

In this work the design of a critical phase point where reversible transition from the ergodic relaxor state to an unstable long-range ordered ferroelectric state takes place is reported.

Correlative multiscale microCT-SBF-SEM imaging of resin-embedded tissue.

Three-dimensional biological microscopy presents a trade-off between spatial resolution and field of view. Correlative approaches applying multiple imaging techniques to the same sample can therefore mitigate against these trade-offs. Here, we present a workflow for correlative microscopic X-ray microfocus computed tomography (microCT) and serial block face scanning electron microscopy (SBF-SEM) imaging of resin-embedded tissue, using mammalian placental tissue samples as an example. This correlative X-ray and electron microscopy (CXEM) workflow allows users to image the same sample at multiple resolutions, and target the region of interest (ROI) for SBF-SEM based on microCT. We detail the protocols associated with this workflow and demonstrate its application in multiscale imaging of horse placental villi and ROI selection in the labyrinthine zone of a mouse placenta. These examples demonstrate how the protocol may need to be adapted for tissues with different densities.

Unexpected Photocatalytic Degeneration of NAD + for Inducing Apoptosis of Hypoxia Cancer Cells

Unexpected Photocatalytic Degeneration of NAD ⁺ for Inducing Apoptosis of Hypoxia Cancer Cells

Single-molecule characterization of a bright and photostable deep-red fluorescent squaraine-figure-eight (SF8) dye

Squaraine Figure Eight (SF8) dyes are a unique class of deep-red fluorescent dyes with self-threaded molecular architecture that provides structural rigidity while simultaneously encapsulating and protecting the emissive fluorochrome. Previous cell microscopy and bulk phase studies of SF8 dyes indicated order of magnitude enhancements in photostability over conventional pentamethine cyanine dyes such as Cy5. Studies conducted at the single molecule level now reveal that these ensemble level enhancements carry over to the single molecule level in terms of enhanced emission quantum yields, longer times to photobleaching, and enhanced total photon yields. When compared to Cy5, the SF8-based dye SF8(D4)2 possesses a three-fold larger single molecule emission quantum yield, exhibits order of magnitude longer average times before photobleaching, and exhibits twenty times larger photon yields. Additional features such as water solubility, fluorochrome encapsulation to protect it against nucleophilic attack, and selective biomarker targeting capability make SF8-based dyes promising candidates for biological labeling and microscopy applications and single molecule tracking.

New Insights into the Cellular Toxicity of Carbon Quantum Dots to Escherichia coli.

In this study, the cytotoxicity and toxic mechanism of carbon quantum dots (CQDs) to E. coli were evaluated in vitro. The synthetic CQDs were extremely small in size (~2.08 nm) and displayed strong fluorescence. The results demonstrated that CQDs showed good biocompatibility with E. coli within a short culture time. However, when the exposure time exceeded 24 h, the toxicity of CQDs became apparent, and the contents of reactive oxygen species, lactate dehydrogenase, and the crystal violet absorption rate increased significantly. To further explore the cytotoxic mechanism, approaches including confocal laser scanning microscopy, scanning electron microscopy, and biological transmission electron microscopy combined with zeta potential tests, osmotic pressure measurement, and comet assays were performed. On the one hand, the CQDs altered the surface charges of cells and induced lipid peroxidation by adhesion on the surface of E. coli, leading to an increase in the permeability of the cell wall. On the other hand, when the concentration of CQDs reached 200 µg/mL, the osmotic pressure of the extracellular environment was significantly reduced. These are the main factors that lead to cell edema and death. Finally, the comet assays confirmed that CQDs could induce DNA damage, which could inhibit the proliferation of E. coli.

Compressed sensing in photonics: tutorial

Traditional optical imaging and sensing methods capture signals of interest by direct sampling in the domain of interest such as by forming images on pixelated camera sensors or by regular temporal sampling of a waveform. These methods are indispensable in our daily lives and for many scientific disciplines such as microscopy in biology and spectroscopy in chemistry. Using these approaches, the sampling constraints and their impact on the bounds on signal fidelity are well understood through the Nyquist–Shannon sampling theorem. However, the problems of modern science require ever increasing amounts of data at unprecedented temporal and spatial scales and resolutions, which challenges the limits of traditional sensing. The increased availability of computational power combined with recent strides in signal processing promise to surpass many of the problems associated with traditional sensing methods through computational imaging and sensing methods. Within the realm of computational sensing, compressed sensing (CS), in particular, has enabled the capture of signals with lower sampling resources than traditionally required by the Nyquist–Shannon sampling theorem using prior information such as sparsity. In this tutorial, we focus on the operation and impact of such sub-Nyquist sampling schemes through the use of CS in photonic sensing and imaging systems. Emphasis is placed on intuition, but mathematical results are derived or cited where appropriate. Finally, we highlight several applications in macroscopic and microscopic imaging, spectroscopy, and microwave photonic sensing.

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu.

Hampered by the diffraction phenomenon, as expressed in 1873 by Abbe, applications of optical microscopy to image biological structures were for a long time limited to resolutions above the ∼200nm barrier and restricted to the observation of stained specimens. The introduction of fluorescence was a game changer, and since its inception it became the gold standard technique in biological microscopy. The plasma membrane is a tenuous envelope of 4nm-10nm in thickness surrounding the cell. Because of its highly versatile spectroscopic properties and availability of suitable instrumentation, fluorescence techniques epitomize the current approach to study this delicate structure and its molecular constituents. The wide spectral range covered by fluorescence, intimately linked to the availability of appropriate intrinsic and extrinsic probes, provides the ability to dissect membrane constituents at the molecular scale in the spatial domain. In addition, the time resolution capabilities of fluorescence methods provide complementary high precision for studying the behavior of membrane molecules in the time domain. This review illustrates the value of various fluorescence techniques to extract information on the topography and motion of plasma membrane receptors. To this end I resort to a paradigmatic membrane-bound neurotransmitter receptor, the nicotinic acetylcholine receptor (nAChR). The structural and dynamic picture emerging from studies of this prototypic pentameric ligand-gated ion channel can be extrapolated not only to other members of this superfamily of ion channels but to other membrane-bound proteins. I also briefly discuss the various emerging techniques in the field of biomembrane labeling with new organic chemistry strategies oriented to applications in fluorescence nanoscopy, the form of fluorescence microscopy that is expanding the depth and scope of interrogation of membrane-associated phenomena.

Hollow mesoporous structured MnFe2O4 nanospheres: A biocompatible drug delivery system with pH-responsive release for potential application in cancer treatment

In this paper, a novel pH-responsive drug delivery system based on hierarchical hollow mesoporous MnFe2O4 nanospheres was developed for potential application in cancer treatment. The well-defined hollow mesoporous MnFe2O4 nanospheres were directly fabricated via a facile one-step solvothermal approach without the involvement of additional templates. A mechanism of inside-out Ostwald ripening was suggested to account for the formation of hollow mesoporous nanostructures. The morphology, crystal structure, chemical composition, magnetic properties, thermal stability and mesoporous characteristics of the fabricated MnFe2O4 nanospheres were comprehensively assessed based on various analysis techniques. The resulting hollow mesoporous MnFe2O4 nanospheres with a high biocompatibility were able to be served as a nanocarrier for loading and delivering of anticancer drug doxorubicin (DOX). In vitro drug release study revealed that DOX release from DOX-loaded MnFe2O4 nanospheres was significantly restrained in pH 7.4 phosphate buffer solution (PBS), while it was markedly accelerated in pH 4.5 PBS. The results of cytotoxicity assay and inverted biological microscopy demonstrated that the DOX-loaded MnFe2O4 nanospheres were more cytotoxic against HepG2 cells than MnFe2O4 nanospheres due to the evident inhibition effect of released DOX from the constructed drug delivery system. Therefore, the developed pH-responsive delivery system will have a great potential for its practical application in cancer treatment.

Autophagy Is a Crucial Path in Chondrogenesis of Adipose-Derived Mesenchymal Stromal Cells Laden in Hydrogel.

Autophagy is a cellular process that contributes to the maintenance of cell homeostasis through the activation of a specific path, by providing the necessary factors in stressful and physiological situations. Autophagy plays a specific role in chondrocyte differentiation; therefore, we aimed to analyze this process in adipose-derived mesenchymal stromal cells (ASCs) laden in three-dimensional (3D) hydrogel. We analyzed chondrogenic and autophagic markers using molecular biology, immunohistochemistry, and electron microscopy. We demonstrated that ASCs embedded in 3D hydrogel showed an increase expression of typical autophagic markers Beclin 1, LC3, and p62, associated with clear evidence of autophagic vacuoles in the cytoplasm. During ASCs chondrogenic differentiation, we showed that autophagic markers declined their expression and autophagic vesicles were rare, while typical chondrogenic markers collagen type 2, and aggrecan were significantly increased. In line with developmental animal models of cartilage, our data showed that in a 3D hydrogel, ASCs increased their autophagic features. This path is the fundamental prerequisite for the initial phase of differentiation that contributes to fueling the cells with energy and factors necessary for chondrogenic differentiation.

A perspective of fluorescence microscopy for cellular structural biology with EGFR as witness.

The epidermal growth factor receptor (EGFR) is a poster child for the understanding of receptor behaviour, and of paramount importance to cell function and human health. Cloned almost forty years ago, the interest in EGFR's structure/function relationships remains unabated, not least because changes in oncogenic EGFR mutants are key drivers of the formation of lung and brain tumours. The structure of the assemblies formed by EGFR have been comprehensibly investigated by techniques such as high-resolution X-ray crystallography, NMR and all-atom molecular dynamics (MD) simulations. However, the complexity embedded in the portfolio of EGFR states that are only possible in the physiological environment of cells has often proved refractory to cell-free structural methods. Conversely, some key inroads made by quantitative fluorescence microscopy and super-resolution have depended on exploiting the wealth of structures available. Here, a brief personal perspective is provided on how quantitative fluorescence microscopy and super-resolution methods have cross-fertilised with cell-free-derived EGFR structural information. I primarily discuss areas in which my research group has made a contribution to fill gaps in EGFR's cellular structural biology and towards developing new tools to investigate macromolecular assemblies in cells.

Self-supervised machine learning for live cell imagery segmentation

Segmenting single cells is a necessary process for extracting quantitative data from biological microscopy imagery. The past decade has seen the advent of machine learning (ML) methods to aid in this process, the overwhelming majority of which fall under supervised learning (SL) which requires vast libraries of pre-processed, human-annotated labels to train the ML algorithms. Such SL pre-processing is labor intensive, can introduce bias, varies between end-users, and has yet to be shown capable of robust models to be effectively utilized throughout the greater cell biology community. Here, to address this pre-processing problem, we offer a self-supervised learning (SSL) approach that utilizes cellular motion between consecutive images to self-train a ML classifier, enabling cell and background segmentation without the need for adjustable parameters or curated imagery. By leveraging motion, we achieve accurate segmentation that trains itself directly on end-user data, is independent of optical modality, outperforms contemporary SL methods, and does so in a completely automated fashion—thus eliminating end-user variability and bias. To the best of our knowledge, this SSL algorithm represents a first of its kind effort and has appealing features that make it an ideal segmentation tool candidate for the broader cell biology research community.

Study on Synergistic Demulsification Mechanism of Microwaves and Ionic Liquids on Heavy Oil Emulsions

Summary Heavy oil-in-water (O/W) emulsions must be treated before refinement and transportation, and microwave and ionic liquid (IL) synergistic effects are stable, efficient, and environmentally friendly methods of demulsification. In this study, the synergistic effects of various IL anion and cation types and concentrations upon microwave emulsion demulsification were investigated. The interfacial tension (IFT) and zeta potential of the ILs and the distribution of oil droplets were measured before and after microwave radiation using a spinning drop tensiometer, zeta potential analyzer, and biological microscope to elucidate the synergistic mechanism of microwave and IL demulsification. The results indicate that microwave radiation parameters that are set too high or low can have an inhibitory effect on demulsification. When [C16MIM]Br with a concentration of 1 mmol/L was used as the IL, and the microwave radiation parameters were set to 300 W for 30 seconds, the demulsification efficiency reached 89.29%, which was 3.23 times and 2.08 times higher than the individual demulsification efficiency of microwave radiation and IL, respectively. This study provides a novel approach for examining high-performance demulsification, and it extends the applicability of microwave-IL synergy.