ABSTRACT
Recent key technological developments, such as super-resolution microscopy and microfabrication, enabled investigation of biological processes, including macroautophagy/autophagy, with unprecedented spatiotemporal resolution and control over experimental conditions. Such disruptive innovations deepened our capability to provide mechanistic understandings of the autophagic process and its causes. This addendum aims to expand the guidelines on autophagy in three key directions: optical methods enabling visualization of autophagic machinery beyond the diffraction-limited resolution; bioengineering enabling accurate designs and control over experimental conditions; and theoretical advances in mechanobiology connecting autophagy and mechanical processes of the cell. Abbreviation: 3D: three-dimensional; SIM: structured illumination microscopy; STORM: stochastic optical reconstruction microscopy.
ABSTRACT
Here we apply the SUPPOSe algorithm on images acquired using Stimulated Emission Depletion (STED) microscopy with the aim of improving the resolution limit achieved. We processed images of the nuclear pore complex (NPC) from cell lines in which the Nup96 nucleoporin was endogenously labeled. This reference protein forms a ring whose diameter is â¼107 nm with 8 corners â¼42 nm apart from each other. The stereotypic arrangement of proteins in the NPC has been used as reference structures to characterize the performance of a variety of microscopy techniques. STED microscopy images resolve the ring arrangement but not the eightfold symmetry of the NPC. After applying the SUPPOSe algorithm to the STED images, we were able to solve the octagonal structure of the NPC. After processing 562 single NPC, the average radius of the NPC was found to beR= 54.2 ± 2.9 nm, being consistent with the theoretical distances of this structure. To verify that the solutions obtained are compatible with a NPC-type geometry, we rotate the solutions to optimally fit an eightfold-symmetric pattern and we count the number of corners that contain at least one localization. Fitting a probabilistic model to the histogram of the number of bright corners gives an effective labeling efficiency of 31%, which is in agreement with the values reported in for other cell lines and ligands used in Single Molecule Localization microscopy, showing that SUPPOSe can reliably retrieve sub-resolution, nanoscale objects from single acquisitions even in noisy conditions.
ABSTRACT
Nowadays, the use of super-resolution microscopy (SRM) is increasing globally due to its potential application in several fields of life sciences. However, a detailed and comprehensive guide is necessary for understanding a single-frame image's resolution limit. This study was performed to provide information about the structural organisation of isolated cellulose fibres from garlic and agave wastes through fluorophore-based techniques and image analysis algorithms. Confocal microscopy provided overall information on the cellulose fibres' microstructure, while techniques such as total internal reflection fluorescence microscopy facilitated the study of the plant fibres' surface structures at a sub-micrometric scale. Furthermore, SIM and single-molecule localisation microscopy (SMLM) using the PALM reconstruction wizard can resolve the network of cellulose fibres at the nanometric level. In contrast, the mean shift super-resolution (MSSR) algorithm successfully determined nanometric structures from confocal microscopy images. Atomic force microscopy was used as a microscopy technique for measuring the size of the fibres. Similar fibre sizes to those evaluated with SIM and SMLM were found using the MSSR algorithm and AFM. However, the MSSR algorithm must be cautiously applied because the selection of thresholding parameters still depends on human visual perception. Therefore, this contribution provides a comparative study of SRM techniques and MSSR algorithm using cellulose fibres as reference material to evaluate the performance of a mathematical algorithm for image processing of bioimages at a nanometric scale. In addition, this work could act as a simple guide for improving the lateral resolution of single-frame fluorescence bioimages when SRM facilities are unavailable.
ABSTRACT
Fluorescence Resonance Energy Transfer (FRET)-based approaches are unique tools for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and Fluorescence Lifetime Imaging Microscopy (FLIM) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction-limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, an approach to obtain super-resolved FRET imaging based on single-molecule localization microscopy using an early prototype of a commercial time-resolved confocal microscope is demonstrated. DNA Points Accumulation for Imaging in Nanoscale Topography with fluorogenic probes provides a suitable combination of background reduction and binding kinetics compatible with the scanning speed of usual confocal microscopes. A single laser is used to excite the donor, a broad detection band is employed to retrieve both donor and acceptor emission, and FRET events are detected from lifetime information.
Subject(s)
DNA , Fluorescence Resonance Energy Transfer , Fluorescence Resonance Energy Transfer/methods , Microscopy, Fluorescence/methods , DNA/chemistry , Microscopy, Confocal , Single Molecule ImagingABSTRACT
The cell-surface topography and density of nicotinic acetylcholine receptors (nAChRs) play a key functional role in the synapse. Here we employ in parallel two labeling and two super-resolution microscopy strategies to characterize the distribution of this receptor at the plasma membrane of the mammalian clonal cell line CHO-K1/A5. Cells were interrogated with two targeted techniques (confocal microscopy and stimulated emission depletion (STED) nanoscopy) and single-molecule nanoscopy (stochastic optical reconstruction microscopy, STORM) using the same fluorophore, Alexa Fluor 647, tagged onto either α-bungarotoxin (BTX) or the monoclonal antibody mAb35. Analysis of the topography of nanometer-sized aggregates ("nanoclusters") was carried out using STORMGraph, a quantitative clustering analysis for single-molecule localization microscopy based on graph theory and community detection, and ASTRICS, an inter-cluster similarity algorithm based on computational geometry. Antibody-induced crosslinking of receptors resulted in nanoclusters with a larger number of receptor molecules and higher densities than those observed in BTX-labeled samples. STORM and STED provided complementary information, STED rendering a direct map of the mesoscale nAChR distribution at distances ~10-times larger than the nanocluster centroid distances measured in STORM samples. By applying photon threshold filtering analysis, we show that it is also possible to detect the mesoscale organization in STORM images.
Subject(s)
Receptors, Nicotinic , Animals , Antibodies, Monoclonal/metabolism , Bungarotoxins , Cell Membrane/metabolism , Mammals/metabolism , Microscopy, Fluorescence/methods , Receptors, Nicotinic/metabolismABSTRACT
Due to the wave nature of light, optical microscopy has a lower-bound lateral resolution limit of approximately half of the wavelength of visible light, that is, within the range of 200 to 350 nm. Fluorescence fluctuation-based super-resolution microscopy (FF-SRM) is a term used to encompass a collection of image analysis techniques that rely on the statistical processing of temporal variations of the fluorescence signal. FF-SRM aims to reduce the uncertainty of the location of fluorophores within an image, often improving spatial resolution by several tens of nanometers. FF-SRM is suitable for live-cell imaging due to its compatibility with most fluorescent probes and relatively simple instrumental and experimental requirements, which are mostly camera-based epifluorescence instruments. Each FF-SRM approach has strengths and weaknesses, which depend directly on the underlying statistical principles through which enhanced spatial resolution is achieved. In this review, the basic concepts and principles behind a range of FF-SRM methods published to date are described. Their operational parameters are explained and guidance for their selection is provided.
Due to light's wave nature, an optical microscope's resolution range is 200 to 350 nanometers. Several techniques enhance resolution; this work encompasses several fluorescence fluctuation super-resolution (FF-SMR) methods capable of achieving nanoscopic scales. FF-SRM is known to be suitable for fixed or live-cell imaging and compatible with most conventional microscope setups found in a laboratory. However, each FF-SRM approach has its strengths and weaknesses, which depend directly on the underlying principles through which enhanced spatial resolution is achieved. Therefore, the basic concepts and principles behind diverse FF-SRM methods are revisited in this review. In addition, their operational parameters are explained, and guidance for their selection is provided for microscopists interested in FF-SRM.
Subject(s)
Fluorescent Dyes , Image Processing, Computer-Assisted , Microscopy, Fluorescence/methodsABSTRACT
Single molecule fluorescence localization microscopy provides molecular localization with a precision in the tens of nanometer range in the plane perpendicular to the light propagation. This opens the possibility to count molecules and correlate their locations, starting from a map of the actual positions in a single molecule super resolution image. Considering molecular pair correlation as an indication of interaction, and a way to discern them from free molecules, we describe a method to calculate thermodynamic equilibrium constants. In this work, we use as a test system two complementary homo-oligonucleotides, one strand marked with Cyanine 3.5 and the other with Alexa Fluor 647. Hybridization is controlled by the amount of each strand, temperature, and the ionic force, and measured in steady state emission. The same samples are examined in Stochastic Optical Reconstruction Microscopy (STORM) experiments with split-field simultaneous two-colour detection. The effect of multiblinking, labelling-detection efficiency, and determination of the critical distance for association are discussed. We consistently determine values in STORM coincident with those of the bulk experiment.
Subject(s)
Nanotechnology , Oligonucleotides , Microscopy, Fluorescence/methodsABSTRACT
The use of a variety of techniques based on super-resolution (SR) microscopy unveiled a close and complex relationship between cytoskeleton reorganization and SOCE. By using SR microscopy many new proteins involved in SOCE regulation have been identified over the last few years. Many enigmas remain unsolved in this highly dynamic field, however, recent developments in SR microscopy promise new answers soon. In the present review, we describe the most relevant findings in SOCE components and SOCE modulation using different methods derived from SR microscopy.
Subject(s)
Calcium , Microscopy , Calcium/metabolism , Calcium Signaling/physiology , ORAI1 Protein/metabolism , Stromal Interaction Molecule 1/metabolismABSTRACT
This paper presents a transfer domain strategy to tackle the limitations of low-resolution thermal sensors and generate higher-resolution images of reasonable quality. The proposed technique employs a CycleGAN architecture and uses a ResNet as an encoder in the generator along with an attention module and a novel loss function. The network is trained on a multi-resolution thermal image dataset acquired with three different thermal sensors. Results report better performance benchmarking results on the 2nd CVPR-PBVS-2021 thermal image super-resolution challenge than state-of-the-art methods. The code of this work is available online.
Subject(s)
Algorithms , Image Processing, Computer-Assisted , Image Processing, Computer-Assisted/methods , Magnetic Resonance Imaging/methodsABSTRACT
Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+ -dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.
Subject(s)
Calcium Channels/metabolism , Spermatozoa/metabolism , cdc42 GTP-Binding Protein/metabolism , Animals , Calcium/metabolism , Calcium Channels/deficiency , Calcium Channels/genetics , Calcium Signaling , Cyclic AMP/metabolism , Female , Fertilization in Vitro , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Signal Transduction , Sperm Capacitation/physiology , Sperm Motility/physiology , Sperm Tail/metabolism , Spermatozoa/drug effects , Spermatozoa/ultrastructure , cdc42 GTP-Binding Protein/antagonists & inhibitorsABSTRACT
The spectrum and incidence of gene fusions in papillary thyroid carcinoma (PTC) can differ significantly depending on the age of onset, histological subtype or radiation exposure history. In sporadic pediatric PTC, RET/PTC1-3 and AGK-BRAF fusions are common genetic alterations. The role of RET/PTC as a prognostic marker in pediatric PTC is still under investigation. We recently showed that AGK-BRAF fusion is prevalent in young patients (mean 10 years) and associated with specific and aggressive pathological features such as multifocality and lung metastasis. In this pilot study, we report a unique patient harboring three different foci: the first was positive for AGK-BRAF fusion, the second was positive for just RET/PTC3 fusion and the third was negative for both rearrangements. To investigate whether AGK-BRAF and RET/PTC3 are associated with genomic instability and chromatin modifications, we performed quantitative fluorescence in situ hybridization (Q-FISH) of telomere repeats followed by 3D imaging analysis and 3D super-resolution Structured Illumination Microscopy (3D-SIM) to analyze the DNA structure from the foci. We demonstrated in this preliminary study that AGK-BRAF is likely associated with higher levels of telomere-related genomic instability and chromatin remodeling in comparison with RET/PTC3 foci. Our results suggest a progressive disruption in chromatin structure in AGK-BRAF-positive cells, which might explain a more aggressive disease outcome in patients harboring this rearrangement.
ABSTRACT
Fluorescence nanoscopy represented a breakthrough for the life sciences as it delivers 20-30 nm resolution using far-field fluorescence microscopes. This resolution limit is not fundamental but imposed by the limited photostability of fluorophores under ambient conditions. This has motivated the development of a second generation of fluorescence nanoscopy methods that aim to deliver sub-10 nm resolution, reaching the typical size of structural proteins and thus providing true molecular resolution. In this review, we present common fundamental aspects of these nanoscopies, discuss the key experimental factors that are necessary to fully exploit their capabilities, and discuss their current and future challenges.
ABSTRACT
We introduce p-MINFLUX, a new implementation of the highly photon-efficient single-molecule localization method with a simplified experimental setup and additional fluorescence lifetime information. In contrast to the original MINFLUX implementation, p-MINFLUX uses interleaved laser pulses to deliver the doughnut-shaped excitation foci at a maximum repetition rate. Using both static and dynamic DNA origami model systems, we demonstrate the performance of p-MINFLUX for single-molecule localization nanoscopy and tracking, respectively. p-MINFLUX delivers 1-2 nm localization precision with 2000-1000 photon counts. In addition, p-MINFLUX gives access to the fluorescence lifetime enabling multiplexing and super-resolved lifetime imaging. p-MINFLUX should help to unlock the full potential of innovative single-molecule localization schemes.
Subject(s)
Nanotechnology , Photons , DNA , Lasers , Microscopy, FluorescenceABSTRACT
Abstract The proficiency of image processing is of extreme importance in perceiving and collecting information from the images, which includes the process of changing or interpreting existing images. In medical image processing, imaging with more accuracy plays a crucial role in better diagnosis or for the posterior analysis of treatment. Magnetic Resonance Imaging (MRI) is a medicinal creative tool for studying the internal structures and functionalities of human brain, knee, heart, liver, etc. Typical MR scans are essential now for better diagnosis but, limited resolution that is often inadequate for extracting detailed and reliable information. So, for the super resolution (SR) of MR brain images concepts of compressive sensing (CS) & fuzzy logical rules to improve data quality are proposed in this paper. Usually, reconstruction of an SR image is the formation of high resolution (HR) image which is obtained from one or few low resolution (LR) images. In the proposed method, with the help of compressive sensing a very limited number of images are considered even though it's a challenging task and fuzzy logical rules for a specific membership function are applied to improve the resolution of the image. To assess the performance of the proposal, different metrics are evaluated and achieved better results.
Subject(s)
Magnetic Resonance Imaging , Fuzzy Logic , Data Compression , Cerebrum/diagnostic imagingABSTRACT
Spectral information provided by multispectral and hyperspectral sensors has a great impact on remote sensing studies, easing the identification of carbonate outcrops that contribute to a better understanding of petroleum reservoirs. Sensors aboard satellites like Landsat series, which have data freely available usually lack the spatial resolution that suborbital sensors have. Many techniques have been developed to improve spatial resolution through data fusion. However, most of them have serious limitations regarding application and scale. Recently Super-Resolution (SR) convolution neural networks have been tested with encouraging results. However, they require large datasets, more time and computational power for training. To overcome these limitations, this work aims to increase the spatial resolution of multispectral bands from the Landsat satellite database using a modified artificial neural network that uses pixel kernels of a single spatial high-resolution RGB image from Google Earth as input. The methodology was validated with a common dataset of indoor images as well as a specific area of Landsat 8. Different downsized scale inputs were used for training where the validation used the ground truth of the original size images, obtaining comparable results to the recent works. With the method validated, we generated high spatial resolution spectral bands based on RGB images from Google Earth on a carbonated outcrop area, which were then properly classified according to the soil spectral responses making use of the advantage of a higher spatial resolution dataset.
ABSTRACT
Single-particle tracking with quantum dots (QDs) constitutes a powerful tool to track the nanoscopic dynamics of individual cell membrane components unveiling their membrane diffusion characteristics. Here, the nano-resolved population dynamics of QDs is exploited to reconstruct the topography and structural changes of the cell membrane surface with high temporal and spatial resolution. For this proof-of-concept study, bright, small, and stable biofunctional QD nanoconstructs are utilized recognizing the endogenous neuronal cannabinoid receptor 1, a highly expressed and fast-diffusing membrane protein, together with a commercial point-localization microscope. Rapid QD diffusion on the axonal plasma membrane of cultured hippocampal neurons allows precise reconstruction of the membrane surface in less than 1 min with a spatial resolution of tens of nanometers. Access of the QD nanoconstructs to the synaptic cleft enables rapid 3D topological reconstruction of the entire presynaptic component. Successful reconstruction of membrane nano-topology and deformation at the second time-scale is also demonstrated for HEK293 cell filopodia and axons. Named "nanoPaint," this super-resolution imaging technique amenable to any endogenous transmembrane target represents a versatile platform to rapidly and accurately reconstruct the cell membrane nano-topography, thereby enabling the study of the rapid dynamic phenomena involved in neuronal membrane plasticity.
Subject(s)
Cell Membrane/chemistry , Imaging, Three-Dimensional , Nanoparticles/chemistry , Nanotechnology/methods , Animals , HEK293 Cells , Humans , Membrane Proteins/metabolism , Quantum DotsABSTRACT
Rotavirus genome replication and assembly take place in cytoplasmic electron dense inclusions termed viroplasms (VPs). Previous conventional optical microscopy studies observing the intracellular distribution of rotavirus proteins and their organization in VPs have lacked molecular-scale spatial resolution, due to inherent spatial resolution constraints. In this work we employed super-resolution microscopy to reveal the nanometric-scale organization of VPs formed during rotavirus infection, and quantitatively describe the structural organization of seven viral proteins within and around the VPs. The observed viral components are spatially organized as five concentric layers, in which NSP5 localizes at the center of the VPs, surrounded by a layer of NSP2 and NSP4 proteins, followed by an intermediate zone comprised of the VP1, VP2, VP6. In the outermost zone, we observed a ring of VP4 and finally a layer of VP7. These findings show that rotavirus VPs are highly organized organelles.
Subject(s)
Epithelial Cells/virology , Rotavirus/growth & development , Viral Proteins/analysis , Virus Replication , Animals , Cell Line , Macaca mulatta , Microscopy, Fluorescence , Spatial AnalysisABSTRACT
The synaptonemal complex is an evolutionarily conserved, supramolecular structure that holds the homologous chromosomes together during the pachytene stage of the first meiotic prophase. Among vertebrates, synaptonemal complex dynamics has been analyzed in mouse spermatocytes following the assembly of its components from leptotene to pachytene stages. With few exceptions, a detailed study of the disassembly of SCs and the behavior of SC components at recombination sites at the onset of diplotene has not been accomplished. Here, we describe for the first time the progressive disassembly of the SC in chicken oocytes during the initial steps of desynapsis using immunolocalization of specific SC proteins and super-resolution microscopy. We found that transverse filament protein SYCP1 and central element component SYCE3 remain associated with the lateral elements at the beginning of chromosomal axis separation. As the separation between lateral elements widens, these proteins eventually disappear, without any evidence of subsequent association. Our observations support the idea that post-translational modifications of the central region components have a role at the initial phases of the SC disassembly. At the crossover sites, signaled by persistent MLH1 foci, the central region proteins are no longer detected when the SYCP3-positive lateral elements are widely separated. These findings are indicative that SC disassembly follows a general pattern along the desynaptic bivalents. The present work shows that the use of avian oocytes at prophase I provides a valuable model to explore the time course and chromosomal localization of SC proteins and its relationship with local changes along meiotic bivalents.
Subject(s)
Chickens/genetics , Microscopy, Confocal , Oocytes/metabolism , Synaptonemal Complex/metabolism , Animals , Biomarkers , Chromosome Segregation , Female , Fluorescent Antibody Technique , Genetic Loci , MeiosisABSTRACT
Neurons are the most asymmetric cell types, with their axons commonly extending over lengths that are thousand times longer than the diameter of the cell soma. Fluorescence nanoscopy has recently unveiled that actin, spectrin and accompanying proteins form a membrane-associated periodic skeleton (MPS) that is ubiquitously present in mature axons from all neuronal types evaluated so far. The MPS is a regular supramolecular protein structure consisting of actin "rings" separated by spectrin tetramer "spacers". Although the MPS is best organized in axons, it is also present in dendrites, dendritic spine necks and thin cellular extensions of non-neuronal cells such as oligodendrocytes and microglia. The unique organization of the actin/spectrin skeleton has raised the hypothesis that it might serve to support the extreme physical and structural conditions that axons must resist during the lifespan of an organism. Another plausible function of the MPS consists of membrane compartmentalization and subsequent organization of protein domains. This review focuses on what we know so far about the structure of the MPS in different neuronal subdomains, its dynamics and the emerging evidence of its impact in axonal biology.
ABSTRACT
Nuclear Epidermal Growth Factor Receptor (EGFR) has been associated with worse prognosis and treatment resistance for several cancer types. After Epidermal Growth Factor (EGF) binding, the ligand-receptor complex can translocate to the nucleus where it functions in oncological processes. By three-dimensional quantification analysis of super-resolution microscopy images, we verified the translocation kinetics of fluorescent conjugated EGF to the nucleus in two mesenchymal cell types: human adipose tissue-derived stem cells (hASC) and SK-HEP-1 tumor cells. The number of EGF clusters in the nucleus does not change after 10â¯min of stimulation with EGF in both cells. The total volume occupied by EGF clusters in the nucleus of hASC also does not change after 10â¯min of stimulation with EGF. However, the total volume of EGF clusters increases only after 20â¯min in SK-HEP-1 cells nuclei. In these cells the nuclear volume occupied by EGF is 3.2 times higher than in hASC after 20â¯min of stimulation, indicating that translocation kinetics of EGF differs between these two cell types. After stimulation, EGF clusters assemble in larger clusters in the cell nucleus in both cell types, which suggests specific sub-nuclear localizations of the receptor. Super-resolution microscopy images show that EGF clusters are widespread in the nucleoplasm, and can be localized in nuclear envelope invaginations, and in the nucleoli. The quantitative study of EGF-EGFR complex translocation to the nucleus may help to unravel its roles in health and pathological conditions, such as cancer.