scholarly journals Efficient and Accurate Synapse Detection With Selective Structured Illumination Microscopy on the Putative Regions of Interest of Ultrathin Serial Sections

2021 ◽  
Vol 15 ◽  
Author(s):  
Gyeong Tae Kim ◽  
Sangkyu Bahn ◽  
Nari Kim ◽  
Joon Ho Choi ◽  
Jinseop S. Kim ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Neural Circuit ◽  
Molecular Characteristics ◽  
Imaging Method ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Gold Standard Method ◽  
Array Tomography ◽  
Synapse Detection

Critical determinants of synaptic functions include subcellular locations, input sources, and specific molecular characteristics. However, there is not yet a reliable and efficient method that can detect synapses. Electron microscopy is a gold-standard method to detect synapses due to its exceedingly high spatial resolution. However, it requires laborious and time-consuming sample preparation and lengthy imaging time with limited labeling methods. Recent advances in various fluorescence microscopy methods have highlighted fluorescence microscopy as a substitute for electron microscopy in reliable synapse detection in a large volume of neural circuits. In particular, array tomography has been verified as a useful tool for neural circuit reconstruction. To further improve array tomography, we developed a novel imaging method, called “structured illumination microscopy on the putative region of interest on ultrathin sections”, which enables efficient and accurate detection of synapses-of-interest. Briefly, based on low-magnification conventional fluorescence microscopy images, synapse candidacy was determined. Subsequently, the coordinates of the regions with candidate synapses were imaged using super-resolution structured illumination microscopy. Using this system, synapses from the high-order thalamic nucleus, the posterior medial nucleus in the barrel cortex were rapidly and accurately imaged.

scholarly journals Three-dimensional residual channel attention networks denoise and sharpen fluorescence microscopy image volumes

2020 ◽  
Author(s):  
Jiji Chen ◽  
Hideki Sasaki ◽  
Hoyin Lai ◽  
Yijun Su ◽  
Jiamin Liu ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Network Performance ◽  
Resolution Enhancement ◽  
Three Dimensional ◽  
Ground Truth ◽  
Time Lapse ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Attention Networks

Abstract We demonstrate residual channel attention networks (RCAN) for restoring and enhancing volumetric time-lapse (4D) fluorescence microscopy data. First, we modify RCAN to handle image volumes, showing that our network enables denoising competitive with three other state-of-the-art neural networks. We use RCAN to restore noisy 4D super-resolution data, enabling image capture over tens of thousands of images (thousands of volumes) without apparent photobleaching. Second, using simulations we show that RCAN enables class-leading resolution enhancement, superior to other networks. Third, we exploit RCAN for denoising and resolution improvement in confocal microscopy, enabling ~2.5-fold lateral resolution enhancement using stimulated emission depletion (STED) microscopy ground truth. Fourth, we develop methods to improve spatial resolution in structured illumination microscopy using expansion microscopy ground truth, achieving improvements of ~1.4-fold laterally and ~3.4-fold axially. Finally, we characterize the limits of denoising and resolution enhancement, suggesting practical benchmarks for evaluating and further enhancing network performance.

scholarly journals Total Internal Reflection Fluorescence Microscopy (TIRFM) – novel techniques and applications

2020 ◽  
Vol 8 (11) ◽  
Author(s):  
Verena Richter ◽  
Michael Wagner ◽  
Herbert Schneckenburger
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Total Internal Reflection ◽  
Plasma Membranes ◽  
Focal Adhesions ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Thin Layers ◽  
Structured Illumination ◽  
Structured Illumination Microscopy

Total Internal Reflection Fluorescence Microscopy (TIRFM) has been established almost 40 years ago for studies of plasma membranes or membrane proximal sites of living cells. The method is based on light incidence at an angle above the critical angle of total internal reflection and generation of an evanescent electromagnetic field penetrating about 100 nm into a sample and permitting selective excitation of membrane proximal fluorophores. Two techniques are presented here: prism-type TIRFM and objective-type TIRFM with high aperture microscope objective lenses. Furthermore, numerous applications are summarized, e.g. measurement of focal adhesions, cell-substrate topology, endocytosis or exocytosis of vesicles as well as single molecule detection within thin layers. Finally, highly innovative combinations of TIRFM with Förster Resonance Energy Transfer (FRET) measurements as well as with Structured Illumination Microscopy (SIM) and fluorescence reader technologies are presented.

scholarly journals Automated Synapse Detection and Validation by Correlated Array Tomography and Scanning Electron Microscopy

2014 ◽  
Vol 106 (2) ◽  
pp. 399a
Author(s):  
David Lenzi ◽  
Juan G. Cueva ◽  
Nenad Amodaj ◽  
Richard J. Weinberg ◽  
Jay K. Trautman
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Array Tomography ◽  
Synapse Detection ◽  
Scanning Electron

scholarly journals Resolving the internal morphology of core–shell microgels with super-resolution fluorescence microscopy

2020 ◽  
Vol 2 (1) ◽  
pp. 323-331 ◽  
Author(s):  
Pia Otto ◽  
Stephan Bergmann ◽  
Alice Sandmeyer ◽  
Maxim Dirksen ◽  
Oliver Wrede ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Internal Structure ◽  
Single Molecule ◽  
Fluorescent Dyes ◽  
Super Resolution ◽  
Core Shell ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Internal Morphology

We investigate the internal structure of smart core–shell microgels by super-resolution fluorescence microscopy by combining of 3D single molecule localization and structured illumination microscopy using freely diffusing fluorescent dyes.

scholarly journals Alpha-Granules in Resting Human Platelets Are a Spatially Clustered, Single Major Population

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 865-865
Author(s):  
Irina D Pokrovskaya ◽  
Brian Storrie ◽  
Shilpi Yadav ◽  
Amith Rao ◽  
Emma McBride ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Compositional Data ◽  
Conflicts Of Interest ◽  
Spatial Clustering ◽  
Spatial Arrangement ◽  
Human Platelets ◽  
Immunogold Electron Microscopy ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Dense Granules

Abstract We interrogated in 3D-space the arrangement of alpha-granules and their proteins within 30 human platelets in order to address two central structure/function questions. First, are alpha-granules based on their size, shape and protein content a single population or not, and second, what implications could organelle frequency and organization within the resting platelet have for platelet biogenesis and membrane fusion events in activated platelets? We used serial block face scanning electron microscopy (SBF-SEM) to render in full platelet volume the ultrastructure of alpha-granules, dense granules, mitochondria, canalicular system (CS), and plasma membrane (PM) in 30 platelets, 10 each from 3 donors. Size and shape were measured for 1488 a-granules. Compositional data were assessed for multiple proteins over hundreds of granules by 3D-structured illumination microscopy (SIM) and serial section cryo-immunogold electron microscopy. Data analysis led to 3 conclusions: 1) Based on size, shape and protein composition, there was one major population of alpha-granules in resting human platelets, 2) Alpha-granules clustered tightly together while dense granules were more peripherally located and distal from one another suggesting a spatial arrangement that in the one case supports compound granule fusion and in the other case rapid fusion with the PM, and 3) Based on the weak relationship between platelet size and organelle number and volume, we inferred that platelet biogenesis is likely relatively imprecise with alpha-granule inclusion being more precisely metered than that of other organelles,. These results provide a strong, informative baseline for a-granule structural properties and suggest a spatial clustering of organelles within the resting platelet that may be functionally significant during platelet activation. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Three-dimensional residual channel attention networks denoise and sharpen fluorescence microscopy image volumes

2020 ◽  
Author(s):  
Jiji Chen ◽  
Hideki Sasaki ◽  
Hoyin Lai ◽  
Yijun Su ◽  
Jiamin Liu ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Network Performance ◽  
Resolution Enhancement ◽  
Three Dimensional ◽  
Ground Truth ◽  
Time Lapse ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Attention Networks

AbstractWe demonstrate residual channel attention networks (RCAN) for restoring and enhancing volumetric time-lapse (4D) fluorescence microscopy data. First, we modify RCAN to handle image volumes, showing that our network enables denoising competitive with three other state-of-the-art neural networks. We use RCAN to restore noisy 4D super-resolution data, enabling image capture over tens of thousands of images (thousands of volumes) without apparent photobleaching. Second, using simulations we show that RCAN enables class-leading resolution enhancement, superior to other networks. Third, we exploit RCAN for denoising and resolution improvement in confocal microscopy, enabling ∼2.5-fold lateral resolution enhancement using stimulated emission depletion (STED) microscopy ground truth. Fourth, we develop methods to improve spatial resolution in structured illumination microscopy using expansion microscopy ground truth, achieving improvements of ∼1.4-fold laterally and ∼3.4-fold axially. Finally, we characterize the limits of denoising and resolution enhancement, suggesting practical benchmarks for evaluating and further enhancing network performance.

scholarly journals Chlamydomonas FAP70 is a component of the previously uncharacterized ciliary central apparatus projection C2a

2021 ◽  
Author(s):  
Yuqing Hou ◽  
Lei Zhao ◽  
Tomohiro Kubo ◽  
Xi Cheng ◽  
Nathan McNeill ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Chlamydomonas Reinhardtii ◽  
Model Organism ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Cryo Electron Microscopy ◽  
N Terminus ◽  
Central Apparatus

Cilia are essential organelles required for cell signaling and motility. Nearly all motile cilia have a “9+2” axoneme composed of 9 outer doublet microtubules plus 2 central microtubules; the central microtubules together with their projections is termed the central apparatus (CA). In Chlamydomonas reinhardtii, a model organism for studying cilia, 30 proteins are known CA components, and ∼36 more are predicted to be CA proteins. Among the candidate CA proteins is the highly conserved FAP70, which also has been reported to be associated with the doublet microtubules. Here we determined by super-resolution structured illumination microscopy that FAP70 is located exclusively in the CA, and show by cryo-electron microscopy that its N-terminus is located at the base of the CA's C2a projection. We also found that fap70-1 mutant axonemes lack most of the C2a projection. Mass spectrometry revealed that fap70-1 axonemes lack not only FAP70 but two other conserved candidate CA proteins, FAP65 and FAP147. Finally, FAP65 and FAP147 co-immunoprecipitated with HA-tagged FAP70. Taken together, these data identify FAP70, FAP65, and FAP147 as the first defining components of the C2a projection.

scholarly journals OPA1 and MICOS Regulate mitochondrial crista dynamics and formation

2020 ◽  
Vol 11 (10) ◽  
Author(s):  
Chao Hu ◽  
Li Shu ◽  
Xiaoshuai Huang ◽  
Jianglong Yu ◽  
liuju Li ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Morphological Changes ◽  
Ion Beam ◽  
Tomographic Reconstruction ◽  
Three Dimensional ◽  
Living Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Main Site

Abstract Mitochondrial cristae are the main site for oxidative phosphorylation, which is critical for cellular energy production. Upon different physiological or pathological stresses, mitochondrial cristae undergo remodeling to reprogram mitochondrial function. However, how mitochondrial cristae are formed, maintained, and remolded is still largely unknown due to the technical challenges of tracking mitochondrial crista dynamics in living cells. Here, using live-cell Hessian structured illumination microscopy combined with transmission electron microscopy, focused ion beam/scanning electron microscopy, and three-dimensional tomographic reconstruction, we show, in living cells, that mitochondrial cristae are highly dynamic and undergo morphological changes, including elongation, shortening, fusion, division, and detachment from the mitochondrial inner boundary membrane (IBM). In addition, we find that OPA1, Yme1L, MICOS, and Sam50, along with the newly identified crista regulator ATAD3A, control mitochondrial crista dynamics. Furthermore, we discover two new types of mitochondrial crista in dysfunctional mitochondria, “cut-through crista” and “spherical crista”, which are formed due to incomplete mitochondrial fusion and dysfunction of the MICOS complex. Interestingly, cut-through crista can convert to “lamellar crista”. Overall, we provide a direct link between mitochondrial crista formation and mitochondrial crista dynamics.

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