Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

10.3791/60003 ◽  
2020 ◽  
Author(s):  
Visnja Jevtic ◽  
Petra Kindle ◽  
Sergiy V. Avilov
Keyword(s):  
Time Lapse ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Mitochondrial Nucleoids

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 High-resolution imaging of mitochondria and mitochondrial nucleoids in differentiated SH-SY5Y cells

2021 ◽  
Author(s):  
Emily Annuario ◽  
Kristal Y-W Ng ◽  
Alessio Vagnoni
Keyword(s):  
Cell Function ◽  
Complex Dynamics ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Mitochondrial Nucleoids ◽  
Morphological Transitions ◽  
Mitochondrial Motility ◽  
Mtdna Replication ◽  
Resolution Imaging ◽  
The Relationship

Abstract Mitochondria are highly dynamic organelles which form intricate networks with complex dynamics. Mitochondrial transport and distribution are essential to ensure proper cell function, especially in cells with an extremely polarised morphology such as neurons. A layer of complexity is added when considering mitochondria have their own genome, packaged into nucleoids. Major mitochondrial morphological transitions, for example mitochondrial division, often occur in conjunction with mitochondrial DNA (mtDNA) replication and changes in the dynamic behaviour of the nucleoids. However, the relationship between mtDNA dynamics and mitochondrial motility in the processes of neurons has been largely overlooked. In this chapter, we describe a method for live imaging of mitochondria and nucleoids in differentiated SH-SY5Y cells by instant structured illumination microscopy (iSIM). We also include a detailed protocol for the differentiation of SH-SY5Y cells into cells with a pronounced neuronal-like morphology and show examples of coordinated mitochondrial and nucleoid motility in the long processes of these cells.

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 Multi-color imaging of sub-mitochondrial structures in living cells using structured illumination microscopy

Nanophotonics ◽  
2018 ◽  
Vol 7 (5) ◽  
pp. 935-947 ◽  
Author(s):  
Ida S. Opstad ◽  
Deanna L. Wolfson ◽  
Cristina I. Øie ◽  
Balpreet S. Ahluwalia
Keyword(s):  
Optical Imaging ◽  
Mitochondrial Dynamics ◽  
Imaging Techniques ◽  
Three Dimensional ◽  
Time Lapse ◽  
Living Cells ◽  
Intermembrane Space ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Microscopy Techniques

AbstractThe dimensions of mitochondria are close to the diffraction limit of conventional light microscopy techniques, making the complex internal structures of mitochondria unresolvable. In recent years, new fluorescence-based optical imaging techniques have emerged, which allow for optical imaging below the conventional limit, enabling super-resolution (SR). Possibly the most promising SR and diffraction-limited microscopy techniques for live-cell imaging are structured illumination microscopy (SIM) and deconvolution microscopy (DV), respectively. Both SIM and DV are widefield techniques and therefore provide fast-imaging speed as compared to scanning based microscopy techniques. We have exploited the capabilities of three-dimensional (3D) SIM and 3D DV to investigate different sub-mitochondrial structures in living cells: the outer membrane, the intermembrane space, and the matrix. Using different mitochondrial probes, each of these sub-structures was first investigated individually and then in combination. We describe the challenges associated with simultaneous labeling and SR imaging and the optimized labeling protocol and imaging conditions to obtain simultaneous three-color SR imaging of multiple mitochondrial regions in living cells. To investigate both mitochondrial dynamics and structural details in the same cell, the combined usage of DV for long-term time-lapse imaging and 3D SIM for detailed, selected time point analysis was a useful strategy.

scholarly journals Superresolution microscopy of the β-carboxysome reveals a homogeneous matrix

2017 ◽  
Vol 28 (20) ◽  
pp. 2734-2745 ◽  
Author(s):  
Matthew J. Niederhuber ◽  
Talley J. Lambert ◽  
Clarence Yapp ◽  
Pamela A. Silver ◽  
Jessica K. Polka
Keyword(s):  
Carbon Fixation ◽  
Three Dimensional ◽  
Population Level ◽  
Automated Analysis ◽  
Time Lapse ◽  
Wide Field ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Bisphosphate Carboxylase ◽  
Shell Protein

Carbon fixation in cyanobacteria makes a major contribution to the global carbon cycle. The cyanobacterial carboxysome is a proteinaceous microcompartment that protects and concentrates the carbon-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in a paracrystalline lattice, making it possible for these organisms to fix CO2 from the atmosphere. The protein responsible for the organization of this lattice in beta-type carboxysomes of the freshwater cyanobacterium Synechococcus elongatus, CcmM, occurs in two isoforms thought to localize differentially within the carboxysome matrix. Here we use wide-field time-lapse and three-dimensional structured illumination microscopy (3D-SIM) to study the recruitment and localization of these two isoforms. We demonstrate that this superresolution technique is capable of distinguishing the localizations of the outer protein shell of the carboxysome and its internal cargo. We develop an automated analysis pipeline to analyze and quantify 3D-SIM images and generate a population-level description of the carboxysome shell protein, RuBisCO, and CcmM isoform localization. We find that both CcmM isoforms have similar spatial and temporal localization, prompting a revised model of the internal arrangement of the β-carboxysome.

scholarly journals Constriction rate modulation can drive cell size control and homeostasis inC. crescentus

2018 ◽  
Author(s):  
Ambroise Lambert ◽  
Aster Vanhecke ◽  
Anna Archetti ◽  
Seamus Holden ◽  
Felix Schaber ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Size ◽  
Caulobacter Crescentus ◽  
Size Control ◽  
Time Lapse ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Daughter Cells ◽  
Division Site ◽  
Cell Size Control

AbstractRod-shaped bacteria typically grow first via sporadic and dispersed elongation along their lateral walls, then via a combination of zonal elongation and constriction at the division site to form the poles of daughter cells. Although constriction comprises up to half of the cell cycle, its impact on cell size control and homeostasis has rarely been considered. To reveal the roles of cell elongation and constriction in bacterial size regulation during cell division, we captured the shape dynamics ofCaulobacter crescentuswith time-lapse structured illumination microscopy and used molecular markers as cell-cycle landmarks. We perturbed constriction rate using a hyperconstriction mutant or fosfomycin inhibition. We report that constriction rate contributes to both size control and homeostasis, by determining elongation during constriction, and by compensating for variation in pre-constriction elongation on a single-cell basis.

scholarly journals Rapid 3D enhanced resolution microscopy reveals the dynamics of cortical dendritic spinules

2019 ◽  
Author(s):  
CR Zaccard ◽  
K Myczek ◽  
MD Martin-de-Saavedra ◽  
P Penzes
Keyword(s):  
Pyramidal Neurons ◽  
Super Resolution ◽  
Time Lapse ◽  
Fast Time ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Fixed Tissue ◽  
Volumetric Imaging ◽  
Enhanced Resolution ◽  
Cortical Pyramidal Neurons

SummaryDendritic spinules are thin, membranous protrusions formed by neuronal dendritic spines that are not adequately resolved by diffraction-limited light microscopy. Hence, our understanding of spinules is inferred predominantly from fixed-tissue electron microscopy (EM). Super-resolution modalities have enabled live-cell nanoscopic imaging, but their utility for fast, time-lapse, volumetric imaging has been restricted. Herein, we utilized rapid structured illumination microscopy (SIM) and ‘enhanced resolution’ confocal microscopy to study spatiotemporal spinule dynamics in live cultured cortical pyramidal neurons. Spinules on mushroom spines typically recurred at the same topographical locations and most were short-lived, originating near simple post-synaptic densities (PSDs), while a subset was long-lived and elongated, emerging from complex PSDs. Ca2+ puncta within spinules synchronized with spine head transients and Ca2+ depletion drastically decreased spinule number. Moreover, we uncovered evidence of differential Ca2+-mediated regulation of short-lived and long-lived spinules. Thus, we identified unique spinule classes divergent in lifespan, dynamics, morphology, relationship to the PSD, and regulation. These data suggest distinct synaptic functions of spinule classes, informing future studies, while demonstrating a new application for enhanced resolution microscopy.

scholarly journals Cellular Architecture Mediates DivIVA Ultrastructure and Regulates Min Activity in Bacillus subtilis

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Prahathees Eswaramoorthy ◽  
Marcella L. Erb ◽  
James A. Gregory ◽  
Jared Silverman ◽  
Kit Pogliano ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Spatial Organization ◽  
Time Lapse ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Content Type ◽  
Temporal Regulation ◽  
Min Proteins

ABSTRACTThe assembly of the cell division machinery at midcell is a critical step of cytokinesis. Many rod-shaped bacteria position septa using nucleoid occlusion, which prevents division over the chromosome, and the Min system, which prevents division near the poles. Here we examined thein vivoassembly of theBacillus subtilisMinCD targeting proteins DivIVA, a peripheral membrane protein that preferentially localizes to negatively curved membranes and resembles eukaryotic tropomyosins, and MinJ, which recruits MinCD to DivIVA. We used structured illumination microscopy to demonstrate that both DivIVA and MinJ localize as double rings that flank the septum and first appear early in septal biosynthesis. The subsequent recruitment of MinCD to these double rings would separate the Min proteins from their target, FtsZ, spatially regulating Min activity and allowing continued cell division. Curvature-based localization would also provide temporal regulation, since DivIVA and the Min proteins would localize to midcell after the onset of division. We use time-lapse microscopy and fluorescence recovery after photobleaching to demonstrate that DivIVA rings are highly stable and are constructed from newly synthesized DivIVA molecules. After cell division, DivIVA rings appear to collapse into patches at the rounded cell poles of separated cells, with little or no incorporation of newly synthesized subunits. Thus, changes in cell architecture mediate both the initial recruitment of DivIVA to sites of cell division and the subsequent collapse of these rings into patches (or rings of smaller diameter), while curvature-based localization of DivIVA spatially and temporally regulates Min activity.IMPORTANCEThe Min systems ofEscherichia coliandBacillus subtilisboth inhibit FtsZ assembly, but one key difference between these two species is that whereas theE. coliMin proteins localize to the poles, theB. subtilisproteins localize to nascent division sites by interaction with DivIVA and MinJ. It is unclear how MinC activity at midcell is regulated to prevent it from interfering with FtsZ engaged in medial cell division. We used superresolution microscopy to demonstrate that DivIVA and MinJ, which localize MinCD, assemble double rings that flank active division sites and septa. This curvature-based localization mechanism holds MinCD away from the FtsZ ring at midcell, and we propose that this spatial organization is the primary mechanism by which MinC activity is regulated to allow division at midcell. Curvature-based localization also conveys temporal regulation, since it ensures that MinC localizes after the onset of division.

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