scholarly journals Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Viktoria Liss ◽  
Britta Barlag ◽  
Monika Nietschke ◽  
Michael Hensel
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Super Resolution ◽  
Super Resolution Microscopy

scholarly journals Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

2020 ◽  
Author(s):  
Fabian U. Zwettler ◽  
Sebastian Reinhard ◽  
Davide Gambarotto ◽  
Toby D. M. Bell ◽  
Virginie Hamel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Super Resolution ◽  
Reference Structure ◽  
Positional Error ◽  
Localization Microscopy ◽  
Resolution Imaging ◽  
Endogenous Proteins ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractExpansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining expansion microscopy (ExM) with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

scholarly journals A molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei

2017 ◽  
Author(s):  
Anneliese Hoffmann ◽  
Sandro Käser ◽  
Martin Jakob ◽  
Simona Amodeo ◽  
Camille Peitsch ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mitochondrial Genome ◽  
Trypanosoma Brucei ◽  
De Novo ◽  
Molecular Model ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Kinetoplast Dna ◽  
Exclusion Zone ◽  
Super Resolution Microscopy

AbstractIn almost all eukaryotes mitochondria maintain their own genome. Despite the discovery more than 50 years ago still very little is known about how the genome is properly segregated during cell division. The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure proper segregation of the mitochondrial genome via the basal bodies movement, during cell cycle. Using super-resolution microscopy we precisely localize each of the currently known unique TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum towards the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on biochemical analysis the TAC consists of several non-overlapping subcomplexes suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC has an impact on mitochondrial organelle positioning however is not required for proper organelle biogenesis or segregation.Significance StatementMitochondrial genome replication and segregation are essential processes in most eukaryotic cells. While replication has been studied in some detail much less is known about the molecular machinery required distribute the replicated genomes. Using super-resolution microscopy in combination with molecular biology and biochemistry we show for the first time in which order the segregation machinery is assembled and that it is assembled de novo rather than in a semi conservative fashion in the single celled parasite Trypanosoma brucei. Furthermore, we demonstrate that the mitochondrial genome itself is not required for assembly to occur. It seems that the physical connection of the mitochondrial genome to cytoskeletal elements is a conserved feature in most eukaryotes, however the molecular components are highly diverse.Abbreviation(EZF)Exclusion zone filaments(ULF)Unilateral filament(TAC)tripartite attachment complex(OM)outer mitochondrial(IM)inner mitochondrial(BSF)bloodstream form(PCF)procyclic form(kDNA)kinetoplast DNA(gRNA)guide RNA(SBFSEM)Serial block face-scanning electron microscopy(Tet)tetracyclin(STED)Stimulated Emission Depletion

scholarly journals Graphene-Enabled Electron Microscopy and Correlated Super-Resolution Microscopy of Wet Cells

2016 ◽  
Vol 110 (3) ◽  
pp. 155a
Author(s):  
Michal Wojcik ◽  
Margaret Hauser ◽  
Wan Li ◽  
Seonah Moon ◽  
Ke Xu
Keyword(s):  
Electron Microscopy ◽  
Super Resolution ◽  
Super Resolution Microscopy

Detection by fluorescence microscopy of N-aminopeptidases in bacteria using an ICT sensor with multiphoton excitation: Usefulness for super-resolution microscopy

2020 ◽  
Vol 321 ◽  
pp. 128487
Author(s):  
Javier Valverde-Pozo ◽  
Jose M. Paredes ◽  
Carmen Salto-Giron ◽  
Pilar Herrero-Foncubierta ◽  
María D. Giron ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Super Resolution ◽  
Multiphoton Excitation ◽  
Super Resolution Microscopy

scholarly journals Imaging the kidney: from light to super-resolution microscopy

2019 ◽  
Vol 36 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Maria Lucia Angelotti ◽  
Giulia Antonelli ◽  
Carolina Conte ◽  
Paola Romagnani
Keyword(s):  
Electron Microscopy ◽  
Kidney Diseases ◽  
Imaging Techniques ◽  
Imaging Mass Spectrometry ◽  
Multiphoton Microscopy ◽  
Three Dimensional ◽  
Super Resolution ◽  
Classification Systems ◽  
Fluorescence Lifetime Imaging ◽  
Super Resolution Microscopy

Abstract The important achievements in kidney physiological and pathophysiological mechanisms can largely be ascribed to progress in the technology of microscopy. Much of what we know about the architecture of the kidney is based on the fundamental descriptions of anatomic microscopists using light microscopy and later by ultrastructural analysis provided by electron microscopy. These two techniques were used for the first classification systems of kidney diseases and for their constant updates. More recently, a series of novel imaging techniques added the analysis in further dimensions of time and space. Confocal microscopy allowed us to sequentially visualize optical sections along the z-axis and the availability of specific analysis software provided a three-dimensional rendering of thicker tissue specimens. Multiphoton microscopy permitted us to simultaneously investigate kidney function and structure in real time. Fluorescence-lifetime imaging microscopy allowed to study the spatial distribution of metabolites. Super-resolution microscopy increased sensitivity and resolution up to nanoscale levels. With cryo-electron microscopy, researchers could visualize the individual biomolecules at atomic levels directly in the tissues and understand their interaction at subcellular levels. Finally, matrix-assisted laser desorption/ionization imaging mass spectrometry permitted the measuring of hundreds of different molecules at the same time on tissue sections at high resolution. This review provides an overview of available kidney imaging strategies, with a focus on the possible impact of the most recent technical improvements.

scholarly journals Highly biocompatible super-resolution fluorescence imaging using the fast photoswitching fluorescent protein Kohinoor and SPoD-ExPAN with L p-regularized image reconstruction

Microscopy ◽  
2018 ◽  
Vol 67 (2) ◽  
pp. 89-98
Author(s):  
Tetsuichi Wazawa ◽  
Yoshiyuki Arai ◽  
Yoshinobu Kawahara ◽  
Hiroki Takauchi ◽  
Takashi Washio ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Time Lapse ◽  
Live Cells ◽  
Polarization Angle ◽  
Microscopy Technique ◽  
Present Technique ◽  
Super Resolution Microscopy ◽  
Power Densities

Abstract Far-field super-resolution fluorescence microscopy has enabled us to visualize live cells in great detail and with an unprecedented resolution. However, the techniques developed thus far have required high-power illumination (102–106 W/cm2), which leads to considerable phototoxicity to live cells and hampers time-lapse observation of the cells. In this study we show a highly biocompatible super-resolution microscopy technique that requires a very low-power illumination. The present technique combines a fast photoswitchable fluorescent protein, Kohinoor, with SPoD-ExPAN (super-resolution by polarization demodulation/excitation polarization angle narrowing). With this technique, we successfully observed Kohinoor-fusion proteins involving vimentin, paxillin, histone and clathrin expressed in HeLa cells at a spatial resolution of 70–80 nm with illumination power densities as low as ~1 W/cm2 for both excitation and photoswitching. Furthermore, although the previous SPoD-ExPAN technique used L1-regularized maximum-likelihood calculations to reconstruct super-resolved images, we devised an extension to the Lp-regularization to obtain super-resolved images that more accurately describe objects at the specimen plane. Thus, the present technique would significantly extend the applicability of super-resolution fluorescence microscopy for live-cell imaging.

scholarly journals Surface marker expression in small and medium/large mesenchymal stromal cell-derived extracellular vesicles in naive or apoptotic condition using orthogonal techniques

2021 ◽  
Author(s):  
Renata Skovronova ◽  
Cristina Grange ◽  
Veronica Dimuccio ◽  
Maria Chiara Deregibus ◽  
Giovanni Camussi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Extracellular Vesicles ◽  
Super Resolution ◽  
Analytical Techniques ◽  
Surface Marker ◽  
Bulk Analysis ◽  
Surface Marker Expression ◽  
Marker Expression ◽  
Super Resolution Microscopy ◽  
Single Vesicle

Extracellular vesicles released by mesenchymal stromal cells (MSC EVs) are a promising resource for regenerative medicine. In particular, small MSC EVs represent the active EV fraction for therapeutic applications. A bulk analysis is applied to characterize MSC EVs identity and purity, coupled with the assessment of single EV morphology, size and integrity using electron microscopy. We here applied different orthogonal methods to provide a quantitative analysis of size and surface marker expression in medium/large and small fractions, namely 10k and 100k fractions, of MSC EVs obtained by sequential ultracentrifugations. Bone marrow, adipose tissue, and umbilical cord MSC EVs were compared, in naive and apoptotic conditions. The 100k EV size <100 nm, as detected by electron microscopy, was confirmed by super-resolution microscopy and ExoView. Quantitative single vesicle imaging using super-resolution microscopy revealed heterogeneous patterns of tetraspanin expressions, being all MSC EV fractions single, double and triple positive, in variable proportions, for CD63, CD81 and CD9. Moreover, ExoView analysis allowed a comparative multiplex screening of single MSC EV tetraspanin and mesenchymal marker levels. Finally, a semiquantitative bead based cytofluorimetric analysis showed the segregation of immunological and pro-coagulative markers on the 10k MSC EV fraction. Apoptotic MSC EVs were released in higher number, without significant differences from the naive fractions in surface marker expression. These results indicate that a consistent profile of MSC EV fractions among the different MSC sources, and a safer profile of the 100k MSC EV population for clinical application. Finally, our study identified suitable applications for different EV analytical techniques.

scholarly journals Surface Marker Expression in Small and Medium/Large Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Naive or Apoptotic Condition Using Orthogonal Techniques

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2948
Author(s):  
Renata Skovronova ◽  
Cristina Grange ◽  
Veronica Dimuccio ◽  
Maria Chiara Deregibus ◽  
Giovanni Camussi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Extracellular Vesicles ◽  
Super Resolution ◽  
Analytical Techniques ◽  
Surface Marker ◽  
Bulk Analysis ◽  
Surface Marker Expression ◽  
Marker Expression ◽  
Super Resolution Microscopy ◽  
Different Sources

Extracellular vesicles released by mesenchymal stromal cells (MSC-EVs) are a promising resource for regenerative medicine. Small MSC-EVs represent the active EV fraction. A bulk analysis was applied to characterise MSC-EVs’ identity and purity, with the assessment of single EV morphology, size and integrity using electron microscopy. We applied different methods to quantitatively analyse the size and surface marker expression in medium/large and small fractions, namely 10k and 100k fractions, of MSC-EVs obtained using sequential ultracentrifugation. Bone marrow, adipose tissue and umbilical cord MSC-EVs were compared in naive and apoptotic conditions. As detected by electron microscopy, the 100k EV size < 100 nm was confirmed by super-resolution microscopy and ExoView. Single-vesicle imaging using super-resolution microscopy revealed heterogeneous patterns of tetraspanins. ExoView allowed a comparative screening of single MSC-EV tetraspanin and mesenchymal markers. A semiquantitative bead-based cytofluorimetric analysis showed the segregation of immunological and pro-coagulative markers on the 10k MSC-EVs. Apoptotic MSC-EVs were released in higher numbers, without significant differences in the naive fractions in surface marker expression. These results show a consistent profile of MSC-EV fractions among the different sources and a safer profile of the 100k MSC-EV population for clinical application. Our study identified suitable applications for EV analytical techniques.

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