scholarly journals Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Errin Johnson ◽  
Elena Seiradake ◽  
E. Yvonne Jones ◽  
Ilan Davis ◽  
Kay Grünewald ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Freeze Substitution ◽  
Microscopy Imaging ◽  
Localization Accuracy ◽  
Culture Cells ◽  
Structural Preservation ◽  
Key Aspects

Abstract We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. We identified key aspects of the sample preparation procedure of high pressure freezing, freeze substitution and resin embedding that are critical for preserving fluorescence and photo-switching of standard fluorescent proteins, such as mGFP, mVenus and mRuby2. This enabled us to combine single molecule localization microscopy with transmission electron microscopy imaging of standard fluorescent proteins in cryo-fixed resin embedded cells. We achieved a structural resolution of 40–50 nm (~17 nm average single molecule localization accuracy) in the fluorescence images without the use of chemical fixation or special fluorophores. Using this approach enabled the correlation of fluorescently labeled structures to the ultrastructure in the same cell at the nanometer level and superior structural preservation.

scholarly journals Precision of fiducial marker alignment for correlative super‐resolution fluorescence and transmission electron microscopy

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Adeeba Fathima ◽  
César Augusto Quintana-Cataño ◽  
Christoph Heintze ◽  
Michael Schlierf
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Specific Protein ◽  
Fiducial Markers ◽  
Transmission Electron ◽  
Microscopy Techniques

AbstractRecent advances in microscopy techniques enabled nanoscale discoveries in biology. In particular, electron microscopy reveals important cellular structures with nanometer resolution, yet it is hard, and sometimes impossible to resolve specific protein localizations. Super-resolution fluorescence microscopy techniques developed over the recent years allow for protein-specific localization with ~ 20 nm precision are overcoming this limitation, yet it remains challenging to place those in cells without a reference frame. Correlative light and electron microscopy (CLEM) approaches have been developed to place the fluorescence image in the context of a cellular structure. However, combining imaging methods such as super resolution microscopy and transmission electron microscopy necessitates a correlation using fiducial markers to locate the fluorescence on the structures visible in electron microscopy, with a measurable precision. Here, we investigated different fiducial markers for super-resolution CLEM (sCLEM) by evaluating their shape, intensity, stability and compatibility with photoactivatable fluorescent proteins as well as the electron density. We further carefully determined limitations of correlation accuracy. We found that spectrally-shifted FluoSpheres are well suited as fiducial markers for correlating single-molecule localization microscopy with transmission electron microscopy.

scholarly journals Erratum: Corrigendum: Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Errin Johnson ◽  
Elena Seiradake ◽  
E. Yvonne Jones ◽  
Ilan Davis ◽  
Kay Grünewald ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Proteins ◽  
Super Resolution

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.

Electron microscopy of rapidly frozen lungs: evaluation on the basis of standard criteria

1982 ◽  
Vol 53 (2) ◽  
pp. 516-527 ◽  
Author(s):  
E. R. Weibel ◽  
W. Limacher ◽  
H. Bachofen
Keyword(s):  
Electron Microscopy ◽  
Lung Tissue ◽  
Wide Spectrum ◽  
Freeze Substitution ◽  
Internal Standards ◽  
Tissue Samples ◽  
Physiological Conditions ◽  
Good Preservation ◽  
Structural Preservation ◽  
Narrow Area

In recent years there has been a debate about the validity of the various methods for fixing lung tissue for electron microscopy in a state that faithfully reflects the physiological conditions prevailing at the time of fixation. Mazzone et al. (J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 45: 32513;333, 1978) introduced a method of rapid freezing followed by freeze-substitution fixation and found good preservation of fine structure; they claimed this method to be superior to others because it allowed careful control of physiological conditions. We have tested the suitability of this method for morphometric studies, where random sampling requires homogeneity of tissue preservation. The results are discussed on the basis of some standard criteria for the faithfulness of structural preservation. In terms of external standards, it was confirmed that one can find parts of the lung tissue samples to show a picture that is compatible with the established equivalent image of eucaryotic cells and tissues; however, the structure of blood was poorly preserved. In terms of the internal standards the method was found to yield inconsistent results; the specimens showed a wide spectrum of images of alveolar septa and capillaries, with “good preservation” limited to a very narrow area; furthermore, the method has a low level of reproducibility. We conclude that for the lung the method of freeze-substitution fixation is not suitable for morphometric work.

scholarly journals Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Maarten W. Tuijtel ◽  
Abraham J. Koster ◽  
Stefan Jakobs ◽  
Frank G. A. Faas ◽  
Thomas H. Sharp
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution

scholarly journals Choosing the right label for single-molecule tracking in live bacteria: Side-by-side comparison of photoactivatable fluorescent protein and Halo tag dyes

2018 ◽  
Author(s):  
Nehir Banaz ◽  
Jarno Mäkelä ◽  
Stephan Uphoff
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Single Molecule Tracking ◽  
Advantages And Disadvantages ◽  
Fluorescent Ligands ◽  
The Right

AbstractVisualizing and quantifying molecular motion and interactions inside living cells provides crucial insight into the mechanisms underlying cell function. This has been achieved by super-resolution localization microscopy and single-molecule tracking in conjunction with photoactivatable fluorescent proteins. An alternative labelling approach relies on genetically-encoded protein tags with cell-permeable fluorescent ligands which are brighter and less prone to photobleaching than fluorescent proteins but require a laborious labelling process. Either labelling method is associated with significant advantages and disadvantages that should be taken into consideration depending on the microscopy experiment planned. Here, we describe an optimised procedure for labelling Halo-tagged proteins in live Escherichia coli cells. We provide a side-by-side comparison of Halo tag with different fluorescent ligands against the popular photoactivatable fluorescent protein PAmCherry. Using test proteins with different intracellular dynamics, we evaluated fluorescence intensity, background, photostability, and single-molecule localization and tracking results. Capitalising on the brightness and extended spectral range of fluorescent Halo ligands, we also demonstrate high-speed and dual-colour single-molecule tracking.

scholarly journals Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo

2017 ◽  
Vol 199 (2) ◽  
pp. 120-131 ◽  
Author(s):  
Christopher J. Peddie ◽  
Marie-Charlotte Domart ◽  
Xenia Snetkov ◽  
Peter O'Toole ◽  
Banafshe Larijani ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Fluorescence And Electron Microscopy

scholarly journals A New Solution of Non-integrated Correlative Light and Electron Microscopy Based on High-vacuum Optical Platform

2016 ◽  
Vol 22 (S3) ◽  
pp. 248-249
Author(s):  
Shuoguo Li ◽  
Gang Ji ◽  
Xiaojun Huang ◽  
Lei Sun ◽  
Jianguo Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Vacuum ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Fluorescent Imaging ◽  
Three Dimensions ◽  
Large Field ◽  
Correlative Imaging ◽  
Structural Preservation ◽  
High Level

Abstract Correlative light and electron microscopy (CLEM) offers a means of guiding the search for the unique or rare events by fluorescence microscopy (FM) and allows electron microscopy (EM) to zoom in on them for subsequent EM examination in three-dimensions (3D) and with nanometer-scale resolution. FM visualizes the localization of specific antigens by using fluorescent tags or proteins in a large field-of-view to study their cellular function, whereas EM provides the high level of resolution for complex structures. And cryo CLEM combines the advantages of maintaining structural preservation in a near-native state throughout the entire imaging process and by avoiding potentially harmful pre-treatments, such as chemical fixation, dehydration and staining with heavy metals. Besides for frozen-hydrated biological samples, CLEM combines the advantages of a close-to-life preservation of biological materials by keeping them embedded in vitreous ice throughout the entire imaging process and the frozen-hydrated condition is very suitable to maintain fluorescent signals. In recent years, many new instruments and software which intended to optimize the workflow and to obtain better experimental results of CLEM have been presented or even commoditized. While, the specimen damage during transfer from FM to EM and the resolution of CLEM were still need to be improved. Here we set up a High-vacuum Optical Platform to develop CLEM imaging technology (HOPE), which was designed to realize high-vacuum optical ( fluorescent) imaging for cryo-sample on EM cryo-holder (e.g. Gatan 626). A non-integrated high-vacuum cryo-optical stage, which adapted to the EM cryo holder, was fixed on epi-fluorescence microscope (or super-resolution microscope) to obtain fluorescent images. And then the EM cryo holder would be transferred to EM for collection of EM data. This protocol was aimed to minimize the specimen damage during transfer from FM to EM and it was versatile to expend to different types of light microscopy or electron microscopy. Our HOPE had already passed correlative imaging test, and the results showed that it was convenient and effective.

Sign in / Sign up

Export Citation Format

Share Document