scholarly journals Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

2017 ◽  
Author(s):  
Stephen D. Carter ◽  
Shrawan K. Mageswaran ◽  
Zachary J. Farino ◽  
João I. Mamede ◽  
Catherine M. Oikonomou ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Secretory Granules ◽  
Light And Electron Microscopy ◽  
Low Contrast ◽  
Fluorescent Protein Tags ◽  
Protein Tags

AbstractCryogenic correlated light and electron microscopy (cryo-CLEM) is a valuable tool for studying biological processes in situ. In cryo-CLEM, a target protein of interest is tagged with a fluorophore and the location of the corresponding fluorescent signal is used to identify the structure in low-contrast but feature-rich cryo-EM images. To date, cryo-CLEM studies of mammalian cells have relied on very bright organic dyes or fluorescent protein tags concentrated in virus particles. Here we describe a method to expand the application of cryo-CLEM to cells harboring genetically-encoded fluorescent proteins. We discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80K). Compared to fluorescent protein tags, these sources of autofluorescence exhibit a broader spectrum of fluorescence, which we exploited to develop a simple, robust approach to discriminate between the two. We validate this method in INS-1 E cells using a mitochondrial marker, and apply it to study the ultrastructural variability of secretory granules in a near-native state within intact INS-1E pancreatic cells by high-resolution 3D electron cryotomography.

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 Possibilities of subunit localization with fluorescent protein tags and electron microscopy examplified by a cyanobacterial NDH-1 study

2010 ◽  
Vol 1797 (9) ◽  
pp. 1681-1686 ◽  
Author(s):  
Mariam Birungi ◽  
Mihaela Folea ◽  
Natalia Battchikova ◽  
Min Xu ◽  
Hualing Mi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Protein ◽  
Fluorescent Protein Tags ◽  
Protein Tags

scholarly journals Comparative assessment of fluorescent proteins forin vivoimaging in an animal model system

2016 ◽  
Author(s):  
Jennifer K Heppert ◽  
Daniel J Dickinson ◽  
Ariel M Pani ◽  
Christopher D Higgins ◽  
Annette Steward ◽  
...  
Keyword(s):  
Animal Model ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Model System ◽  
C Elegans ◽  
Red Fluorescent Proteins ◽  
Fluorescent Protein Tags ◽  
Protein Tags

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamicsin vivo. Recent advances in genome editing have enabled precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential ofin vivoimaging experiments, and they facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been madein vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap we quantitatively assessed fluorescent protein propertiesin vivoin an animal model system. We generated transgenicC. elegansstrains expressing green, yellow, or red fluorescent proteins in embryos, and we imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not brightin vivoas predicted based onin vitrodata, but that mNeonGreen is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imagingin vivoinC. elegansembryos, and they suggest good candidate fluorescent proteins to test in other animal model systems.

scholarly journals Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system

2016 ◽  
Vol 27 (22) ◽  
pp. 3385-3394 ◽  
Author(s):  
Jennifer K. Heppert ◽  
Daniel J. Dickinson ◽  
Ariel M. Pani ◽  
Christopher D. Higgins ◽  
Annette Steward ◽  
...  
Keyword(s):  
Animal Model ◽  
In Vivo Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Model System ◽  
Red Fluorescent Proteins ◽  
Fluorescent Protein Tags ◽  
Protein Tags

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments.

The Effect of Fluorescent Protein Tags on Phosphoglycerate Kinase Stability Is Nonadditive

2016 ◽  
Vol 120 (11) ◽  
pp. 2878-2885 ◽  
Author(s):  
Kapil Dave ◽  
Hannah Gelman ◽  
Chu Thi Hien Thu ◽  
Drishti Guin ◽  
Martin Gruebele
Keyword(s):  
Fluorescent Protein ◽  
Phosphoglycerate Kinase ◽  
Fluorescent Protein Tags ◽  
Protein Tags

scholarly journals Development and Applications of Fluorescent Proteins for Correlative Light and Electron Microscopy

2018 ◽  
Vol 24 (S1) ◽  
pp. 2318-2319
Author(s):  
Maria G. Paez-Segala ◽  
Yalin Wang ◽  
Nirmala Iyer ◽  
Wei-Ping Li ◽  
Patricia K. Rivlin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Proteins ◽  
Light And Electron Microscopy

scholarly journals Optimal fluorescent protein tags for quantifying protein oligomerization in living cells

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Valentin Dunsing ◽  
Madlen Luckner ◽  
Boris Zühlke ◽  
Roberto A. Petazzi ◽  
Andreas Herrmann ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Living Cells ◽  
Protein Oligomerization ◽  
Fluorescent Protein Tags ◽  
Protein Tags

scholarly journals Correlative 3D imaging of whole mammalian cells with light and electron microscopy

2011 ◽  
Vol 176 (3) ◽  
pp. 268-278 ◽  
Author(s):  
Gavin E. Murphy ◽  
Kedar Narayan ◽  
Bradley C. Lowekamp ◽  
Lisa M. Hartnell ◽  
Jurgen A.W. Heymann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
3D Imaging ◽  
Light And Electron Microscopy

FRET Studies Between CdTe Capped by Small-Molecule Ligands and Fluorescent Protein

2014 ◽  
Vol 13 (05n06) ◽  
pp. 1460013
Author(s):  
Yue Zhang ◽  
Dejian Zhou ◽  
Junhui He
Keyword(s):  
Fluorescent Protein ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Semiconductor Nanocrystals ◽  
Resonance Energy ◽  
Emission Spectra ◽  
Binding Constants ◽  
Dipole Interaction ◽  
Water Soluble ◽  
Donor Acceptor

Water-soluble luminescent semiconductor nanocrystals also known as quantum dots (QDs) that have prominent photostability, wide absorption cross sections and tunable narrow emission, have been shown as promising probes in immunoassays. QDs are often used as donors in fluorescence resonance energy transfer (FRET) based sensors using organic dyes or fluorescent proteins as acceptors. Here, the FRET between a QD donor and fluorescent protein acceptors has been studied. The fluorescent protein (FP)mCherry appended with a hexa-histidine-tag could effectively self-assemble onto CdTe to produce small donor-acceptor distances and hence highly efficient FRET (efficiency > 80%) at relatively low FP: CdTe copy numbers (ca.1). Using the Förster dipole–dipole interaction formula, the Förster radius (R0) and respective donor-acceptor distances for the CdTe -FP FRET systems have been calculated. The binding constants (Kd) of the QD-FP systems have also been evaluated by the emission spectra.

scholarly journals Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in C. elegans

2018 ◽  
Author(s):  
Gregoriy A. Dokshin ◽  
Krishna S. Ghanta ◽  
Katherine M. Piscopo ◽  
Craig C. Mello
Keyword(s):  
Green Fluorescent Protein ◽  
Cost Effectiveness ◽  
Genome Editing ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
C Elegans ◽  
Multiple Loci ◽  
Green Fluorescent ◽  
Fluorescent Protein Tags ◽  
Protein Tags

AbstractCRISPR-based genome editing using ribonucleoprotein (RNP) complexes and synthetic single stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs – such as green fluorescent protein (GFP) tags remains challenging in many systems. Here we describe a streamlined and optimized editing protocol for the nematode C. elegans. We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging all twelve of the Worm-specific Argonaute (WAGO) proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based partially single-stranded “hybrid” donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.

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