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 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 Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Pieter Baatsen ◽  
Sergio Gabarre ◽  
Katlijn Vints ◽  
Rosanne Wouters ◽  
Dorien Vandael ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Fluorescent Proteins ◽  
Fluorescent Microscopy ◽  
Light And Electron Microscopy ◽  
High Resolution Electron Microscopy ◽  
Fluorescence Signal ◽  
Microscopy Imaging ◽  
Tissue Samples ◽  
Rapid Acquisition

Life science research often needs to define where molecules are located within the complex environment of a cell or tissue. Genetically encoded fluorescent proteins and or fluorescence affinity-labeling are the go-to methods. Although recent fluorescent microscopy methods can provide localization of fluorescent molecules with relatively high resolution, an ultrastructural context is missing. This is solved by imaging a region of interest with correlative light and electron microscopy (CLEM). We have adopted a protocol that preserves both genetically-encoded and antibody-derived fluorescent signals in resin-embedded cell and tissue samples and provides high-resolution electron microscopy imaging of the same thin section. This method is particularly suitable for dedicated CLEM instruments that combine fluorescence and electron microscopy optics. In addition, we optimized scanning EM imaging parameters for samples of varying thicknesses. These protocols will enable rapid acquisition of CLEM information from samples and can be adapted for three-dimensional EM.

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 Optimization of Fluorescent Proteins and Techniques for In-resin Correlative Light and Electron Microscopy

2020 ◽  
Vol 26 (S2) ◽  
pp. 1036-1039
Author(s):  
Maria Paez-Segala ◽  
Yalin Wang ◽  
Nirmala Iyer ◽  
Wei-Ping Li ◽  
Patricia Rivlin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Proteins ◽  
Light And Electron Microscopy

scholarly journals ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy

2016 ◽  
Vol 1 ◽  
pp. 26 ◽  
Author(s):  
Elisabeth Brama ◽  
Christopher J. Peddie ◽  
Gary Wilkes ◽  
Yan Gu ◽  
Lucy M. Collinson ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Proteins ◽  
Light And Electron Microscopy ◽  
Large Cell ◽  
Fluorescence Microscope ◽  
Correlative Imaging ◽  
Array Tomography ◽  
Electron Image ◽  
Fluorescent Cells ◽  
Electron Microscopes

In-resin fluorescence (IRF) protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables ‘smart collection’ of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables ‘smart tracking’ of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes.

The Morphology of Vacuolated Cells in the Liver Following Administration of Vitamin A.

1973 ◽  
Vol 31 ◽  
pp. 408-409
Author(s):  
Odell T. Minick ◽  
Hidejiro Yokoo ◽  
Fawzia Batti
Keyword(s):  
Electron Microscopy ◽  
Body Weight ◽  
Vitamin A ◽  
Light And Electron Microscopy ◽  
Liver Cells ◽  
Prominent Feature ◽  
Vascular Space ◽  
Vacuolated Cell ◽  
Vacuolated Cells ◽  
Vacuolated Cytoplasm

Vacuolated cells in the liver of young rats were studied by light and electron microscopy following the administration of vitamin A (200 units per gram of body weight). Their characteristics were compared with similar cells found in untreated animals.In rats given vitamin A, cells with vacuolated cytoplasm were a prominent feature. These cells were found mostly in a perisinusoidal location, although some appeared to be in between liver cells (Fig. 1). Electron microscopy confirmed their location in Disse's space adjacent to the sinusoid and in recesses between liver cells. Some appeared to be bordering the lumen of the sinusoid, but careful observation usually revealed a tenuous endothelial process separating the vacuolated cell from the vascular space. In appropriate sections, fenestrations in the thin endothelial processes were noted (Fig. 2, arrow).

Control of Autogenous Vein Grafts Prior to Reimplantation, By Electron Microscopy

1972 ◽  
Vol 30 ◽  
pp. 106-107
Author(s):  
John H. L. Watson ◽  
John L. Swedo ◽  
M. Vrandecic
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Physiological Saline ◽  
Experimental Conditions ◽  
Vein Grafts ◽  
Arterial Blood ◽  
Saphenous Veins ◽  
Autogenous Vein ◽  
Control Of Parameters ◽  
Post Implantation

The ambient temperature and the nature of the storage fluids may well have significant effects upon the post-implantation behavior of venus autografts. A first step in the investigation of such effects is reported here. Experimental conditions have been set which approximate actual operating room procedures. Saphenous veins from dogs have been used as models in the experiments. After removal from the dogs the veins were kept for two hours under four different experimental conditions, viz at either 4°C or 23°C in either physiological saline or whole canine arterial blood. At the end of the two hours they were prepared for light and electron microscopy. Since no obvious changes or damage could be seen in the veins by light microscopy, even with the advantage of tissue specific stains, it was essential that the control of parameters for successful grafts be set by electron microscopy.

Ultrastructure of two neoplasms in culture compared with original biopsies

1984 ◽  
Vol 42 ◽  
pp. 50-51
Author(s):  
Joseph M. Harb ◽  
James T. Casper ◽  
Vlcki Piaskowski
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Biopsy Specimen ◽  
Cultured Cells ◽  
Light And Electron Microscopy ◽  
Human Tumor ◽  
Soft Agar ◽  
Human Tumor Cells ◽  
Morphological Distinction ◽  
Tumor Types

The application of tissue culture and the newer methodologies of direct cloning and colony formation of human tumor cells in soft agar hold promise as valuable modalities for a variety of diagnostic studies, which include morphological distinction between tumor types by electron microscopy (EM). We present here two cases in which cells in culture expressed distinct morphological features not apparent in the original biopsy specimen. Evaluation of the original biopsies by light and electron microscopy indicated both neoplasms to be undifferentiated sarcomas. Colonies of cells propagated in soft agar displayed features of rhabdomyoblasts in one case, and cultured cells of the second biopsy expressed features of Ewing's sarcoma.

The Effect of Vitamin A on the Intermittent Nitrogen Dioxide Gas Exposure in Hamsters

1976 ◽  
Vol 34 ◽  
pp. 176-177
Author(s):  
J.C.S. Kim ◽  
M.G. Jourden ◽  
E.S. Carlisle
Keyword(s):  
Electron Microscopy ◽  
Vitamin A ◽  
Nitrogen Dioxide ◽  
Chronic Exposure ◽  
Light And Electron Microscopy ◽  
Specific Effect ◽  
Deficient Diet ◽  
Intermittent Exposure ◽  
Hypovitaminosis A ◽  
Gas Exposure

Chronic exposure to nitrogen dioxide in rodents has shown that injury reaches a maximum after 24 hours, and a reparative adaptive phase follows (1). Damage occurring in the terminal bronchioles and proximal portions of the alveolar ducts in rats has been extensively studied by both light and electron microscopy (1).The present study was undertaken to compare the response of lung tissue to intermittent exposure to 10 ppm of nitrogen dioxide gas for 4 hours per week, while the hamsters were on a vitamin A deficient diet. Ultrastructural observations made from lung tissues obtained from non-gas exposed, hypovitaminosis A animals and gas exposed animals fed a regular commercially prepared diet have been compared to elucidate the specific effect of vitamin A on nitrogen dioxide gas exposure. The interaction occurring between vitamin A and nitrogen dioxide gas has not previously been investigated.

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