Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins

2021 ◽  
Vol 221 (1) ◽  
Author(s):  
Jan van der Beek ◽  
Cecilia de Heus ◽  
Nalan Liv ◽  
Judith Klumperman
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Functional Organization ◽  
Light And Electron Microscopy ◽  
Correlative Microscopy ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endocytic Vesicles ◽  
Endosomal System

The key endosomal regulators Rab5, EEA1, and APPL1 are frequently applied in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as a marker for late endosomes and lysosomes. However, endogenous levels of these proteins localize poorly in immuno-EM, and systematic studies on their native ultrastructural distributions are lacking. To address this gap, we here present a quantitative, on-section correlative light and electron microscopy (CLEM) approach. Using the sensitivity of fluorescence microscopy, we label hundreds of organelles that are subsequently visualized by EM and classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 colocalizes with Rab5 on early endosomes, but unexpectedly also labels Rab5-negative late endosomes, which are positive for PI(3)P but lack Rab7. APPL1 is restricted to small Rab5-positive, tubulo-vesicular profiles. Rab7 primarily labels late endosomes and lysosomes. These data increase our understanding of the structural–functional organization of the endosomal system and introduce quantitative CLEM as a sensitive alternative for immuno-EM.

scholarly journals Morpho-functional characterization of the endo-lysosomal system by high-throughput correlative light-electron microscopy

2021 ◽  
Author(s):  
Jan A van der Beek ◽  
Cecilia de Heus ◽  
Nalan A Liv ◽  
Judith Klumperman
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
High Throughput ◽  
Functional Characterization ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Immuno Electron Microscopy ◽  
Lysosomal System ◽  
Endocytic Vesicles

Rab5, EEA1 and APPL1 are frequently used in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as marker for late endosomes and lysosomes. However, since these proteins localize poorly in immuno-electron microscopy, systematic studies on their ultrastructural distributions are lacking. Here we address this gap by presenting a quantitative, high-throughput, on-section correlative light-electron microscopy (CLEM) approach using the sensitivity of fluorescence microscopy to infer label to hundreds of organelles classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 co-localizes with Rab5 on especially early endosomes, but unexpectedly also labels Rab5-negative late endosomes and even lysosomes. APPL1 is restricted to small Rab5-positive, vesicular profiles without any visible content or ultrastructural marks. Rab7 primarily labels late endosomes and lysosomes. Our studies reveal the first ultrastructural distribution of key endosomal proteins at their endogenous levels and introduce CLEM as sensitive alternative for quantitative immuno-EM.

Gold nanoparticle printed coverslips to facilitate fluorescence-TEM correlative microscopy

Microscopy ◽  
2017 ◽  
Vol 67 (1) ◽  
pp. 51-54
Author(s):  
Neeraj Prabhakar ◽  
Anni Määttänen ◽  
Jouko Peltonen ◽  
Pekka Hänninen ◽  
Markus Peurla ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Gold Nanoparticle ◽  
Cell Tracking ◽  
Light And Electron Microscopy ◽  
High Contrast ◽  
Correlative Microscopy ◽  
Glass Coverslip ◽  
Nano Gold ◽  
Commercial Glass

Abstract Correlative light and electron microscopy (CLEM) allows combining the advantages of fluorescence microscopy and electron microscopy for cell imaging. Rare phenomenon expressing cells can be studied by specifically tagged fluorophores with fluorescence microscopy. Subsequently, cells can be fixed and ultra-structural details can be studied with transmission electron microscopy (TEM) at a higher resolution. However, precise landmarks are necessary to track the same cell throughout the CLEM process. In this technical report, we present a high contrast inkjet-printed gold nanoparticle patterns over commercial glass coverslip to facilitate cell tracking with correlative microscopy. High contrast and strong reflection from nano gold pattern can be used as a fixed landmark for cell identification with fluorescence microscopy. Nano gold printed letters over coverslips are visible in resin blocks, which can be further used to identify the cell of interest for performing sectioning of embedded cell blocks for TEM.

scholarly journals Bimodal Endocytic Probe for Three-Dimensional Correlative Light and Electron Microscopy

2021 ◽  
Author(s):  
Job Fermie ◽  
Leanne de Jager ◽  
Helen Foster ◽  
Tineke Veenendaal ◽  
Cecilia de Heus ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Bovine Serum Albumin ◽  
Fluorescence Microscopy ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Fiducial Marker ◽  
Regions Of Interest ◽  
Dynamic Information ◽  
Endosomal System ◽  
2D And 3D

Correlative light and electron microscopy (CLEM) can infer molecular, functional and dynamic information to ultrastructure by linking information of different imaging modalities. One of the main challenges, especially in 3D-CLEM, is the accurate registration of fluorescent signals to electron microscopy (EM). Here, we present fluorescent BSA-gold (fBSA-Au), a bimodal endocytic tracer as fiducial marker for 2D and 3D CLEM applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent bovine serum albumin (BSA). The conjugate is efficiently endocytosed and distributed throughout the 3D endo-lysosomal network of the cells, and has an excellent visibility both in fluorescence microscopy (FM) and EM. We demonstrate the use of fBSA-Au in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryo-EM. As a fiducial marker, fBSA-Au facilitates rapid registration of regions of interest between FM and EM modalities and enables accurate (50-150 nm) correlation of fluorescence to EM data. Endocytosed fBSA-Au benefits from a homogenous 3D distribution throughout the endosomal system within the cell, and does not obscure any cellular ultrastructure. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo-)CLEM applications.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

Characterization of Endocytic Pathways by Quantitative Fluorescence Microscopy

1998 ◽  
Vol 4 (S2) ◽  
pp. 1024-1025
Author(s):  
Frederick R. Maxfield ◽  
Richik N. Ghosh ◽  
William G. Mallet ◽  
Thwe Thwe Soe ◽  
Philip L. Leopold ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Light And Electron Microscopy ◽  
Endocytic Pathway ◽  
Early Endosomes ◽  
Pass Through ◽  
Golgi Network ◽  
Quantitative Fluorescence ◽  
Endocytic Pathways

We have used light and electron microscopy to analyze endocytic trafficking pathways. In one set of studies, we have used fluorescently labeled antibodies to trace an endocytic pathway from the cell surface to the trans- Golgi network (TGN). Cells were transfected with a construct consisting of the transmembrane and cytoplasmic domains of TGN38 and the extracellular domain of Tac. TGN38 is predominantly in the TGN, but a small fraction is found on the cell surface. We used FITC-labeled anti-Tac monoclonal IgG to analyze the pathway from the surface to the TGN. We compared the distribution of internalized Tac-TGN38 to internalized transferrin. We found that most Tac-TGN38 enters the same early endosomes as transferrin. Furthermore, most Tac-TGN38 returns to the cell surface from the endocytic recycling compartment (ERC) at the same rate as transferrin. However, on each pass through the cell approximately 18% of Tac-TGN is retained, and this Tac-TGN38 is delivered to the TGN.

scholarly journals Dynamic-ultrastructural cell volume (3D) correlative microscopy facilitated by intracellular fluorescent nanodiamonds as multi-modal probes

2019 ◽  
Author(s):  
Neeraj Prabhakar ◽  
Ilya Belevich ◽  
Markus Peurla ◽  
Xavier Heiligenstein ◽  
Huan-Cheng Chang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Volume ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Correlative Microscopy ◽  
Straightforward Method ◽  
Functional Aspects ◽  
A Cell ◽  
Block Face ◽  
Fluorescent Nanodiamonds

ABSTRACTThree-dimensional correlative light and electron microscopy (3D CLEM) are attaining popularity as a potential technique to explore the functional aspects of a cell together with high-resolution ultrastructural details across the cell volume. In order to perform such a 3D CLEM experiment, there is an imperative requirement for multi-modal probes that are both fluorescent and electron-dense. These multi-modal probes will serve as landmarks in matching up the large full cell volume datasets acquired by different imaging modalities. Fluorescent nanodiamonds (FNDs) are a unique nanosized, fluorescent, and electron-dense material from the nanocarbon family. We hereby propose a novel and straightforward method for executing 3D CLEM using FNDs as multi-modal landmarks. We demonstrate that FNDs is biocompatible and easily identified both in living cell fluorescence imaging and in serial block-face scanning electron microscopy (SB-EM). We illustrate the 3D CLEM method by registering multi-modal datasets.

scholarly journals Synaptic Vesicles Form by Budding from Tubular Extensions of Sorting Endosomes in PC12 Cells

1999 ◽  
Vol 10 (12) ◽  
pp. 4163-4176 ◽  
Author(s):  
Heidi de Wit ◽  
Yael Lichtenstein ◽  
Hans J. Geuze ◽  
Regis B. Kelly ◽  
Peter van der Sluijs ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Transferrin Receptor ◽  
T Antigen ◽  
Marker Proteins ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Quantitative Immunoelectron Microscopy ◽  
Recycling Pathway ◽  
Endocytic Vesicles

The putative role of sorting early endosomes (EEs) in synaptic-like microvesicle (SLMV) formation in the neuroendocrine PC12 cell line was investigated by quantitative immunoelectron microscopy. By BSA-gold internalization kinetics, four distinct endosomal subcompartments were distinguished: primary endocytic vesicles, EEs, late endosomes, and lysosomes. As in other cells, EEs consisted of vacuolar and tubulovesicular subdomains. The SLMV marker proteins synaptophysin and vesicle-associated membrane protein 2 (VAMP-2) localized to both the EE vacuoles and associated tubulovesicles. Quantitative analysis showed that the transferrin receptor and SLMV proteins colocalized to a significantly higher degree in primary endocytic vesicles then in EE-associated tubulovesicles. By incubating PC12 cells expressing T antigen-tagged VAMP (VAMP-TAg) with antibodies against the luminal TAg, the recycling pathway of SLMV proteins was directly visualized. At 15°C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37°C, the labeling shifted to the tubular part of EEs and to newly formed SLMVs. Our data delineate a pathway in which SLMV proteins together with transferrin receptor are delivered to EEs, where they are sorted into SLMVs and recycling vesicles, respectively.

scholarly journals Efficient Immunocytochemical Labeling of Leukocyte Microtubules with FluoroNanogold: An Important Tool for Correlative Microscopy

1997 ◽  
Vol 45 (5) ◽  
pp. 631-642 ◽  
Author(s):  
John M. Robinson ◽  
Dale D. Vandré
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Gold Particle ◽  
Model System ◽  
The Other ◽  
Secondary Antibody ◽  
Correlative Microscopy ◽  
Silver Enhancement ◽  
Fab Fragment ◽  
Gold Particles

We tested the immunoprobe FluoroNanogold (FNG) for its utility as an immunocytochemical labeling reagent. This immunoprobe consists of a 1.4-nm gold particle to which a specific Fab' fragment and a fluorochrome are conjugated. We employed the microtubules (MTs) of human phagocytic leukocytes as a model system for testing the usefulness of FNG as a secondary antibody for immunocytochemistry. We show that these fluorescently labeled ultrasmall immunogold particles are very efficient for labeling MTs in these cells. The signal from FNG can be detected directly by fluorescence microscopy or indirectly by other modes of optical microscopy and electron microscopy, after silver-enhancement of the gold. The spatial resolution of immunolabeled MTs obtained with FNG and silver enhancement was comparable to that of conventional immunofluorescence detection. Colloidal gold (5-nm and 10-nm in diameter), on the other hand, failed to label MTs in cells prepared in a similar manner. This difference in labeling was due in large part to greater penetration of 1.4-nm gold into aldehyde-fixed cells than either 5-nm or 10-nm gold particles. The fluorescent 1.4-nm immunoprobe was shown to be an important new tool for general use in correlative microscopy.

scholarly journals Cell Volume (3D) Correlative Microscopy Facilitated by Intracellular Fluorescent Nanodiamonds as Multi-Modal Probes

Nanomaterials ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 14
Author(s):  
Neeraj Prabhakar ◽  
Ilya Belevich ◽  
Markus Peurla ◽  
Xavier Heiligenstein ◽  
Huan-Cheng Chang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Volume ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Correlative Microscopy ◽  
Straightforward Method ◽  
Functional Aspects ◽  
A Cell ◽  
Block Face ◽  
Fluorescent Nanodiamonds

Three-dimensional correlative light and electron microscopy (3D CLEM) is attaining popularity as a potential technique to explore the functional aspects of a cell together with high-resolution ultrastructural details across the cell volume. To perform such a 3D CLEM experiment, there is an imperative requirement for multi-modal probes that are both fluorescent and electron-dense. These multi-modal probes will serve as landmarks in matching up the large full cell volume datasets acquired by different imaging modalities. Fluorescent nanodiamonds (FNDs) are a unique nanosized, fluorescent, and electron-dense material from the nanocarbon family. We hereby propose a novel and straightforward method for executing 3D CLEM using FNDs as multi-modal landmarks. We demonstrate that FND is biocompatible and is easily identified both in living cell fluorescence imaging and in serial block-face scanning electron microscopy (SB-EM). We illustrate the method by registering multi-modal datasets.

scholarly journals Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells

2002 ◽  
Vol 159 (4) ◽  
pp. 625-635 ◽  
Author(s):  
Jyoti K. Jaiswal ◽  
Norma W. Andrews ◽  
Sanford M. Simon
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Cell Surface ◽  
Total Internal Reflection ◽  
Cytosolic Calcium ◽  
Internal Reflection ◽  
Secretory Cells ◽  
Calcium Dependent ◽  
Late Endosomes ◽  
Early Endosomes

Similar to its role in secretory cells, calcium triggers exocytosis in nonsecretory cells. This calcium-dependent exocytosis is essential for repair of membrane ruptures. Using total internal reflection fluorescence microscopy, we observed that many organelles implicated in this process, including ER, post-Golgi vesicles, late endosomes, early endosomes, and lysosomes, were within 100 nm of the plasma membrane (in the evanescent field). However, an increase in cytosolic calcium led to exocytosis of only the lysosomes. The lysosomes that fused were predominantly predocked at the plasma membrane, indicating that calcium is primarily responsible for fusion and not recruitment of lysosomes to the cell surface.

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