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.

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 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 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 Just Seeing Is Not Enough for Believing: Immunolabelling as Indisputable Proof of SARS-CoV-2 Virions in Infected Tissue

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1816
Author(s):  
Andreja Erman ◽  
Karmen Wechtersbach ◽  
Daniel Velkavrh ◽  
Jerica Pleško ◽  
Maja Frelih ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Protein Identification ◽  
Light And Electron Microscopy ◽  
Immunogold Labelling ◽  
Correlative Microscopy ◽  
Nucleocapsid Proteins ◽  
Similar Morphology ◽  
Cell Structures ◽  
Transmission Electron ◽  
Nasopharyngeal Tissue

Background: There is increasing evidence that identification of SARS-CoV-2 virions by transmission electron microscopy could be misleading due to the similar morphology of virions and ubiquitous cell structures. This study thus aimed to establish methods for indisputable proof of the presence of SARS-CoV-2 virions in the observed tissue. Methods: We developed a variant of the correlative microscopy approach for SARS-CoV-2 protein identification using immunohistochemical labelling of SARS-CoV-2 proteins on light and electron microscopy levels. We also performed immunogold labelling of SARS-CoV-2 virions. Results: Immunohistochemistry (IHC) of SARS-CoV-2 nucleocapsid proteins and subsequent correlative microscopy undoubtedly proved the presence of SARS-CoV-2 virions in the analysed human nasopharyngeal tissue. The presence of SARS-CoV-2 virions was also confirmed by immunogold labelling for the first time. Conclusions: Immunoelectron microscopy is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Furthermore, we developed a variant of correlative microscopy that allows pathologists to check the results of IHC performed first on routinely used paraffin-embedded samples, followed by semithin, and finally by ultrathin sections. Both methodological approaches indisputably proved the presence of SARS-CoV-2 virions in cells.

scholarly journals 3aB_MB-3Pre-embedding correlative light and electron microscopy using FluoroNanogold combined gold nanoparticle and new fluorescent dye, Fluolid NSL Orange

Microscopy ◽  
2018 ◽  
Vol 67 (suppl_2) ◽  
pp. i30-i30
Author(s):  
Takaaki Kanemaru ◽  
Teruyoshi Kondo ◽  
Kentaro Nishi ◽  
Takashi Yazumi ◽  
Kei-ichiro Nakamura ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Gold Nanoparticle ◽  
Light And Electron Microscopy ◽  
Fluorescent Dye

scholarly journals Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging

2020 ◽  
Author(s):  
Felix Lange ◽  
Paola Agüi-Gonzalez ◽  
Dietmar Riedel ◽  
Nhu T.N. Phan ◽  
Stefan Jakobs ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Fluorescence Microscopy ◽  
High Performance ◽  
Secondary Ion Mass Spectrometry ◽  
Cell Structure ◽  
Light And Electron Microscopy ◽  
Epifluorescence Microscopy ◽  
Ion Mass Spectrometry ◽  
Secondary Ion

AbstractElectron microscopy (EM) has been employed for decades to analyze cell structure. To also analyze the positions and functions of specific proteins, one typically relies on immuno-EM or on a correlation with fluorescence microscopy, in the form of correlated light and electron microscopy (CLEM). Nevertheless, neither of these procedures is able to also address the isotopic composition of cells. To solve this, a correlation with secondary ion mass spectrometry (SIMS) would be necessary. SIMS has been correlated in the past to EM or to fluorescence microscopy in biological samples, but not to CLEM. We achieved this here, using a protocol based on transmission EM, conventional epifluorescence microscopy and nanoSIMS. The protocol is easily applied, and enables the use of all three technologies at high performance parameters. We suggest that CLEM-SIMS will provide substantial information that is currently beyond the scope of conventional correlative approaches.

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 Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0240768
Author(s):  
Felix Lange ◽  
Paola Agüi-Gonzalez ◽  
Dietmar Riedel ◽  
Nhu T. N. Phan ◽  
Stefan Jakobs ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Fluorescence Microscopy ◽  
High Performance ◽  
Secondary Ion Mass Spectrometry ◽  
Cell Structure ◽  
Light And Electron Microscopy ◽  
Epifluorescence Microscopy ◽  
Ion Mass Spectrometry ◽  
Secondary Ion

Electron microscopy (EM) has been employed for decades to analyze cell structure. To also analyze the positions and functions of specific proteins, one typically relies on immuno-EM or on a correlation with fluorescence microscopy, in the form of correlated light and electron microscopy (CLEM). Nevertheless, neither of these procedures is able to also address the isotopic composition of cells. To solve this, a correlation with secondary ion mass spectrometry (SIMS) would be necessary. SIMS has been correlated in the past to EM or to fluorescence microscopy in biological samples, but not to CLEM. We achieved this here, using a protocol based on transmission EM, conventional epifluorescence microscopy and nanoSIMS. The protocol is easily applied, and enables the use of all three technologies at high performance parameters. We suggest that CLEM-SIMS will provide substantial information that is currently beyond the scope of conventional correlative approaches.

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