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Abstract

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Protocol

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Materials

References

Bioengineering

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

Published: August 7th, 2016

DOI:

10.3791/54307

1South Western Sydney Clinical School, Faculty of Medicine, University of New South Wales Australia, 2School of Medicine, Western Sydney University, 3Correlative Microscopy Group, Ingham Institute for Applied Medical Research, 4Electron Microscopy Laboratory, Department of Anatomical Pathology, Sydney South West Pathology Service, New South Wales Health Pathology, 5School of Medical Sciences, Faculty of Medicine, University of New South Wales Australia

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

Correlative light- and electron microscopy (CLEM) is a powerful approach for the analysis of transient dynamic events1, rare events2, 3 and complex systems4. There are many different technical permutations available5 depending on the question being asked however a common requirement is that the same structure in a single sample6 is imaged by multiple microscopy modalities. Our particular approach to CLEM was developed for the study of archival human pathology tissue and the case used here has been well characterized and published previously7. The aim was firstly, to maximize the analytical data from a s....

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1. Tissue Dissection and Fixation

  1. Tissue Dissection
    1. Dissect tissue pieces from a surgically resected tumor specimen or tissue biopsy.
      Note: The tissue used in this study was routinely fixed in formalin but fresh tissue is also suitable. We selected an area confirmed and reported by an anatomical pathologist to contain somatostatinoma tumor after routine histological staining and anti-somatostatin immunostaining (not shown).
    2. Use tissue pieces no larger than appro.......

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The somatostatinoma tumor specimen used for this study comprised tumor cells forming ductal structures mixed with collagenous stomal tissue. By fluorescence light microscopy, individual tumor cells that contained abundant secretory granules showed positive labeling for the somatostatin hormone. Nuclei appeared as dark holes with minimal non-specific labeling detectable (Figure 1). At low magnifications, variably intense granular orange fluorescence was seen in the cytopla.......

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This study has demonstrated the potential utility of QDs as universal probes for CLEM studies. The 585 nm QD nanoparticles used showed bright and stable fluorescence when viewed by widefield light microscopy and were readily observed by TEM. A previous study by one of the present authors has shown QDs also to be suitable for super-resolution light microscopy7. Their photostability was particularly useful for extended viewing periods and long imaging exposures. QDs can also be used for multiplex immunohistochem.......

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The authors wish to acknowledge the support of Xiao Juan Wu (Immunohistochemistry Laboratory) and the Department of Anatomical Pathology, Sydney South West Pathology Service (SSWPS), NSW Health Pathology, Liverpool, New South Wales, Australia.

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Name Company Catalog Number Comments
Sodium cacodylate Proscitech C0205 Harmful chemical
Osmium tetroxide Proscitech C010 Use only in fume hood
Uranyl acetate Univar-Ajax 569 Hazardous chemical
Ethanol 100% Fronine JJ008
Acetone 100% Fronine JJ006
ERL 4221 Proscitech C056
DER 732 Proscitech C047
NSA Proscitech C059
DMAE Proscitech C050
Sodium metaperiodate Analar BDH 10259
anti-somatostatin antibody Dako A0566
Antibody diluent Dako S3022
Qdot 585 Streptavidin Conjugate  Invitrogen Q10113MP
Biotinylated goat anti-rabbit IgG antibody Sigma B7389-1ML
Glutaraldehyde 50% EMS 16320
Normal goat serum Invitrogen PCN5000
PBS "Dulbecco A" Oxoid  BR0014G
BSAc (10%) Aurion 900.022
Parafilm Pechiney PP M
pH indicator strips (pH 2.0 - 9.0) Merck 1.09584.0001
Micromoulds Proscitech RL063
Diamond knife Diatome Ultra 45
Transmission electron microscope FEI Morgagni 268D
Fluorescence light microscope Carl Zeiss Axioscope A1
Grids 300 mesh nickel (thin bar) Agar Scientific G2740N
Ultramicrotome RMC Powertome
TEM camera control software Soft Imaging System AnalySIS Version 3.0
Image processing software Adobe Systems Incorporated Photoshop CS2

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