Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

Summary

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.

Abstract

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.

Introduction

Correlative light- and electron microscopy (CLEM) is a powerful approach for the analysis of transient dynamic events¹, rare events^{2, 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....

Protocol

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 approximately 1.0 mm³.
2. Cacodylate Buffer Preparation
   1. Prepare a stock buffer solution (1 M) ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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