Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

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.

Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage

“…The second approach uses a fluorophore or molecular probe that is visible by both fluorescence imaging and another imaging modality, such as TEM and SEM. Examples include quantum dots (Hanifi et al, 2019), which can be conjugated to antibodies (Giepmans et al, 2005;Killingsworth and Bobryshev, 2016;Peckys and de Jonge, 2015), and a fluorophore and a gold cluster conjugated to antibodies (FluoroNanogold) (Takizawa et al, 2015). This approach has multiple variants.…”

Transmission Electron Microscopy as a Powerful Tool to Investigate the Interaction of Nanoparticles with Subcellular Structures

“…Fluorescence microscopy and STEM tomography allowed the analysis of 900 nm-thick sample sections, thus providing the 3D distribution of rhodamine B-labelled gold nanoparticles in human retinal pigment epithelial cells [ 129 ]. Similarly, quantum dots were identified inside fibroblasts [ 130 ] and human pathological tissues [ 131 ]. STEM in combination with light microscopy allowed the visualization of molybdenum-based nanoparticles into human hepatoma cells cultured in 2D (cell monolayers) and 3D (spheroids) models [ 132 ].…”

Discussions of Fluorescence in Selenium Chemistry: Recently Reported Probes, Particles, and a Clearer Biological Knowledge

“…Widefield fluorescence light microscopy revealed QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. TEM showed that QD probes attached to amorphous material contained in individual secretory granules [ 161 ].…”

“…Widefield fluorescence light microscopy revealed QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. TEM showed that QD probes attached to amorphous material contained in individual secretory granules[161].On the topic of Se nanocrystals and quantum dots, Mir et al reported the synthesis of CuInGaSe (CIGC) nanocrystals at room temperature by a sol-gel method which can be conducted at room temperature; the authors obtained a uniform cluster size of 75 nm. After analysis, using TEM, SEM, XRD, EDX the authors concluded the stoichiometry was Cu 0.19, In 0.24, Ga 0.76, and Se 2.7.…”