Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots

Abstract

The importance of locating proteins in their context within cells has been heightened recently by the accomplishments in molecular structure and systems biology. Although light microscopy (LM) has been extensively used for mapping protein localization, many studies require the additional resolution of the electron microscope. Here we report the application of small nanocrystals (Quantum dots; QDs) to specifically and efficiently label multiple distinct endogenous proteins. QDs are both fluorescent and electron dense, facilitating their use for correlated microscopic analysis. Furthermore, QDs can be discriminated optically by their emission wavelength and physically by size, making them invaluable for multilabeling analysis. We developed pre-embedding labeling criteria using QDs that allows optimization at the light level, before continuing with electron microscopy (EM). We provide examples of double and triple immunolabeling using light, electron and correlated microscopy in rat cells and mouse tissue. We conclude that QDs aid precise high-throughput determination of protein distribution.

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

“…Fixation of brain and nerve tissue-A 21 day old rat was perfused with a solution of 2% of paraformaldehyde/2.5% of glutaraldehyde in 0.15 M cacodylate buffer according to the protocol described in (Giepmans et al, 2005). Brain and spinal roots were taken out and post-fixed in same fixative for 2 hrs at 4°C.…”

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

“…There has been growing interest in the use of quantum dots as fluorescent tags in biology (2,3). One popular method for attaching dots to biological molecules involves the use of streptavidin conjugated quantum dots that can be readily attached to biotinylated targets (4)(5)(6)(7)(8). Generally there are 8-10 streptavidin molecules per quantum dot, and the size is 10-15nm in diameter, which is similar to the size of an antibody (∼14 nm) or a large protein (i.e.…”

A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed Nanogold-labeled molecules

“…Various labeling methods have or could be used to label ligands for EM studies of endocytosis, including attachment of large, electron-dense labels such as colloidal gold (Harding et al, 1983;Murk et al, 2003), ferritin (Rodewald, 1973), or cadmium selenide quantum dots (Glepmans et al, 2005). Other methods involve attachment of labels that are visualized after photooxidation of diaminobenzidine (DAB) and metal deposition (e.g., peroxidase (Rodewald, 1980) and fluorescent labels for correlative light and electron microcopy (Deerinck et al, 1994;Gaietta et al, 2002)).…”

“…The large diameter of ferritin (∼14 nm) and its multimeric nature promote undefined ligand-ferritin stoichiometries and cross-linking, which can interfere with the proper trafficking of a ligand (Slade and Wild, 1971). Quantum dots, although promising for correlated fluorescent and electron microscopic studies (Glepmans et al, 2005), are often large (≥10 nm) and most methods for attaching them to proteins of interest either lead to random attachment or, in the case of streptavidin quantum dots, require biotinylation of the ligand and can cause cross-linking resulting from binding to tetrameric streptavidin. Although the labels visualized after photooxidation and metal deposition are generally smaller than colloidal gold, ferritin or quantum dots, the stain that develops from these tags tends to be diffuse, reducing their resolution.…”