Abstract
Fluorescent nanodiamonds (fNDs) represent an emerging class of nanomaterials offering great opportunities for ultrahigh resolution imaging, sensing and drug delivery applications. Their biocompatibility, exceptional chemical and consistent photostability renders them particularly attractive for correlative light-electron microscopy studies providing unique insights into nanoparticle-cell interactions. Herein, we demonstrate a stringent procedure to image and quantify fNDs with a high contrast down to the single particle level in cells. Individual fNDs were directly visualized by energy-filtered transmission electron microscopy, that is, inside newly forming, early endosomal vesicles during their cellular uptake processes as well as inside cellular organelles such as a mitochondrion. Furthermore, we demonstrate the unequivocal identification, localization, and quantification of individual fNDs in larger fND clusters inside intracellular vesicles. Our studies are of great relevance to obtain quantitative information on nanoparticle trafficking and their various interactions with cells, membranes, and organelles, which will be crucial to design-improved sensors, imaging probes, and nanotherapeutics based on quantitative data.
Highlights
D uring the past decades, nanoparticles have transformed biomedicine as traceable drug carriers and sensitive probes for therapy and diagnostics.[1]
Individual Fluorescent nanodiamonds (fNDs) were directly visualized by energy-filtered transmission electron microscopy, that is, inside newly forming, early endosomal vesicles during their cellular uptake processes as well as inside cellular organelles such as a mitochondrion
Quantum dots (QDs) and gold nanoparticles have been applied as imaging probes for correlative light-electron microscopy (CLEM) but they are limited by their weak fluorescence,[18] blinking problems of QDs,[19] and their inherent cytotoxicity, raising various concerns for long-term in vitro and in vivo studies.[20,21]
Summary
Of TEM (below 1 nm) and surrounding structures could not be imaged.[11,12] In contrast, electron microscopy provides morphological visualization at unmatched resolution without the need to apply specific marker molecules. The negatively charged nitrogen vacancy (NV−) centers in fNDs emit light at a wavelength of 680 nm after excitation with a 561 nm laser Their emission intensity depends on the number, size, and shape of the fNDs. about 15 NV− on average were present statistically distributed within the fNDs and their optical properties were not affected by the biopolymer coating (Figure S1e). Since z-stack images of the sample were recorded with CLSM first (Figure 1d), spatial information on the sample as well as the fluorescence signals of the fNDs were obtained before the EM preparation Cellular structures such as mitochondria remained well preserved, and they could be imaged with high resolution and contrast. Background fluorescence was largely suppressed for an improved colocalization and the CLSM and EM images appeared only slightly shifted (white arrows, Figure 1f), which was mainly due to small changes in the position or shape changes of the living cells before fixation
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