KEYWORDS: Luminescence, Near infrared, Quantum dots, Tissues, In vivo imaging, Signal detection, Pulsed laser operation, Fluorescence spectroscopy, Tissue optics, Microscopy
In vivo cell tracking is a promising tool to improve our understanding of certain biological processes (circulating tumor cell migration, immune cell activity). Several cell tracking techniques have been developed like MRI or PET but remain ill adapted to detect rare and individual cells because of their low spatial resolution and limited sensitivity. Fluorescence detection is a promising alternative. Its sensitivity is however limited by the high tissue autofluorescence and poor visible light penetration depth. To overcome these limitations, we have developed a novel cell imaging modality, based on nearinfrared quantum dots (QDs) allowing long term cell labeling and a sensitive detection based on time-gated wide field fluorescence microscopy. We present the synthesis and characterization of Zn-Cu-In-Se / ZnS (core/shell) QDs composed of low toxicity materials. These QDs exhibit a bright emission centered around 800 nm, where absorption and scattering of tissues are minimal. These nanocrystals are coated with a new surface chemistry, which yields small, stable, bright and individual probes in the cell cytoplasm for several days after the labeling. These QDs also present a fluorescence lifetime much longer (150-200 ns) than tissue autofluorescence (5-10 ns). By combining a pulsed excitation source to a time-gated fluorescence imaging system, we show that we can efficiently discriminate the QD signal from autofluorescence and thus increase the detection sensitivity of labeled cells into tissues.
Fluorescence imaging is a promising technique for the detection of individual cell migration. Its sensitivity is, however, limited by a high tissue autofluorescence and a poor visible light penetration depth. In order to solve this problem, the fluorescence signal peak wavelength should lie in an absorption and diffusion free region and should be distinguishable, either spectrally or temporally, from the autofluorescence background. We present, here, the synthesis and characterization of low toxicity Zn–Cu–In–Se/ZnS core/shell quantum dots. Their fluorescence emission wavelength peaks around 800 nm, where the absorption and scattering of tissues are minimal. They are coated with a new ligand, which yields small, stable, and bright individual probes in the live cell cytoplasm, even 48 h after the labeling. Furthermore, these near-infrared-emitting quantum dots have a long fluorescence lifetime component (around 150 ns) compared to autofluorescence (<5 ns ). Taking the advantage of this property and coupling these probes to a time-gated detection, we demonstrate efficiently the discrimination between the signal and short lifetime fluorescence such as the autofluorescence. This technique is supported by a method we developed, to massively stain cells that preserves the quantum dot stability and brightness for 48 h.
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