Quantum dots for detection, identification and tracking of single biomolecules in tissue and cells (original) (raw)
The study of basic cell ultrastructure and intracellular physiological functions has been greatly aided by detection and identifi cation of single macromolecules. Since the current in situ labeling methods for directly correlative (light and electron) microscopy observations have a number of substantial limitations, the semiconductor nanocrystals quantum dots gain distinguished with long-term imaging and high photostability. The quantum dots (Qdots) have quickly fi lled in the role, being found to be superior to traditional organic dyes on several counts. It has been estimated that Qdots are 20 times brighter and 100 times more stable than traditional fl uorescent reporters. Nowadays, a wide variety of quantum dots conjugated to secondary antibodies suitable for multiple labeling have become commercially available. They make possible the study of biological processes, both in the membrane or in the cytoplasm, at a truly molecular scale and with high spatial and temporal resolutions. By applying Qdots with different size and color light we might achieve multiple labeling of proteins. However, the use of particles with different size is problematic for high-resolution imaging, semi-quantitative measurement of epitope numbers, or when epitope density is high. Recently we report new electron microscopy method for immunolabeling, where two approaches are performed to distinguish yet unattainable spatial resolution: i) for fi rst time as small as 1 nm nanoparticles were applied and observed and ii) the scanning transmission electron microscope (STEM) equipped with an energy dispersive X-ray (EDX) detector were used to distinguish equal in size small labels. We prove that various quantum dots in the range between 1–5 nm can be observed and identified at these conditions. Our method is not limited by the necessity of using labels of different sizes and, therefore could open a number of new biological applications requiring small labels. Because the only requirement is that labels have different chemical compositions that can be differentiated by an EDX spectrometer, this method is not restricted to only two labels. The number of different labels depends on the number of species with sufficiently different X-ray spectra that can be produced.
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