Gold Nanoparticle as a Marker for Precise Localization of Nano-objects Within Intracellular Sub-domains
Delivery of nano-objects to certain intracellular sub-domains is crucial for nanomedicine. Therefore delivery of nano-object to desirable cellular compartment has to be confirmed. The most valuable confirmation of the delivery comes from direct visualizat
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Introduction Therapeutic delivery of genes and drugs to intracellular sub-domains with nanocarriers requires the creation of reliable delivery systems (1, 2). The methods available for monitoring the delivery of nanoobjects could be divided into retrieval of the products of the nanoobject delivery and visualization of nano-objects. The former is especially important in the case of gene delivery when the level of corresponding proteins clearly confirms the delivery. The latter, visualization, has to show the location of nano-objects directly. The direct visualization of the nano-object requires use of microscope and corresponding probe which has to be conjugated with the nano-object. There are two most popular methods of the visualization: confocal and electron microscopy. They require correspondingly fluorescent or electron-dense probe to be tagged to nano-objects. Unfortunately, confocal microscopy could be used
Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 991, DOI 10.1007/978-1-62703-336-7_4, © Springer Science+Business Media New York 2013
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Valeriy Lukyanenko and Vadim Salnikov
mostly for in vitro experiments. Also, the precise localization of nano-objects with confocal microscopy is significantly complicated due to diffraction limited resolution of confocal systems and threedimensional convolution of fluorescence (3). This along with folding of cellular membranes and clamping of nanoparticles makes practically impossible precise localization of nano-objects within structures smaller than 0.5 μm. For example, diameter of isolated mitochondria is about 1 μm, and mitochondrial inner membrane makes multiple invaginations. It is clear that confocal microscopy cannot resolve between fluorescent dot located inside mitochondrial matrix and another dot located in the mitochondrial intermembrane space. Hence, for purposes of localization of nanoparticles more accurate confocal microscopy methods, such as Förster resonance energy transfer (FRET) or more accurate systems, as superresolution structured illumination microscopy (SSIM) or electron microscopy (EM), should be used. However, both FRET and SSIM has some limitations of confocal microscopy and cannot be used for localization of nanoparticles in tissue. The EM allows studying of tissue fragments after in vivo experiments and precise localization of electron-dense objects >10 nm in diameter within ultrathin sections of the tissue (50–90 nm). To make this localization of nanogold particles more precise, we slightly modified the usual procedure of EM preparation (4, 5). Namely, we employed water-soluble resin for cell polymerization and silver enhancement within ultrathin sections (4, 5). Figure 1 shows the major steps of the method. The silver enhancement significantly increases size of gold nanoparticles and makes their distribution obvious. In addition, the silver enhancement is more effective closer to the surface of the slice; therefore, the size of silver grains allows
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