Polymersomes-Mediated Delivery of Fluorescent Probes for Targeted and Long-Term Imaging in Live Cell Microscopy
Fluorescent microscopy becomes an essential tool for live imaging analysis of complex biological pathways and events as it enables noninvasive real-time/real-space imaging. The design of fluorescent probes to provide dynamic information and long-term trac
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Introduction The use of fluorescence microscopy has become essential in biology and biomedical sciences for the study of functional and structural aspects at the cellular, tissue, or whole animal level. Due to recent technical advances (1, 2) and compared to many other imaging techniques, fluorescence microscopy today achieves high spatial resolution; it is very sensitive and specific with safe and relatively easy detection procedures. One of the biggest advantages is perhaps that it allows compatibility with living specimens, thus providing realtime dynamic studies of biological events. However, live imaging is a fairly recent technology prompted by the introduction of video signal techniques in the 1980s. Fluorescence imaging has traditionally been performed on fixed samples, and at present, improvements are very much needed, especially in the design of fluorescent probes and chemo-sensors suitable to perform live imaging in cells.
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_31, © Springer Science+Business Media New York 2013
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Irene Canton and Giuseppe Battaglia
The ability to bring together multi-functionalities within a single particle gives an advantage to nanotechnology in the creation of such revolutionary imaging devices. These functionalities mainly include coatings for biocompatibility (3) and/or prolonged half-life (4), targeting sequences (5) combined with drug encapsulation for theragnostic applications (6), powerful bioimaging fluorescent dyes (7), activating fluorescent probes in response to bio-stimuli (8), and also combination of fluorescent and magnetic labels (9). To gain the most relevant information in live cellular imaging, the probe has often to target the inside of cells. Small hydrophobic molecules can permeate cell membranes with relative ease, but hydrophilic molecules and especially large macromolecules such as proteins and nucleic acids require a vector to assist their transport across the cell membrane. Nanotechnologists have exploited endocytosis as a successful and reliable gate to enter cells in live bioimaging (10, 11). Understanding the uptake process of the fluorescent nanoparticle from the particular endocytic mechanism to intracellular dynamics of fluorescent nanoparticles is imperative to its biological application. Furthermore, to enable rational design of nanovectors, interactions of the probe with the cell as well as with the biological environment that surrounds the cells need to be properly understood (i.e., effect of cell seeding density, effect of cell trypsinization on receptor-mediated uptake, or presence of serum in the media). Indeed, there are a number of underlying mechanisms that govern the success/failure of the fluorescent probe. Efficient uptake characterization protocols must demonstrate three important parameters in nanoformulations: how fast the signal is detected, how much of the formulation is required to obtain a good e
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