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סיכום השיעור

Quantum dots (QDs) are man-made nanoscale crystals that that can transport electrons. When UV light hits these semiconducting nanoparticles, they can emit light of various colors. These artificial semiconductor nanoparticles that have found applications in composites, solar cells and fluorescent biological labels.

If semiconductor particles are made small enough, quantum effects come into play, which limit the energies at which electrons and holes (the absence of an electron) can exist in the particles. As energy is related to wavelength (or color), this means that the optical properties of the particle can be finely tuned depending on its size. Thus, particles can be made to emit or absorb specific wavelengths (colors) of light, merely by controlling their size.

Fluorescent proteins are members of a structurally homologous class of proteins that share the unique property of being self-sufficient to form a visible wavelength chromophore from a sequence of 3 amino acids within their own polypeptide sequence. It is common research practice for biologists to introduce a gene (or a gene chimera) encoding an engineered fluorescent protein into living cells and subsequently visualize the location and dynamics of the gene product using fluorescence microscopy.

The most popular applications of fluorescent proteins involve exploiting them for imaging of the localization and dynamics of specific organelles or recombinant proteins  in live cells. For imaging of a specific organelle, standard molecular biology techniques are used to fuse the gene encoding the fluorescent protein to a cDNA encoding a protein or peptide known to localize to that specific organelle. This fusion is done such that the chimeric gene will be expressed as a single polypeptide, creating a covalent link between the targeting motif and the fluorescent protein. A plasmid containing the chimeric gene under control of a suitable promoter is used to transfect mammalian cells that then express the gene to produce the corresponding chimeric protein. The chimera localizes to the target organelle and thus renders it fluorescent. Through the use of fluorescence microscopy, the morphology, dynamics, and distribution of the organelle can be imaged as a function of time. The procedure for imaging of a fusion between a fluorescent protein and a specific protein-of-interest (in order to gain insight into its localization and dynamics) is identical  The availability of a broad selection of colors of fluorescent protein has provided researchers with the means to image the localization of multiple organelles and/or proteins-of-interest, simultaneously.

Fluorescence Resonance Energy Transfer (FRET) Microscopy

The process of resonance energy transfer (RET) can take place when a donor fluorophore in an electronically excited state transfers its excitation energy to a nearby chromophore, the acceptor. In principle, if the fluorescence emission spectrum of the donor molecule overlaps the absorption spectrum of the acceptor molecule, and the two are within a minimal spatial radius, the donor can directly transfer its excitation energy to the acceptor through long-range dipole-dipole intermolecular coupling. A theory proposed by Theodor Förster in the late 1940s initially described the molecular interactions involved in resonance energy transfer, and Förster also developed a formal equation defining the relationship between the transfer rate, interchromophore distance, and spectral properties of the involved chromophores.

Resonance energy transfer is a non-radiative quantum mechanical process that does not require a collision and does not involve production of heat. When energy transfer occurs, the acceptor molecule quenches the donor molecule fluorescence, and if the acceptor is itself a fluorochrome, increased or sensitized fluorescence emission is observed (see Figure 3). The phenomenon can be observed by exciting a specimen containing both donor and acceptor molecules with light of wavelengths corresponding to the absorption maximum of the donor fluorophore, and detecting light emitted at wavelengths centered near the emission maximum of the acceptor. An alternative detection method, growing rapidly in popularity, is to measure the fluorescence lifetime of the donor fluorophore in the presence and absence of the acceptor.

Photobleaching is the light-induced destruction of fluorophores, which can be a particular problem for imaging biological samples using time-lapse studies for long periods of time or where high laser powers are required, as is the case with STED microscopy.

Vital bleaching techniques involve the application of peroxide solutions to increase the value (whiteness) of teeth that are unusually dark. Peroxide bleaching methods appear to work best on teeth that are mildly discolored, predominantly yellow, and from which the discoloration originates in enamel rather than dentin. The basic methods for vital bleaching are: in office power bleaching, custom-fabricated tray bleaching, and over-the-counter bleaching strips.29

Power bleaching is an in-office procedure in which a concentrated hydrogen peroxide solution is applied to rubber dam–isolated teeth while heating the teeth, usually with an electric lamp or laser.30 This method of bleaching may require numerous office visits and often causes temporary tooth sensitivity. Typically, patients who have had this treatment require periodic retreatment to maintain the desired color.

Custom tray or over-the-counter vital bleaching is an at-home treatment. This method of vital bleaching uses a milder peroxide solution (usually 10% carbamide peroxide) that the patient applies and wears outside the dental office, often at night during sleep, for approximately 2 to 3 weeks. At-home bleaching appears to work as well as power bleaching and causes less sensitivity. Concerns were initially raised about the potentially hazardous soft tissue effects of applying peroxide solutions in this manner, but long-term studies of the safety and efficacy of this approach are demonstrating no harmful effects.31 Vital bleaching also appears to cause limited damage to existing restorations, although these may no longer be a color match after the teeth have been whitened.32 Over-the-counter bleaching strips can achieve similar whitening effects but take longer to accomplish.

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