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Stensberg et al. Page 12 superior to conventional dye molecules in both emission intensity and photostability. Variations in chemistry enable Ag NCs to be used as fluorescent labels for various cellular components, such as actin, microtubule filaments and specific surface proteins [138]. The emission wavelength of Ag NCs is highly size dependent and can even be tuned to near- infrared (NIR) wavelengths. For example, Ag NCs synthesized in the presence of oligocytosine DNA can be used as NIR-active biomarkers to monitor their transfection of live cells [139]. Colloidal Ag NPs are also capable of photoluminesence, although their quantum yields are much smaller than that of Ag NCs. Nevertheless, they can still be used as fluorescent labels if the excitation intensity is sufficiently high. For instance, 36-nm Ag NPs encapsulated in polymer shells have been used as drug delivery vehicles and tracked by fluorescence imaging inside of B16F10 cells [140]. Metallic nanoshells with 50-nm silica cores and 10- nm Ag shells have also been reported as fluorescent labels for detecting CXCR4 chemokine receptors on the surfaces of T lymphocytes [141]. Metal nanoparticles can also produce a two-photon excited luminescence (TPL) by ultrafast pulsed laser excitation. This has been particularly well studied in NIR-resonant NPs, such as Au nanorods [142], but the TPL activity of Ag NPs has also been reported with applications toward biological imaging [143,144]. For example, TPL has been used to monitor the nonspecific uptake of Ag–Fe3O4 NPs into macrophages (Figure 8C & 8D) [145]. Ag NPs as small as 10 nm could produce strong TPL signals with femto-second NIR laser excitation, whereas the magnetic component allowed cells to be manipulated by external magnetic field gradients. Third-harmonic generation (THG) is another nonlinear optical technique for imaging Ag NPs, one that is more efficient than TPL because it does not require excited states for energy conversion. Ag NPs are ideal contrast agents for THG owing to their large third-order susceptibility and the overlap of their plasmon resonance with the tripling of the NIR frequencies used to excite THG signals [146]. THG imaging has been applied toward in vitro cancer cell detection, using antibody-labeled Ag NPs incubated with mouse bladder carcinoma cells (Figure 9) [14]. While TPL and THG are excellent imaging tools for in vitro studies involving Ag NPs, their short working distances are a drawback for whole-animal imaging, so their application toward nanotoxicology is best served at the cellular level. Surface-enhanced fluorescence & Raman imaging Although colloidal Ag NPs are less efficient than Ag NCs as fluorescent markers, they can indirectly support fluorescence imaging by enhancing the emission rates of nearby dye molecules by a process termed surface-enhanced fluorescence. Ag NP–dye conjugates have been demonstrated as fluorescent probes for cellular imaging by conjugating fluorescently labeled lectins onto 20-nm Ag NPs, which were then incubated with HEK 293A cells (Figure 8e) [147,148]. Cells labeled with the Ag NP-coupled probes produced fluorescence signals 20–30-times brighter than those labeled with organic dyes alone (Figure 8F & 8g). Further studies demonstrated that the lifetime of the coupled Ag NP–dye is significantly modified compared with uncoupled dye molecules, which extends the application of Ag NP–dye conjugates to fluorescence lifetime imaging (Figure 8H & 8i). The plasmon resonances of Ag NPs can also be applied toward imaging modalities based on SERS. Close-packed or aggregated metal NPs form electromagnetic ‘hot spots’ that can enhance Raman signals by several orders of magnitude, to the extent that their emissions are comparable to fluorescence. Bacteria adsorbed onto Ag nanostructures can be detected by their characteristic Raman vibrational spectra using SERS microscopy, with limits of detection as low as ten bacteria/ml [149,150]. SERS-active Ag NP ‘tags’ can also be Nanomedicine (Lond). Author manuscript; available in PMC 2012 May 24. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptPDF Image | Toxicological studies on silver nanoparticles
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