Polymer Based Fluorophore Nanoparticles

Nanoparticles prepared by the incorporation of fluorophores with a polymeric matrix have been developed to circumvent the drawbacks of bare fluorophore molecules for the study of the dynamics of subcellular events in live cells [14, 15].

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Figure 2. Representative snapshots of AABM cell uptake and efflux of SEGNPs using dark-field microscopy through a coverslip with a 100 x objective, equipped with a PID 1030 x 1300-pixel CCD camera (Micromax, 5 MHz-Interline), at 100 ms exposure time with a temporal resolution of 1.17 s [8]. The time shown in each image represents real time with 1 min as a unit. A blue nanoparticle (~50 nm in diameter) participated in several uptake/efflux events in the AABM cell: first uptake at 7.170 min with efflux at 14.313 min; second uptake at 25.050 min and efflux at 25.386 min; third uptake at 25.601 min and efflux at 25.855 min; fourth uptake at 26.948 min and efflux at 27.085 min; and fifth uptake at 27.182 min and efflux at 27.241 min. The bright yellow particle was

Figure 2. Representative snapshots of AABM cell uptake and efflux of SEGNPs using dark-field microscopy through a coverslip with a 100 x objective, equipped with a PID 1030 x 1300-pixel CCD camera (Micromax, 5 MHz-Interline), at 100 ms exposure time with a temporal resolution of 1.17 s [8]. The time shown in each image represents real time with 1 min as a unit. A blue nanoparticle (~50 nm in diameter) participated in several uptake/efflux events in the AABM cell: first uptake at 7.170 min with efflux at 14.313 min; second uptake at 25.050 min and efflux at 25.386 min; third uptake at 25.601 min and efflux at 25.855 min; fourth uptake at 26.948 min and efflux at 27.085 min; and fifth uptake at 27.182 min and efflux at 27.241 min. The bright yellow particle was

Two primary types of fluorophore polymer-based nanoparticles are fluorescence FluoSphere beads (20-100 nm) [35] and PEBBLEs (probes encapsulated by biologically localized embedding) (20-200 nm) [14, 15]. Like fluorophores, these polymer-based fluorophore nanoparticles use the fluorescence properties of fluorophores as sensing elements and hence are unable to overcome the inherent photobleaching of fluorophores even though the inner reference (free fluo-rophores) has been used to deduct the possible photobleach-ing of the fluorophores. This affects the sensitivity, stability, and lifetime of these nanoparticles. Thus, in general, these nanoparticles have a shorter lifetime than that of QDs and Au and Ag nanoparticles.

FluoSphere beads are prepared by loading polystyrene nanospheres with a variety of fluorescent dyes. These fluorescence nanospheres provide emission spectra ranging from the near ultraviolet to the near infrared and are able to be used in multicomplex analysis [35]. These nanospheres are also easily functionalized for sensing the specific target molecules. This property circumvents the limitation of reactive dyes that easily disrupt the function of biomolecules upon conjugation. Furthermore, these fluorescence nanospheres offer highly intensive fluorescence emission and can be visualized at the single-nanoparticle level. These nanospheres also provide a longer shelf life and can be monitored for a longer lifetime than free fluorophore molecules in solution. Moreover, the polymeric matrix protects the embedded fluorophores from nonspecific interactions and reduces the toxicity of the fluorophores to live cells.

PEBBLEs have been prepared by incorporation of fluorescence indicators with a polyacrylamide matrix and have been used to measure ion flux rates in live cells as described by Kopelman and co-workers [14, 15]. Unlike fluorescent indicators, the fluorophore molecules in PEBBLEs are protected by the polymeric matrix in order to avoid the interaction of fluorophore molecules with proteins in live cells and to eliminate the possibility of false results and the toxicity of dyes. This approach allows the dyes to stay in the live cells for a relatively longer time and to be used for monitoring the dynamics of ion fluxes in live cells more effectively.

taken up at 13.182 min and extruded at 28.317 min by the same AABM cell. This yellow particle (~80 nm) is larger than the blue nanoparticle (~50 nm) and therefore appears brighter. The larger size made it harder for the nanoparticle to enter the cell. The cellular membrane absorbs and scatters the light. This leads to a reduction in the illumination intensity inside the cell. Thus, the nanoparticles look significantly dimmer inside the cells than outside the cells. This allows us to distinguish if the nanoparticles are inside or outside the cells. The nanopar-ticles appear to stick out of the cellular membrane. This is because the size of the nanoparticles and the thickness of the cell membrane are under the optical diffraction limit and the nanoparticles scatter light much more efficiently and appear brighter than the cell membrane. The gray scale was set at 262-500 electron counts. The color of the nanoparticles was directly observed and confirmed with spectroscopy. Samples were prepared by directly mixing 20 pL of the cell solution (10 x dilution of absorbance at 600 nm = 0.1) with 20 pL of a 2.60-pM SEGNPs solution. The timer was started at the point of mixing. Reprinted with permission from [8], X.-H. Xu et al., Nano Lett. 2, 175 (2002). © 2002, American Chemical Society.

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