Nanofiber Probes for Intracellular Fluorescence Measurements

Optical nanofiber probes without antibody probes have been fabricated and used to monitor fluorescence emission from chemical species inside living single cells [17]. In this study, mouse epithelial cells were incubated with a fluorescent dye by incubating the cells in the dye solution and allowing membrane permeabilization to take place. Another procedure for loading cells with fluorophors involved the method called "scrape loading." In the scrape loading procedure, a portion of the cell monolayer was removed by mechanical means, and cells along the boundary of this "scrape" were transiently permeabilized, allowing the dye to enter these cells. The dye was subsequently washed away, and only permeabilized cells retained the dye molecules, as they were not internalized by cells with intact membranes. Following incubation, the fluorescence signal of fluorescent dye molecules in single cells was detected using the optical nanofibers. A photograph of a nanosensor used to monitor clone 9 single cells is shown in Figure 7. Micromanipulators were used to move the optical fiber into contact with the cell membrane of a cell to be monitored. The fiber tip was then gently inserted just inside the cell membrane for fluorescence measurements. An argon-ion laser beam was transmitted through the optical nanofiber, and used for excitation. The dye molecules inside the cell were excited, y y

Coupling Fiber

Micromanipulator

Single Cell Inside Holder

Inverted Microscope

Figure 6. Instrumental system for fluorescence measurements of single cells using nanosensors.

Coupling Fiber

Micromanipulator

Single Cell Inside Holder

Inverted Microscope

Optical Filter

Figure 7. Photograph of single-cell sensing using a fiber-optic nanosensor.

Figure 6. Instrumental system for fluorescence measurements of single cells using nanosensors.

Figure 7. Photograph of single-cell sensing using a fiber-optic nanosensor.

and their fluorescence emission was collected and detected using the microscope instrumental system described previously. Background measurements were performed with cells that were not loaded with the fluorophores. Fluorescence signals were successfully detected inside the fluorophore-loaded cells, and not inside nonloaded cells. As another control, the optical-fiber probe was then moved to an area of the specimen where there were no cells, and the laser light was again passed down the fiber for excitation. No visible fluorescence (at the emission wavelength) was detected for this control measurement, thus demonstrating the successful detection of the fluorescent dye molecules inside single cells. These results demonstrated the capability of optical nanofibers for measurements of fluorophores in intracellular environments of single living cells.

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