Instrumentation

The optical measurement system used for monitoring single cells using the nanosensors is schematically illustrated in Figure 6 [18-23]. Laser excitation light, either the 325 nm line of an HeCd laser (Omnichrome, 8 mW laser power) or the 488 nm line of an argon ion laser (Coherent, 10 mW), was focused onto a 600 /m delivery fiber, which was connected to the nanofiber through an SMA connector. The nanofiber was secured to a micromanipulator on the microscope. The experimental setup used to probe single cells was adapted for this purpose from a standard micromanipulation/microinjection apparatus. A Nikon Diaphot 300 inverted microscope (Nikon, Inc.) with a Diaphot 300/Diaphot 200 Incubator, to maintain the cell cultures at ~37 °C on the microscope stage, was used for these experiments. The micromanipulation equipment used consisted of MN-2 (Narishige Company, Ltd.) Nar-ishige three-dimensional manipulators for coarse adjustment, and Narishige MMW-23 three-dimensional hydraulic micromanipulators for final movements. The optical-fiber nanoprobe was mounted on a micropipette holder (World Precision Instruments, Inc.). The fluorescence emitted from the cells was collected by the microscope objective, and passed through an appropriate long-pass dichroic mirror to

2-D Dis| System

2-D Dis| System eliminate the laser excitation scatter light. The fluorescence beam was then focused onto a photomultiplier tube (PMT) for detection. The output from the PMT was passed through a picoammeter, and recorded on a strip-chart recorder or a personal computer (PC) for further data treatment. To record the fluorescence of analyte molecules binding to antibodies at the fiber tip, a Hamamatsu PMT detector assembly (HC125-2) was mounted in the front port of the Diaphot 300 microscope, and fluorescence was collected via this optical path (80% of available light at the focal plane can be collected through the front port). A charge-coupled device mounted onto another port of the microscope could be used to record images of the nanosensor monitoring single cells.

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