Probe-type near-field confocal having feedback for adjusting probe distance
A method and apparatus for achieving optical microscopic images and monitoring metabolic processes of living biological specimens such as cells at a resolution superior to the diffraction limit is disclosed. A primary difficulty in performing near-field scanning optical microscopy of living cells, e.g., determining the separation between the cell surface and the illuminating probe tip, is overcome by using a photon-density feedback method in which a fluorescent dye signal strength is monitored in the cell as the tip is brought to the cell surface, and registering a maximum value, at which point the tip begins to dimple the cell surface and can get no closer to the dye. Thereafter the tip is either maintained in contact with the membrane for point measurements of metabolic processes or is withdrawn a selected distance from the surface as measured against a corresponding decrease in the fluorescent dye signal strength The signal strength serves as a photon-density feedback for maintaining the probe tip at a constant elevation above the cell surface as scanning is performed or time-series measurements of metabolism are recorded. Advantageously, the apparatus also combines confocal means in the form of a pin-hole or the like for high-fidelity light detection in three dimensions from the cell surface in the near-field of the probe tip.