NEAR-FIELD CONFOCAL OPTICAL SPECTROSCOPY (NCOS) - SUBDIFFRACTION OPTICAL RESOLUTION FOR BIOLOGICAL-SYSTEMS

Optical resolution is limited by diffraction. However, in near-field m icroscopes sample illumination is provided through a subwavelength ape rture to increase optical resolution, In this study we have evaluated the usefulness of this technique for living biological systems and rep ort two significant improvements in this form of microscopy to enhance optical resolution for biological studies, We report a unique feedbac k method, photon-density feedback, which is used to monitor the regist ration of a near-field illumination probe with living cell membranes, In this method, the fluorescence intensity of a uniformly distributed fluorochrome is monitored while the sample is moved in the z-axis towa rds the probe. Upon contact between the cell membrane and the near-fie ld probe a maximum intensity is detected. A problem with near-field mi croscopy is that enhanced optical resolution is only achieved within t he near-field of the illuminating aperture. Thick biological specimens also fluoresce in the far-field reducing optical resolution. To reduc e this problem we incorporated a confocal pinhole together with the ne ar-field probe to enhance the resolution of this form of near-field mi croscopy. Finally, we demonstrate that near-field confocal optical spe ctroscopy does not impair physiological properties of neurons, astrocy tes or mast cells, indicating that this high-resolution optical method ology will permit a new approach to the study of molecular distributio n and action within living specimens.