Photon scanning-tunneling microscopy of unstained Mammalian cells and chromosomes.

ABSTRACT

The photon scanning-tunneling microscope (PSTM) yields optical topographical images of samples that are thin or that are transparent at the wavelength used. A range of sample sizes can be imaged extending to well below the diffraction limit for sufficiently flat samples. But samples of the order of several to many micrometers in size can be analyzed with less-refined resolution if total internal reflection can be made to occur in the sample. We used the PSTM to examine the optical topography of mouse and human cells and of chromosomes that are unstained. Our objectives were to demonstrate the images as an alternative to conventional microscopy and to provide a sample-preparation methodology that will later permit localized, simultaneous fluorescence or absorption spectroscopy with the signals collected by the probe tip. Furthermore, the PSTM's ability to produce optical profiles in air and in water was tested to establish the basis for future investigation of possible abnormalities in the chromosomes. That is, we considered both physical and biological objectives. To this end we utilized the 442-nm line of a He-Cd laser as well as the 633-nm line from a He-Ne laser, the resulting image quality being tested partly to ascertain the increased effects of scattering at the smaller wavelength. It is shown that adequate resolution and signal-to-noise ratio can be obtained with the shorter wavelength even in the presence of intensity fluctuations from the laser, thus showing that fluorescence and absorption studies can be expected to be practicable.