Near Field Scanning Optical Microscopy (NSOM) is the newest member of the Scanning Probe Microscopy (SPM) family, which includes Scanning Tunneling Microscopy (STM) and Atomic Force Microscopy (AFM). Like its predecessors, it uses a sharp tip and a very precise positioning system to scan the surface of samples. The important difference is that the NSOM tip is made of an optical fiber, with a sub-wavelength size aperture at its end, which is brought to near-field proximity to the sample. In the near-field regime, the resolution is only limited by the size of the light source (the aperture), and not by the wavelength of light, thus allowing NSOM to obtain images with subwavelength resolution. Applying NSOM to biological samples is very appealing, since biological research has developed a wide variety of fluorescent dyes which can be directed with high specificity to objects of interest. Moreover, biological research relies to a large extent on conventional fluorescence microscopy, which is diffraction limited. Application of NSOM to biological problems requires the ability to image rough and soft samples in liquid, a task that presents many challenges.
We have developed a NSOM which is capable of imaging cells in liquid with a resolution of ∼150 nm. This has enabled us to image the organization of fluorescently labeled proteins on the membrane of cells in liquid, and demonstrate for the first time that the proteins are clustered.