PHOEBE,a prototype scanning laser-feedback microscope for imaging biological cells in aqueous media

Based on the principle of laser-feedback interferometry (LFI), a laser-feedback microscope (LFM) has been constructed capable of providing an axial (z) resolution of a target surface topography of ～ 1 nm and a lateral (x, y) resolution of ～ 200 nm when used with a high-numerical-aperture oil-immersion microscope objective. LFI is a form of interferometry in which a laser's intensity is modulated by light re-entering the illuminating laser. Interfering with the light circulating in the laser resonant cavity, this back-reflected light gives information about an object's position and reflectivity. Using a 1-mW He–Ne (λ = 632·8 nm) laser, this microscope (PHOEBE) is capable of obtaining 256 × 256-pixel images over fields from (10 μm × 10 μm) to (120 μm × 120 μm) in ～ 30 s. An electromechanical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously. For biological cells, imaging can be accomplished using back-reflected light originating from small refractive-index changes (> 0·02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point-by-point by growing or placing a cell suspension on a higher-reflecting substrate (glass or a silicon wafer). Advantages of the laser-feedback microscope in comparison to other confocal optical microscopes include: the simplicity of the single-axis interferometric design; the confocal property of the laser-feedback microscope (a virtual pinhole), which is achieved by the requirement that only light that re-enters the laser meeting the stringent frequency, spatial (TEM₀₀), and coherence requirements of the laser cavity resonator mode modulate the laser intensity; and the improved axial resolution, which is based on interferometric measurement of optical amplitude and phase rather than by use of a pinhole as in other types of confocal microscopes.