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PHOEBE, a prototype scanning laser‐feedback microscope for imaging biological cells in aqueous media
Author(s) -
WONG T. L.,
SABATO S. L.,
BEARDEN A.
Publication year - 1995
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1995.tb03546.x
Subject(s) - optics , laser , microscope , materials science , numerical aperture , wafer , interferometry , oil immersion , microscopy , optoelectronics , wavelength , physics
SUMMARY 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 (TEM 00 ), 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.