Standing-wave excitation of fluorescence is highly desirable in optical
microscopy because it improves the axial resolution. We demonstrate here that
multiplanar excitation of fluorescence by a standing wave can be produced in a
single-spot laser scanning microscope by placing a plane reflector close to the
specimen. We report that the relative intensities in each plane of excitation
depend on the Stokes shift of the fluorochrome. We show by the use of dyes
specific for the cell membrane how standing-wave excitation can be exploited to
generate precise contour maps of the surface membrane of red blood cells, with
an axial resolution of ~90 nm. The method, which requires only the addition of
a plane mirror to an existing confocal laser scanning microscope, may well
prove useful in studying diseases which involve the red cell membrane, such as
malaria.Comment: 15 pages, 4 figures; changed the discussion of narrow-band detected
fringes (Fig. 3) to describe the phenomenon as a moire pattern between the
excitation and emission standing-wave fields, rather than a beats pattern;
added DiI(5)-labelled red blood cell in Fig. 4 to show that standing-wave
fringes are present even when the dye excitation wavelength is outside the
haemoglobin absorption ban