Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells

TL;DR

This paper demonstrates a simple method to achieve multiplanar fluorescence excitation in a laser scanning microscope using standing waves, enabling detailed 3D imaging of red blood cell membranes with high axial resolution.

Contribution

It introduces a novel, easy-to-implement technique for multiplanar fluorescence excitation using a plane reflector, enhancing 3D imaging capabilities in confocal microscopy.

Findings

Achieved ~90 nm axial resolution in red blood cell membrane imaging.

Demonstrated dependence of excitation intensity on fluorochrome Stokes shift.

Provided a method compatible with existing confocal microscopes.

Abstract

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.