Super-resolution measurements with evanescent-wave fluorescence excitation using variable beam incidence

Area of Science:

• Optics and Photonics
• Biophysics
• Cell Biology

Background:

• Epiillumination microscopy suffers from out-of-focus fluorescence.
• Confocal and two-photon microscopy have limitations in axial optical sectioning.
• Evanescent waves (EW) provide a method for selective excitation at interfaces.

Purpose of the Study:

• To develop and apply evanescent wave microscopy for high-resolution imaging of fluorophore distribution at interfaces.
• To achieve enhanced axial optical sectioning beyond conventional microscopy techniques.
• To investigate the topographical distribution of subcellular organelles near a cell-interface.

Main Methods:

• Utilized total internal reflection of light to generate evanescent waves for selective fluorophore excitation.
• Modified an upright microscope with specialized condenser optics for EW excitation.
• Employed an acousto-optical deflector, telecentric optics, and a hemicylindrical prism to systematically vary the angle of incidence.
• Performed three-dimensional reconstruction of fluorophore distribution from angle-resolved image stacks.

Main Results:

• Achieved a fivefold enhancement of axial optical sectioning near the coverslip compared to confocal and two-photon microscopy.
• Demonstrated the ability to study fluorophore distribution and concentration at interfaces, even with high bulk solution concentrations.
• Obtained topographical information with an axial resolution of tens of nanometers.
• Successfully mapped the axial position of dye-labeled subcellular storage organelles in living neuroendocrine cells.

Conclusions:

• Evanescent wave microscopy is a powerful technique for high-resolution imaging at interfaces.
• The method provides superior axial optical sectioning and topographical information.
• Applied to living cells, EW microscopy enables detailed study of subcellular organelle localization and distribution.