STED microscopy: Simplified superposition of laser beams

Beam superposition is of fundamental importance in STED microscopy: The precise imaging of a sample requires an optimal superposition of two laser beams. An excitation laser activates the fluorescence of the sample, while the excitation laser eliminates the fluorescence. When correctly superimposed, the sample section appears visible and super-resolved in the microscope.

The adjustment of the laser beams places high demands on the accuracy of the system and the operator. A simple, user-friendly method for adjusting the lasers does not exist so far. The users of these technical microscopes require a high degree of experience. Especially fluctuating environmental conditions require repeated calibration, sometimes even during a running measurement.

The invention combines innovative methods in STED microscopy and ensures beam superposition in the focus by a physical process. Excitation of the sample is achieved by superimposing two lasers (utilizing sum frequency excitation). One of the two lasers causes, in addition to the excitation, the stimulated emission. This effect contributes significantly to improving the resolution. The combination of sum-frequency excitation and partial excitation of one of the lasers causes the beams to be superimposed intrinsically; the excitation remains centered relative to the excitation. This enables high resolution despite poor alignment, and the image accuracy is improved. The frequency of the first and second beam can be adjusted so that there is both a small and a large Stokes shift between the excitation and emission frequency. The simplified handling extends the user range to people with little or no experience in handling optical equipment.

The invention gives manufacturers of optical STED systems the opportunity to expand their technology to a wider range of users. In addition, the quality of the images increases.

• Simplified handling
• Reduced time expenditure
• High resolution independent of adjustment
• Extended range of application

Scientific testing was successful: the optical design of the invention showed that the improvements in localization and resolution were experimentally reproducible.

The invention is a result of the research project „Key to Instrinsically Self-aligned STED“ (KISS) which was funded by the German Federal Ministry of Education and Research (BMBF).