Using saturated absorption for superresolution laser scanning transmission microscopy.

RESUMEN

Confocal laser scanning microscopy is a powerful technique for three-dimensional imaging to study structures in a specimen. The use of confocal pinhole provides three-dimensional spatial resolution in various types of optical microscopes, such as fluorescence, reflection and scattering. However, in transmission microscopy, the confocal pinhole cannot provide the same effect because the spatial information on the optical axial is not transferred in the imaging system. Therefore, the three-dimensional distribution of light absorbers cannot be observed by laser scanning transmission microscopy. In this paper, we propose the use of saturated absorption to image the three-dimensional distribution of light absorbers in a sample by laser scanning transmission microscopy. The saturated absorption is induced by the high-intensity light irradiation and occurs prominently at the centre of a focal spot. The information of the saturated absorption signal within the focal spot is transferred to the transmitted light, providing the capability of optical sectioning in transmission imaging. In our research, we theoretically and experimentally confirmed that light absorption by dye molecules is saturable at the high illumination intensity, and the saturated absorption signal can be extracted by harmonic demodulation. We obtained the images of a stained rat kidney tissue by selectively detecting the nonlinear transmission signals induced by saturable absorption of the dye molecules. We confirmed the high depth discrimination capability of our technique clearly visualised the fine structures in the specimen that cannot be observed by a conventional laser scanning absorption microscope.