Datensatz

Zusammenfassung

The image quality of a two−photon microscope is often degraded by wavefront aberrations induced by the specimen. We demonstrate here that resolution and signal size in two−photon microcopy can be substantially improved, even in living biological specimens, by adaptive wavefront correction based on sensing the wavefront of coherence−gated backscattered light (coherence−gated wavefront sensing, CGWS) and wavefront control by a deformable mirror. A nearly diffraction−limited focus can be restored even for strong aberrations. CGWS−based wavefront correction should be applicable to samples with a wide range of scattering properties and it should be possible to perform real−time pixel−by−pixel correction even at fast scan speeds. Resolution, signal, and contrast, especially for confocal (1) and multiphoton microscopy (2) are often degraded by refractive−index inhomogeneities in biological specimens (3−6). This degradation can be reversed if the wavefront of the incoming light is predistorted such as to cancel distortions introduced in the excitation light path, for example by the specimen. Several groups have demonstrated the usefulness of adaptive optics for both confocal and multiphoton microscopy. However, all wavefront measurement schemes (7−10) used so far are based on fluorescence and need strongly and widely stained specimens. Finding the correction parameters usually requires numerous iterations, during which the useful life of fluorophores is consumed by photobleaching and the tissue is exposed to photodynamic damage.

Here we use a wavefront measurement method, coherence−gated wavefront sensing (CGWS), that is independent of fluorescence generation (11). CGWS is instead based on backscattered light, whereby the majority of the light coming back from the sample is rejected by a coherence gate leaving only that light that has been scattered near the focus. The parameters needed for wavefront correction can thus be determined at low laser power levels and for completely nonfluorescent specimens. Here we demonstrate, in various samples, that CGWS−based adaptive wavefront correction can be used to improve signal size and resolution in episcopic two−photon microscopy