WAVEFRONT CONTROL
FOCUS
SEEING DEEPER WITH DYNAMIC WAVEFRONT SHAPING IN OPTICAL MICROSCOPY
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Julian MALOBERTI, Juan MUÑOZ-BOLAÑOS, Eva ERNST, Simon MOSER and Alexander JESACHER * Institute of Biomedical Physics, Medical University of Innsbruck, Innsbruck, Austria * alexander. jesacher @ i-med. ac. at
Optical microscopy is indispensable in biology and medicine. However, tissue scatters the imaging light, which limits how deeply and clearly it can see. Adaptive optics uses dynamic wavefront shaping to compensate for aberrations induced by the sample and can increase imaging depth. We demonstrate this principle using three complementary techniques: multi-photon microscopy for deep imaging, confocal Raman microscopy for label-free chemical contrast and single-molecule localisation microscopy for nanometre-scale imaging. https:// doi. org / 10.1051 / photon / 202614051
Optical microscopy has long been central to exploring the microworld. Variants reveal molecular specificity, chemical composition or structures below the diffraction limit. Yet one obstacle remains common: tissue is optically inhomogeneous and therefore scatters light. The average distance a visible or near-infrared photon travels in biological tissue is often only approximately 100 µ m or less. After a few scattering events, the number of ballistic photons that form a conventional image rapidly declines and the scattered light component, which reduces the image contrast, becomes overwhelmingly strong.
Some microscopes therefore aim to reject scattered light. Confocal microscopy uses a pinhole to select light from the focal region, and optical coherence tomography separates light by its time of flight. Wavefront shaping follows a different idea. If the medium is stable, propagation through it is deterministic: suitable phase engineering can make distorted wavefronts interfere constructively again. Adaptive optics( AO) uses this principle to restore a focus or image.
AO was first developed for astronomy, where deformable mirrors compensate atmospheric turbulence. Since the early 2000s, similar concepts have been adapted to microscopy to compensate aberrations introduced by the sample between the objective lens and the plane of interest [ 1 ]. Figure 1 illustrates the basic idea: scattered photons that would arrive at the wrong points in the image plane are redirected by a spatial light modulator( SLM) so that they contribute to a sharper image.
Microscopic specimens range from nearly transparent embryos to strongly scattering tumour spheroids, and imaging goals span live dynamics to nanometre-scale molecular
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