Abstract
Diffraction-limited deep focusing into biological tissue is challenging due to aberrations that lead to a broadening of the focal spot. The diffraction limit can be restored by employing aberration correction for example with a deformable mirror. However, this results in a bulky setup due to the required beam folding. We propose a bi-actuator adaptive lens that simultaneously enables axial scanning and the correction of specimen-induced spherical aberrations with a compact setup. Using the bi-actuator lens in a confocal microscope, we show diffraction-limited axial scanning up to 340 μm deep inside a phantom specimen. The application of this technique to in vivo measurements of zebrafish embryos with reporter-gene-driven fluorescence in a thyroid gland reveals substructures of the thyroid follicles, indicating that the bi-actuator adaptive lens is a meaningful supplement to the existing adaptive optics toolset.
Highlights
The inertia-free tunability of adaptive lenses leads to their application for the axial scaning in many microscopic techniques such as confocal microscopy[1,2,3,4], two-photon microscopy[5,6], structured illumination microscopy[7,8,9,10], light sheet microscopy[11,12] and standard wide-field microscopy[13,14,15]
Some applications, e.g., measurements of semi-transparent specimens with diameters in the low millimetre range such as zebrafish embryos, do not require sophisticated aberration correction and scattering compensation with a high number of degrees of freedom, as specimen-induced spherical aberrations are the key limitation of axial resolution: Zebrafish embryos and larvae are used as a model organism in developmental biology and toxicology[29]
As the adaptive lens is employed both for axial scanning and spherical aberration compensation, the placement of the adaptive lens in the optical setup is crucial: To ensure axial scanning without inducing additional aberration, the planes of the adaptive lens and the objective lens (OL) of the microscope have to be mapped onto each other by a 4 f configuration
Summary
The inertia-free tunability of adaptive lenses leads to their application for the axial scaning in many microscopic techniques such as confocal microscopy[1,2,3,4], two-photon microscopy[5,6], structured illumination microscopy[7,8,9,10], light sheet microscopy[11,12] and standard wide-field microscopy[13,14,15]. Applying spatial light modulators or deformable mirrors for both aberration correction and axial scanning is possible; the tuning range of their refractive power is usually lower than that of adaptive lenses, and their implementation as a Fresnel lens results in a decrease in optical quality[28] Their high number of degrees of freedom requires complex control or calibration strategies. While this approach supports a high number of degrees of freedom, it does not allow for the quasi-static operation that is required for sensor-less correction of specimen-induced aberrations These adaptive lenses with multiple degrees of freedom can be seen as a special case of transmissive spatial light modulators and as such achieve the requirements for diffraction-limited axial scanning in a semitransparent specimen with extended depth. Fuh et al.[42] achieved a large defocusing tuning range with limited success in spherical aberration correction by adopting a biconvex lens employing two thin polyvinyl chloride membranes whose thicknesses can be varied by the applied pressure
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