Adaptive optics (AO) is used to improve the performance of an optical signal by using information about the environment which it passes [29]. In 1953, Babcock proposed a method for improving the performance of a telescope by compensating for atmospheric distortions using a deformable optical element which is driven by a wavefront sensor (WFS) [30]. In the early 2000s, AO methods were adapted for microscopy to compensate for specimen-induced aberrations instead of atmospheric turbulence [7].
This section will describe the overall principle of adaptive optics in microscopy and lithography. Starting from the source of aberrations, the basic principle of AO is explained. Afterwards a brief overview of AOSs with a focus on devices used in the system’s implementation is provided. Then the two common methods for AO, namely sensor-based AO and sensor-less AO, are discussed.
At the end, a brief overview of the development and state of the art of AO is reviewed.
Aberrations
A microscope’s resolving power is limited by the phenomenon of the diffrac-tion of light [31,32]. However, in practice deviations in the optical system and the specimen cause distortion of the focus from the diffraction-limit. These
de-viations are known as aberrations. Aberrations in microscopy and lithography have at least three main causes. First, the intrinsic optical element’s quality and their misalignment [6,7,8,33]. Second, one of the most studied specimen geometries, a planar refractive index mismatch when passing from the objec-tive to the immersion media to the cover glass [34]. Third, a more significant challenge, the complex aberration introduced by the optically inhomogeneous structure of biological specimens [8,6,7,33]. Small but measurable refractive indices variations of biological materials and organelles have been found in measurements [35]. These variations induce significant aberrations in thick samples, especially during deep tissue imaging [7]. In addition, scattering of specimens also reduces the image quality [33,36]. These aberrations lead to a spreading of the focus in the sample, both in axial and lateral directions, decreasing the contrast of the microscope images [6,8,37]. As a consequence, the resolution and the intensity reduces, ultimately limiting the depth at which imaging is practical [7,33,38]. The induced aberrations can be removed by the introduction of the opposite phase aberration into the optical path, which is done dynamically by AO [6,38].
2.2.1. Adaptive Optics Background
AO is a technique to optimize and in the best case restore the optimum resolution of an optical system.
In order to correct for induced aberrations, the aberration present has to
be determined. To do so, a WFS for measuring the phase of the distorted wavefront is usually implemented. This approach is called sensor-based AO.
Another approach for measuring the aberrated wavefront is an indirect process, which is called sensor-less AO. Both methods will be discussed in detail in the following sections. Compensation is then performed with an adaptive optical element. This adaptive element introduces an equal but opposite aberration to that measured in the optical path. As the sum of these two aberrations is zero, un-aberrated operation, in principle, is restored. The adaptive optics system is driven by a control, typically a computer [7,39].
Figure2.7shows the principle of a conventional closed-loop AOS. Light from the guide star in the region of interest (ROI) is captured by an objective and sampled by the WFS. The control computer then calculates the necessary compensation and applies it to the corrector.
Figure2.7.: A conventional closed-loop adaptive optics system. The non-linear guide star from the ROI is captured by an objective and imaged onto a wavefront sensor. The control computer calculates the compensation and applies it to the corrector. Figure adapted from [39]
2.2.2. Adaptive Optics Systems
Adaptive optics system (AOS) have been developed for many different appli-cations, each taking into consideration the microscope type (e.g. wide-field, confocal, two-photon etc.), as well as the excitation lasers used and the spec-imens being probed. In a TPM for example, aberration correction has to be applied just in the excitation beam path. Aberrations induced in the emission path do not affect image quality due to large area detectors, which act as fluorescent light collectors [6, 33, 40]. In confocal microscopy, on the other
hand, both excitation and collected beams need to be compensated because of the confocal aperture [33,40,41].
Adaptive Optics Elements
Aberrations can in principle be removed in the form of static correction plates, such as binary phase plates [42]. However, aberrations vary among specimens and even among different regions of the same specimen. Thus, dynamic correction elements such as spatial light modulators (SLMs) or deformable mirrors (DMs) are preferable [8].
Liquid Crystal Spatial Light Modulator SLMs modulate the incident light corresponding to optical or electronic properties, performing a modulation of phase and intensity [43]. SLMs are highly motivated by their compactness, high density (e.g.: panel resolution⇠4000x2100, pixel pitch4µm) and low cost.
Therefore, SLMs have been employed in several AOSs [6,44, 45]. However, SLMs liquid crystal devices are polarization and wavelength dependent, which is not compatible with fluorescence microscopy.
Deformable Mirror In microelectromechanical DMs, a reflective membrane is positioned between a transparent electrode and a series of individual elec-trodes, also called actuators, at the back of the mirror, as shown in Figure
2.8. When no voltage is applied, the membrane is assumed to remain flat.
When a voltage is applied to the electrodes, the electrostatic attraction be-tween the electrodes deforms the membrane into the desired shape [43]. DMs have their advantages in the wavelength and polarization independence and additionally ensure high optical efficiency with low optical losses [7]. Further-more, subnanometer positioning precision, repeatability and stability have been demonstrated, which has made them very attractive for high-contrast microscopy applications [46].
Figure2.8.: Implementation of a deformable mirror. A deformable mirror consists of multiple actuators, which are arranged in a grid pattern. When applying a voltage to the several actuators, the shape of the reflective membrane changes. Figure from [43]
Wavefront Sensors
Before aberration correction can be performed in a sensor-based AOS, the wavefront needs to be sensed to measure the induced aberration. Several types of wavefront sensing have been developed for sensor-based AO, the most
prominent being interferometric sensors and the Shack-Hartmann wavefront sensor (SHWFS) [7, 47]. Since a SHWFS detecting a non-linear guide star is incorporated in the implemented AOS, both will be discussed in more detail.
Non-linear guide star In order to determine the aberration introduced by a sample, a light source needs to be present in the ROI enabling its wavefront detection with a wavefront sensor for sensor-based AO. Since TPE provides a small confined volume, the excited fluorescence can be used as an incoherent secondary light source, called nonlinear guide-star (NL-GS), for wavefront sensing. The key point for the NL-GS concept is that fluorescence is an incoherent process and thus does not contain information about the aberration gained by the excitation beam. Therefore, a single pass aberration scheme, where the light source for aberration detection passes the aberrating medium only once, can be implemented with a NL-GS [33,48].
Shack-Hartmann Wavefront Sensor A SHWFS consists of a lenslet array, which is an aperture with small lenses, so called microlenses, and a camera, shown in Figure2.9. The impinging wavefront is focused by the lenslet array onto the camera generating a so-called spot diagram. Thus, the wavefront slope in each subaperture is measured by the deflection of the focused spot, as displayed on the left. When a planar wavefront enters the SHWFS, the wavefront, case B in figure2.9, in each subaperture is parallel to each lenslet’s
lateral axis. Therefore, each plane wavefront will be focused on the lenslet’s center. However, when an aberrated wavefront impinges onto the SHWFS, the wavefront at some subapertures will have a tip/tilt regarding to the lenslet’s lateral axis resulting in a displacement of these focal spots, see case A [47, 49].
Figure2.9.: Principle of a Shack-Hartmann wavefront sensor. The impinging wavefront is sampled by a lenslet array, which focuses the wavefront onto a CCD camera. On the left the front few of the CCD camera is shown. When an aberrated wavefront impinges onto the lenslet array, the spots will be displaced on the camera in regard to the lenslet’s center positions, see case A (red). However, if a planar wavefront impinges, the lenslets will focus the wavefront in their centers and no displacements are present, see case B (green).
2.2.3. Sensor-based Adaptive Optics
In sensor-based AO, the correction is performed by an explicit determination of the optical aberrations through a sensing device such as an interferometer or WFS. These methods need a point-like reference source in the ROI to detect a well-defined wavefront [7,49]. Several reference sources have been developed for wavefront sensing. In some AOS, beads are placed within the specimen either using microinjection needles or negative pressure protocols to create the reference source. However, this is prone to cause sample damage and therefore limiting its potential for invivoimaging [33,50]. As another reference source reflected or scattered light is used. This can cause ambiguity in the wavefront measurement due to coherent interaction between the incident and reflected light [7,51]. In nonlinear microscopy the guide star concept is adopted from astronomy. Thus, the WFS (e.g. SHWFS etc.) can measure the specimen aberrations by the use of a fluorescent guide star excited by TPE [33].
2.2.4. Sensor-less Adaptive Optics
Another common aberration sensing technique is called sensor-less AO. This is an indirect approach, which, in contrast to sensor-based AO, never measures the explicit wavefront. More precisely, it optimizes the photodetector signal via a sequence of images using optimization schemes. Although optimized
algorithms to reduce specimen exposure have been reported, the ROI still has to be exposed a considerable number of times, which might not be feasible for some applications [7,49].
2.2.5. Adaptive Optics in Microscopy and Lithography
Since the adoption of AO from astronomy to microscopy in the early2000s, AO has progressed tremendously in microscopy. One of the first implementations of AO in microscopy was a tip/tilt correction in a transmission confocal microscope [52]. A DM for aberration correction in a confocal fluorescence microscope was successfully implemented in2002[53]. As TPM is normally used for imaging thick specimens, AO was also implemented in TPM early on and is still advancing [6,54]. Newer approaches use NL-GSs as source for wavefront sensing. Since TPE naturally produces a small confined volume, this is utilized in the guide star concept as an incoherent secondary light source. The key is that fluorescence is an incoherent process and does not contain information about the aberrations gained by the excitation beam [33].
Thus, the WFS (e.g. SHWFS etc.) can measure the specimen aberrations by the use of a fluorescent guide star excited by TPE. The latest approaches in TPM minimize bleaching of the WFS signal and correct for an averaged aberration by scanning the ROI during wavefront sensing. Consequently, WFS signal collection is conducted via a de-scanned signal collection [55]. Since the most widely used optical microscopes for high-resolution biomedical imaging are
scanning confocal and two-photon microscopes, AO was in the beginning mainly developed for scanning microscopes. However, more recent research in AO is also focusing on camera-based microscopes [8]. AOS for widefield, structured illumination, light sheet microscopes and micro-endoscopy are reported [56,57,58]. Even AOSs for the expanding field of super-resolution microscopes are studied, enabling better image quality with resolution in the nanometer range [45]. Due to the higher complexity of super-resolution microscopes more elaborate AOSs are developed [59]. In stimulated emission depletion (STED) microscopy, for example, a dual adaptive optics scheme was developed. The scheme makes use of the combination of a sensor-based AOS and a sensor-less AOS, both based on Zernike polynomials, to conduct aberration correction and consequently restore image quality [60].
AO also expanded its horizon from microscopy to other optical and engineer-ing techniques. These techniques include data storage, optical trappengineer-ing and micro/nanofabrication [61,62]. As in microscopy, these methods also suffer from aberrations. Thus, AO can be used to compensate for the induced aber-rations and restore the focal spot quality even when focusing at depth [7,63].
In optical lithography, for example, introduces the refractive index mismatch between the immersion medium, the coverslip and the photoresist mainly spherical aberrations. These introduced aberrations will reduce the focal inten-sity and thus limit the use of the nonlinear, thresholded optical process [61].
Recent developments in AO for ultra UV lithography use multiple-mirror AO to correct for thermally induced aberrations [64].
The microscopy and lithography setup is based on a custom built confocal microscope. A sketch of the setup is shown in Figure3.1. Multiphoton mi-croscopy and lithography are conducted with a titanium:sapphire (Ti:S) laser (MaiTai HP, Spectra-Physics), whose excitation wavelength can be tuned from 690nm to1040nm. Stimulated emission depletion (STED) is performed with a picosecond-pulsed laser (Katana HP, OneFive GmbH) at775nm. The excita-tion laser power is tuned by an electro-optic modulator (EOM), whereas the STED laser power is regulated by an acousto-optic modulator (AOM). Coarse sample adjustment is performed by manually driven stages in3D. Precision positioning can be conducted by a3D piezo-driven stage (P-733.3DD, Physik Instrumente). Scanning of the field of view (FOV) is done by beam scanning with a two-rotating-mirror scanning system (EOPC), which includes a reso-nant scanner in x-direction with a frequency of⇠10kHz and a galvo scanner in y-direction. A25x,1.05NA water dipping objective lens (XLPLN25XWMP2, Olympus) is used for focusing into the sample. Aberration correction is ex-ecuted in closed-loop configuration using a deformable mirror (Multi-5.5,
Boston Micromachines Corporation) and a SHWFS (custom-made: lenslet-array (#64-483, Edmund Optics), EMCCD camera (iXon ultra, Andor)), which are both conjugated to the back focal plane as are the two scanning mirrors.
The wavefront sensor signal from the non-linear guide star is directed to the SHWFS by a dichroic mirror. It has to be emphasized that the adaptive optics element is a DM even though SLMs are more accurate. This is due to the simultaneous correction of the excitation and depletion point spread func-tion (PSF) in STED mode and the fact that in the closed-loop operafunc-tion the DM is also located in the wavefront sensing path and reflects the non-polarized fluorescence light. Although both excitation and depletion wavelengths are in close proximity to each other, a SLM would not be applicable to correct for all wavelengths simultaneously because of the narrow wavelength bandwidth of the SLM.
780nm and810nm. STED wavelength is775nm. Coarse sample adjustment is conducted by a manual3D stage, whereas precision positioning is performed by a3D motorized stage. Scanning of the FOV is executed by two rotating mirrors.
Several AOS have been invented for different microscopes. To optimize specif-ically for their application great importance has to be attached to the selection of the AOS components. In this chapter, the implemented system is explained in detail, especially focusing on the compensation theory of the implemented AOS and the specific implementation. Afterwards a simulation of the com-bined system is outlined to determine the system’s compensation limitations.
Then the combined system is characterized and calibration enhancements are tested. The last section of the chapter will show first results of correcting manually introduced aberrations.