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Adaptive optics is being applied widely to a range of microscopies in order to improve imaging quality in the presence of specimen-induced aberrations. We present here the first implementation of wavefront-sensorless adaptive optics for a laser-free, aperture correlation, spinning disk microscope. This widefield method provides confocal-like optical sectioning through use of a patterned disk in the illumination and detection paths. Like other high-resolution microscopes, its operation is compromised by aberrations due to refractive index mismatch and variations within the specimen. Correction of such aberrations shows improved signal level, contrast and resolution., (© 2020 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

In adaptive optical microscopy of thick biological tissue, strong scattering and aberrations can change the effective pupil shape by rendering some Shack-Hartmann spots unusable. The change of pupil shape leads to a change of wavefront reconstruction or control matrix that should be updated accordingly. Modified slope and modal wavefront control methods based on measurements of a Shack-Hartmann wavefront sensor are proposed to accommodate an arbitrarily shaped pupil. Furthermore, we present partial wavefront control methods that remove specific aberration modes like tip, tilt and defocus from the control loop. The proposed control methods were investigated and compared by simulation using experimentally obtained aberration data. The performance was then tested experimentally through closed-loop aberration corrections using an obscured pupil.

IsoSTED nanoscopy, a variant of stimulated emission depletion (STED) microscopy, utilizes two opposing objective lenses and features the highest three-dimensional resolution of STED nanoscopes currently available. However, this technique is limited by axially repetitive side minima in the interference pattern of the depletion point-spread function (PSF), which can lead to ghost images. Here, we describe novel strategies to further improve the performance of isoSTED nanoscopy by reshaping the PSF. In particular, we propose employing moderate defocus on the depletion beam to reduce the side minima. Furthermore, we demonstrate a simplified alternative based on objective misalignment and quantitatively compare the expected performance between the two approaches.

Adaptive optics has been used to compensate the detrimental effects of aberrations in a range of high-resolution microscopes. We investigate how backscattered laser illumination can be used as the source for direct wavefront sensing using a pinhole-filtered Shack-Hartmann wavefront sensor. It is found that the sensor produces linear response to input aberrations for a given specimen. The gradient of this response is dependent upon experimental configuration and specimen structure. Cross sensitivity between modes is also observed. The double pass nature of the microscope system leads in general to lower sensitivity to odd-symmetry aberration modes. The results show that there is potential for use of this type of wavefront sensing in microscopes.

The scanning two-photon fluorescence microscope produces optically sectioned images from the focal plane. It is sometimes desirable to acquire images from other planes of the specimen that are inclined with respect to the focal plane. In this Letter, we discuss the issues concerned with acquiring such images together with the effects of the inclination angle on image resolution and sectioning strength. To obtain images from oblique planes at high speed, a two-photon system was built wherein a novel optical system is used to provide aberration-free scanning.

Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.

In this work, we demonstrate the use of two-photon polymerization direct laser writing in the production of continuously tuneable optical vortex beam (OV) generators in a liquid crystal (LC) layer sandwiched between glass substrates. Results are presented that show how an OV generator can be inscribed into a 20 μm-thick LC layer and how the order of the OV beam can be tuned with the application of a voltage. Importantly, only a single pair of electrodes is needed to tune the order of the vortex as the required phase profile is generated through the 3D structuring of the polymer network using the laser writing process. Following the design and fabrication of the LC-OV generator, a Mach–Zehnder interferometer is subsequently employed, in conjunction with polarizing optical microscopy, to characterize the devices to confirm the generation of OVs of different orders and to determine the corresponding chirality. The paper concludes by considering whether these LC-OV generators can function at a range of different operation wavelengths. Such devices would be of potential importance in applications ranging from optical communications to quantum physics. [ABSTRACT FROM AUTHOR]

Mechanistic understanding of how the brain gives rise to complex behavioral and cognitive functions is one of science's grand challenges. The technical challenges that we face as we attempt to gain a systems-level understanding of the brain are manifold. The brain's structural complexity requires us to push the limit of imaging resolution and depth, while being able to cover large areas, resulting in enormous data acquisition and processing needs. Furthermore, it is necessary to detect functional activities and 'map' them onto the structural features. The functional activity occurs at multiple levels, using electrical and chemical signals. Certain electrical signals are only decipherable with sub-millisecond timescale resolution, while other modes of signals occur in minutes to hours. For these reasons, there is a wide consensus that new tools are necessary to undertake this daunting task. Optical techniques, due to their versatile and scalable nature, have great potentials to answer these challenges. Optical microscopy can now image beyond the diffraction limit, record multiple types of brain activity, and trace structural features across large areas of tissue. Genetically encoded molecular tools opened doors to controlling and detecting neural activity using light in specific cell types within the intact brain. Novel sample preparation methods that reduce light scattering have been developed, allowing whole brain imaging in rodent models. Adaptive optical methods have the potential to resolve images from deep brain regions. In this roadmap article, we showcase a few major advances in this area, survey the current challenges, and identify potential future needs that may be used as a guideline for the next steps to be taken.

Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

Multielement and multiwavelength switchable Dammann gratings are demonstrated in polymerizable nematic liquid crystal (LC) devices fabricated using two‐photon polymerization direct laser writing. To begin with, a single element 2D Dammann grating, with a diffraction efficiency of around 60%, is fabricated in a thin polymerizable LC layer such that the grating could be activated and deactivated with the application and removal of an external applied voltage. The impact of the fabrication parameters on the resulting intensity distribution in the far‐field diffraction pattern is presented and discussed, along with results for the response times, which are found to be less than that recorded for the regions of the LC device that did not consist of the laser‐written grating. A multielement device is then presented that exhibits two different Dammann gratings that are activated by the application of two different voltage amplitudes. Results for the corresponding far‐field intensity profiles and response times are presented, which demonstrate that high quality laser written switchable Dammann gratings can be fabricated in polymerizable LCs. These switchable elements have potential in programmable beam shaping, microscopy and optical communication devices. [ABSTRACT FROM AUTHOR]

We present a self-calibrating scheme for microscopes using model-based wavefront sensorless adaptive optics. Unlike previous methods, this scheme permits the calibration of system aberration modes without the need for a separate wavefront sensor or interferometer. Basis modes are derived from the deformable mirror influence functions and an image cross-correlation method is used to remove image displacement effects from these modes. Image based measurements are used to derive an optimum modal representation from the displacement-free basis modes. These new modes are insensitive to system misalignments and the shape of the illumination profile. We demonstrate the effectiveness and robustness of these optimal modes in a third harmonic generation (THG) microscope.

Remote focussing microscopy allows sharp, in-focus images to be acquired at high speed from outside of the focal plane of an objective lens without any agitation of the specimen. However, without careful optical alignment, the advantages of remote focussing microscopy could be compromised by the introduction of depth-dependent scaling artifacts. To achieve an ideal alignment in a point-scanning remote focussing microscope, the lateral (XY) scan mirror pair must be imaged onto the back focal plane of both the reference and imaging objectives, in a telecentric arrangement. However, for many commercial objective lenses, it can be difficult to accurately locate the position of the back focal plane. This paper investigates the impact of this limitation on the fidelity of three-dimensional data sets of living cardiac tissue, specifically the introduction of distortions. These distortions limit the accuracy of sarcomere measurements taken directly from raw volumetric data. The origin of the distortion is first identified through simulation of a remote focussing microscope. Using a novel three-dimensional calibration specimen it was then possible to quantify experimentally the size of the distortion as a function of objective misalignment. Finally, by first approximating and then compensating the distortion in imaging data from whole heart rodent studies, the variance of sarcomere length (SL) measurements was reduced by almost 50%. [ABSTRACT FROM AUTHOR]

Adaptive optics has been implemented in a range of high-resolution microscopes in order to overcome the problems of specimen-induced aberrations. Most implementations have used a single aberration correction across the imaged field. It is known, however, that aberrations often vary across the field of view, so a single correction setting cannot compensate all aberrations. Multi-conjugate adaptive optics (MCAO) has been suggested as a possible method for correction of these spatially variant aberrations. MCAO is modelled to simulate the correction of aberrations, both for simple model specimens and using real aberration data from a biological specimen. [ABSTRACT FROM AUTHOR]