Martin J. Booth

Adaptive aberration correction in a confocal microscope

The main advantage of confocal microscopes over their conventional counterparts is their ability to optically “section” thick specimens; the thin image slices thus obtained can be used to reconstruct three-dimensional images, a capability which is particularly useful in biological applications. However, it is well known that the resolution and optical sectioning ability can be severely degraded by system or specimen-induced aberrations. The use of high aperture lenses further exacerbates the problem. Moreover, aberrations can considerably reduce the number of photons that reach the detector, leading to lower contrast. It is rather unfortunate, therefore, that in practical microscopy, aberration-free confocal imaging is rarely achieved. Adaptive optics systems, which have been used widely to correct aberrations in astronomy, offer a solution here but also present new challenges. The optical system and the source of aberrations in a confocal microscope are considerably different and require a novel approach to wavefront sensing. This method, based upon direct measurement of Zernike aberration modes, also exhibits an axial selectivity similar to that of a confocal microscope. We demonstrate an adaptive confocal fluorescence microscope incorporating this modal sensor together with a deformable membrane mirror for aberration correction. Aberration corrected images of biological specimens show considerable improvement in contrast and apparent restoration of axial resolution.

Image-based adaptive optics for two-photon microscopy

We demonstrate wavefront sensorless aberration correction in a two-photon excited fluorescence microscope. Using analysis of the image-formation process, we have developed an optimized correction scheme permitting image-quality improvement with minimal additional exposure of the sample. We show that, as a result, our correction process induces little photobleaching and significantly improves the quality of images of biological samples. In particular, increased visibility of small structures is demonstrated. Finally, we illustrate the use of this technique on various fresh and fixed biological tissues.

Aberration correction for confocal imaging in refractive-index-mismatched media

A major limitation to the use of confocal microscopes to image thick biological tissue lies in the dramatic reduction in both signal level and resolution when focusing deep into a refractive‐index‐mismatched specimen. This limitation may be overcome by measuring the wavefront aberration and pre‐shaping the input beam so as to cancel the effects of aberration. We consider the images of planar and point objects in brightfield, single‐photon fluorescence and two‐photon fluorescence imaging. In all cases, the specimens are imaged using an oil‐immersion objective through various thicknesses of water.

Adaptive optics enables 3D STED microscopy in aberrating specimens

Stimulated emission depletion (STED) microscopy allows fluorescence far-field imaging with diffraction-unlimited resolution. Unfortunately, extending this technique to three-dimensional (3D) imaging of thick specimens has been inhibited by sample-induced aberrations. Here we present the first implementation of adaptive optics in STED microscopy to allow 3D super-resolution imaging in strongly aberrated imaging conditions, such as those introduced by thick biological tissue.

Adaptive aberration correction in a two-photon microscope

We demonstrate aberration correction in two‐photon microscopy. Specimen‐induced aberrations were measured with a modal wavefront sensor, implemented using a ferro‐electric liquid crystal spatial light modulator (FLCSLM). Wavefront correction was performed using the same FLCSLM. Axial scanned (xz) images of fluorescently labelled polystyrene beads using an oil immersion lens show restored sectioning ability at a depth of 28 µm in an aqueous specimen.

New modal wave-front sensor: a theoretical analysis

We present a new design of a modal wave-front sensor capable of measuring directly the Zernike components of an aberrated wave front. The sensor shows good linearity for small aberration amplitudes and is particularly suitable for integration in a closed-loop adaptive system. We introduce a sensitivity matrix and show that it is sparse, and we derive conditions specifying which elements are necessarily zero. The sensor may be temporally or spatially multiplexed, the former using a reconfigurable optical element, the latter using a numerically optimized binary optical element. Different optimization schemes are discussed, and their performance is compared.

The 2015 super-resolution microscopy roadmap.

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio) physical and biomedical research, particularly with respect to ...

Aberration-free optical refocusing in high numerical aperture microscopy

We describe a method of optical refocusing for high numerical aperture (NA) systems that is particularly relevant for confocal and multiphoton microscopy. This method avoids the spherical aberration that is common to other optical refocusing systems. We show that aberration-free images can be obtained over an axial scan range of 70 mum for a 1.4 NA objective lens. As refocusing is implemented remotely from the specimen, this method will enable high axial scan speeds without mechanical interference between the objective lens and the specimen.

Wavefront sensorless adaptive optics for large aberrations

In some adaptive optics systems the aberration is determined not by using a wavefront sensor but by sequential optimization of the adaptive correction element. Efficient schemes for the control of such systems are essential if they are to be effective. A scheme is introduced that permits the efficient measurement of large amplitude wavefront aberrations that are represented by an appropriate series of modes. This scheme uses an optimization metric based on the root-mean-square spot radius (or focal spot second moment) and an aberration expansion using polynomials suited to the representation of lateral aberrations. Experimental correction of N aberration modes is demonstrated with a minimum of N+1 photodetector measurements. The geometrical optics basis means that the scheme can be extended to arbitrarily large aberrations.

Parallel direct laser writing in three dimensions with spatially dependent aberration correction

We propose a hologram design process which aims at reducing aberrations in parallel three-dimensional direct laser writing applications. One principle of the approach is to minimise the diffractive power of holograms while retaining the degree of parallelisation. This reduces focal distortion caused by chromatic aberration. We address associated problems such as the zero diffraction order and aberrations induced by a potential refractive index mismatch between the immersion medium of the microscope objective and the fabrication substrate. Results from fabrication in diamond, fused silica and lithium niobate are presented.