Dean Wilding

Pupil filters for extending the field-of-view in light-sheet microscopy.

Pupil filters, represented by binary phase modulation, have been applied to extend the field of view of a light-sheet fluorescence microscope. Optimization has been used, first numerically to calculate the optimum filter structure and then experimentally, to scale and align the numerically synthesized filter in the microscope. A significant practical extension of the field of view has been observed, making the reported approach a valuable tool on the path to wide-field light-sheet microscopy.

Adaptive illumination based on direct wavefront sensing in a light-sheet fluorescence microscope

A methodology for the adaptive control and correction of phase aberrations in the illumination arm of a light-sheet fluorescence microscope has been developed. The method uses direct wavefront sensing on epi-fluorescent light to detect the aberration present in the sample. Using this signal, the aberrations in the illumination arm are subsequently corrected with a spatial light modulator in a feedforward mode. Adaptive correction, resulting in significant improvement in the axial resolution, has been demonstrated by imaging Tg(fli:GFP) zebrafish embryos.

High speed wavefront sensorless aberration correction in digital micromirror based confocal microscopy

The quality of fluorescence microscopy images is often impaired by the presence of sample induced optical aberrations. Adaptive optical elements such as deformable mirrors or spatial light modulators can be used to correct aberrations. However, previously reported techniques either require special sample preparation, or time consuming optimization procedures for the correction of static aberrations. This paper reports a technique for optical sectioning fluorescence microscopy capable of correcting dynamic aberrations in any fluorescent sample during the acquisition. This is achieved by implementing adaptive optics in a non conventional confocal microscopy setup, with multiple programmable confocal apertures, in which out of focus light can be separately detected, and used to optimize the correction performance with a sampling frequency an order of magnitude faster than the imaging rate of the system. The paper reports results comparing the correction performances to traditional image optimization algorithms, and demonstrates how the system can compensate for dynamic changes in the aberrations, such as those introduced during a focal stack acquisition though a thick sample.

Holographic imaging with a Shack-Hartmann wavefront sensor

A high-resolution Shack-Hartmann wavefront sensor has been used for coherent holographic imaging, by computer reconstruction and propagation of the complex field in a lensless imaging setup. The resolution of the images obtained with the experimental data is in a good agreement with the diffraction theory. Although a proper calibration with a reference beam improves the image quality, the method has a potential for reference-less holographic imaging with spatially coherent monochromatic and narrowband polychromatic sources in microscopy and imaging through turbulence.

Optimal model-based sensorless adaptive optics for epifluorescence microscopy

We report on a universal sample-independent sensorless adaptive optics method, based on modal optimization of the second moment of the fluorescence emission from a point-like excitation. Our method employs a sample-independent precalibration, performed only once for the particular system, to establish the direct relation between the image quality and the aberration. The method is potentially applicable to any form of microscopy with epifluorescence detection, including the practically important case of incoherent fluorescence emission from a three dimensional object, through minor hardware modifications. We have applied the technique successfully to a widefield epifluorescence microscope and to a multiaperture confocal microscope.

Use of digital micromirror devices as dynamic pinhole arrays for adaptive confocal fluorescence microscopy

In this work, we present a new confocal laser scanning microscope capable to perform sensorless wavefront optimization in real time. The device is a parallelized laser scanning microscope in which the excitation light is structured in a lattice of spots by a spatial light modulator, while a deformable mirror provides aberration correction and scanning. A binary DMD is positioned in an image plane of the detection optical path, acting as a dynamic array of reflective confocal pinholes, images by a high performance cmos camera. A second camera detects images of the light rejected by the pinholes for sensorless aberration correction.

Hybrid adaptive and computational light-sheet fluorescence microscopy

The light-sheet fluorescence microscopy is an excellent tool for the investigation of large three dimensional microscopy samples at the cellular level, however, the ability to resolve features is strongly affected by the presence of scattering and aberrations. These effects are two fold in light-sheet microscopy, as the illumination path providing the optical sectioning and the fluorescence detection path are both affected by the aberrations in different ways. To overcome these difficulties, we have developed hybrid adaptive optical and computational microscopy techniques to remove the effect of the aberrations in both the excitation and the fluorescence paths of these microscopes.

Practical guidelines for implementing adaptive optics in fluorescence microscopy

In life sciences, interest in the microscopic imaging of increasingly complex three dimensional samples, such as cell spheroids, zebrafish embryos, and in vivo applications in small animals, is growing quickly. Due to the increasing complexity of samples, more and more life scientists are considering the implementation of adaptive optics in their experimental setups. While several approaches to adaptive optics in microscopy have been reported, it is often difficult and confusing for the microscopist to choose from the array of techniques and equipment. In this poster presentation we offer a small guide to adaptive optics providing general guidelines for successful adaptive optics implementation.

Blind multi-frame deconvolution by tangential iterative projections (TIP)

A methodology for retrieving the unknown object distribution and point-spread functions (PSFs) from a set of images acquired in the presence of temporal phase aberrations is presented in this paper. The method works by finding optimal complimentary linear filters for multi-frame deconvolution. The algorithm uses undemanding computational operations and few a priori, making it simple, fast and robust even at low signal-to-noise ratios. Results of numerical simulations and experimental tests are given as empirical proof, alongside comparisons with other algorithms found in the literature.

Rapid identification of coherent pupil functions from multiple intensity measurements

By taking multiple input-output measurements, it is shown how to determine the input to an optical system that corrects unknown phase aberrations without interferometric measurements or online iterative optimization within a couple of seconds. It is shown to work in simulations and experiment. This technique may also be used to acquire the complex field in the pupil, hereby permitting a complex field image to be acquired.