M. A. A. Neil

Method of obtaining optical sectioning by using structured light in a conventional microscope

We describe a simple method of obtaining optical sectioning in a conventional wide-field microscope by projecting a single-spatial-frequency grid pattern onto the object. Images taken at three spatial positions of the grid are processed in real time to produce optically sectioned images that are substantially similar to those obtained with confocal microscopes.

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 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.

Time-domain whole-field fluorescence lifetime imaging with optical sectioning

A whole‐field time‐domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode‐locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time‐gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time‐gated images, 2‐D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real‐world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all‐solid‐state diode‐pumped laser technology. Whole‐field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.

Dynamic wave-front generation for the characterization and testing of optical systems

We describe a simple method for generating known optical aberrations dynamically, using a ferroelectric liquid-crystal spatial light modulator. Aberrations inherent in the optical system are measured and corrected, and as an example Kolmogorov turbulence is simulated for aperture sizes D/r(0) from 0 to 30, varying at frame rates up to 2.5 kHz. A measure of wave-front generation efficiency is introduced and is shown to be better than 86% for Kolmogorov phase screens with D/r(0) in the range from 0 to 30.

Optimized pupil-plane filters for confocal microscope point-spread function engineering.

We present a new method of superresolving pupil-plane filter design in confocal microscopy in which we specify the properties of the desired point-spread function and use an optimization procedure to determine a suitable pupil-plane filter. A new, flexible method of filter implementation using reconfigurable binary optical elements is described, and experimental results are presented.

A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning

This article describes a wide-field time-domain fluorescence lifetime imaging (FLIM) microscope with optical sectioning. The FLIM system utilizes a wide-field time-gated optical image intensifier, with a minimum gate width of 85 ps, to achieve high temporal resolution of fluorescence decays induced by ultrashort laser pulses. Different configurations, using excitation pulses of picojoule energy at 80 MHz repetition rate and of nanojoule energy at 10 kHz, are compared. The instrument has a temporal dynamic range spanning from 100 ps to tens of μs and is shown to have a temporal discrimination better than 10 ps. When applied to laser dye samples, it has produced FLIM maps demonstrating sensitivity to variations in both chemical species and local environment, e.g., viscosity. Wide-field optical sectioning is achieved using the technique of structured illumination, which is applied to remove out-of-focus light that can result in lifetime artifacts. The sectioning strength, which may be adjusted by choosing an appropriate spatial modulation frequency, is characterized and shown to be comparable to that of a confocal microscope. Practical considerations concerned with improving the quality of sectioned fluorescence lifetime maps, including using a large bit depth camera, are discussed.

Wide-field optically sectioning fluorescence microscopy with laser illumination.

We describe an extremely simple method by which optically sectioned fluorescence images may be obtained with conventional microscopes using laser illumination. A one‐dimensional grid pattern is introduced into the illumination system, together with a rotating ground glass diffuser. This causes an image of the grid pattern to be projected into the specimen. Images taken at three spatial positions of the grid are processed in a simple manner to provide optically sectioned images of fluorescent specimens.

Real time 3D fluorescence microscopy by two beam interference illumination

We describe a method of obtaining optically sectioned fluorescence images in a widefield conventional microscope by interfering two beams on an object so as to illuminate it with a single spatial frequency fringe pattern. Images taken at three spatial positions of the fringe pattern are processed in real time to produce optically sectioned images which are substantially similar to those obtained with confocal microscopes.

Closed-loop aberration correction by use of a modal Zernike wave-front sensor

We describe the practical implementation of a closed-loop adaptive-optics system incorporating a novel modal wave-front sensor. The sensor consists of a static binary-phase computer-generated holographic element, which generates a pattern of spots in a detector plane. Intensity differences between symmetric pairs of these spots give a direct measure of the Zernike mode amplitudes that are present in the input wave front. We use a ferroelectric liquid-crystal spatial light modulator in conjunction with a 4-f system and a spatial filter as a wave-front correction element. We present results showing a rapid increase in Strehl ratio and focal spot quality as the system corrects for deliberately introduced aberrations.