R. Juškaitis

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.

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.

Method for the generation of arbitrary complex vector wave fronts.

We describe an extremely versatile method that permits the accurate generation of arbitrary complex vector wave fields. We implement the scheme using a reconfigurable binary optical element that also permits additional fine tuning, such as aberration correction, to be performed. As examples we demonstrate the generation of both azimuthally and radially polarized beams.

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.

Whole-field optically sectioned fluorescence lifetime imaging

We describe a novel three-dimensional fluorescence lifetime imaging microscope that exploits structured illumination to achieve whole-field sectioned fluorescence lifetime images with a spatial resolution of a few micrometers.

Whole-field five-dimensional fluorescence microscopy combining lifetime and spectral resolution with optical sectioning

We report a novel whole-field three-dimensional fluorescence lifetime imaging microscope that incoporates multispectral imaging to provide five-dimensional (5-D) fluorescence microscopy. This instrument, which can acquire a 5-D data set in less than a minute, is based on potentially compact and inexpensive diode-pumped solid-state laser technology. We demonstrate that spectral discrimination as well as optical sectioning minimize artifacts in lifetime determination and illustrate how spectral discrimination improves the lifetime contrast of biological tissue.

The imaging of dielectric point scatterers in conventional and confocal polarisation microscopes

Abstract We consider the image of a dielectric sub-resolution scatterer in conventional and confocal microscopes. We employ a vector theory and obtain theoretical images in a number of polarisation microscopes. In general the use of linear polarisation leads to asymmetric polarisation images whereas circularly symmetric polarisation images result if circular polarisation is used. Experimental results are presented to support the theoretical predictions.