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We present a reflection confocal microscope incorporating a white light supercontinuum source and spectral detection. The microscope provides images resolved spatially in three-dimensions, in addition to spectral resolution covering the wavelength range 450-650 nm. Images and reflection spectra of artificial and natural specimens are presented, showing features that are not normally revealed in conventional microscopes or confocal microscopes using discrete-line lasers. The specimens include thin film structures on semiconductor chips, iridescent structures in Papilio blumei butterfly scales, nacre from abalone shells and opal gemstones. Quantitative size and refractive index measurements of transparent beads are derived from spectral interference bands.

Presents several abstracts of the articles published in June 2000 issue of 'Proceedings RMS,' journal. 'Iterative Thresholding for Segmentation of Cells From Noisy Images,' by H.-S. Wu, J. Barba and J. Gil; 'A Simplified Method of Cleaning Ixodid Ticks for Microscopy,' by B.R. Dixon, T.N. Petney and R.H. Andrews.

The standard microscope architecture around which confocal microscopes are built imposes fundamental restrictions on the speed with which images (three-dimensional data sets) can be obtained. Commercially available slit scanning confocal microscopes are able to produce optically sectioned images at frame rates well in excess of 100 Hz. However only the focal (x-y) plane can be imaged at this speed. To image a volume specimen it is necessary to physically change the distance between the objective lens and the specimen. This refocusing process is often necessarily slow and represents a bottleneck to the speed of image acquisition. We describe the construction of a slit scanning confocal microscope based on what we know to be a novel microscope architecture, which permits images of other planes and, particular, the meridional (x-z) plane to be acquired in real time.

We describe an optical microscope system whose focal setting can be changed quickly without moving the objective lens or specimen. Using this system, diffraction limited images can be acquired from a wide range of focal settings without introducing optical aberrations that degrade image quality. We combine this system with a real time Nipkow disc based confocal microscope so as to permit the acquisition of extended depth of field images directly in a single frame of the CCD camera. We also demonstrate a simple modification that enables extended depth of field images to be acquired from different angles of perspective, where the angle can be changed over a continuous range by the user in real-time.

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.

The axial position of a laser-trapped particle has been controlled by modification of the wave front by means of a membrane deformable mirror. The mirror gives wave-front modulation in terms of Zernike polynomials. By modulation of the Zernike defocus term we can modulate the particle position under conditions of laser trapping. A polystyrene particle of 1-microm diameter was moved along the optical axis direction for a distance of 2370 nm in minimum steps of 55.4 nm. We also demonstrated particle oscillation along the optical axis by changing the focal position in a sinusoidal manner. From the frequency dependency of the amplitude of particle oscillation we determined the spring constant as 91.7 nN/m.

We describe a simple modification to a confocal microscope, which analyses the state of polarization of light emerging from the specimen so as to permit quantitative polarized light microscopy to be performed. The system uses a novel form of rotating analyser which, together with lock-in detection, permits images to be obtained where the image contrast corresponds to both specimen retardance and orientation (e.g. in the case of a birefringent specimen). Images are presented from a wide range of specimens and the origin of the contrast observed from simple point scatterers is investigated both theoretically and experimentally.

We describe a simple modification to a rigid endoscope so as to provide both high-quality conventional and confocal images of reasonably accessible regions of the body. This versatile system uses a structured illumination approach together with a conventional incoherent illumination source. Images taken in fluorescence are presented using this combined conventional and confocal endoscope.

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.

Rigid endoscopes provide high quality optical images of reasonably accessible regions of the inner body, especially regions such as the aero-digestive and genital tracts. In order to enhance the versatility of these instruments we describe a development that permits confocal endoscopic images to be obtained - along with traditional endoscopic images - in real-time, from within the living patient. The system is based around a host lenslet-array tandem scanning microscope, which is capable of producing images viewed directly by eye. These types of confocal microscope are configured for fluorescence imaging together with laser illumination. Hard and soft tissues in the mouth were imaged using this combined system.

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.

We describe an active optical system that both measures and corrects the aberrations introduced when writing three-dimensional bit-oriented optical memory by a two-photon absorption process. The system uses a ferroelectric liquid-crystal spatial light modulator (FLCSLM) configured as an arbitrary wave-front generator that is reconfigurable at speeds as great as 2.5 kHz. A method of aberration measurement by the FLCSLM wave-front generator is described. The same device is also used to correct the induced aberrations by preshaping the wave fronts with the conjugate phase aberration as well as to scan the focal spot in three dimensions. Experimental results show the correction of both on- and off-axis aberrations, allowing the writing of data at depths as great as 1 mm inside a LiNbO3 crystal.

The tandem scanning microscope permits confocal images to be obtained in real time and viewed directly by eye. The light budget of these instruments may be increased from a few percent to a few tens of percent by incorporating an array of microlenses so as to increase the amount of illumination light that reaches the specimen. These instruments are configured for fluorescence imaging together with laser illumination. We describe how the versatility of the instrument may be enhanced to permit the use of incoherent light sources as well as extending the imaging modes to include bright-field reflection.

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.

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.

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 &mgr;m in an aqueous specimen.

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.

We describe a simple method to produce an arbitrary complex optical field using a ferroelectric liquid crystal spatial light modulator. The system is configured so as to act as a pupil plane filter in a confocal microscope. The ability to tune the complex pupil function permits the system to be used both to modify the imaging performance by effectively engineering the point spread function as well as to remove optical aberrations present in the optical system.

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.

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.

We describe a novel method of characterizing the longitudinal chromatic aberration of microscope objectives by recording a series of axial responses as a function of wavelength as a plane reflector is scanned through the focal region of a confocal microscope. Measurements are presented for a variety of objectives with differing degrees of correction. The use of the chromatic focal shift to measure surface profiles is also discussed.

We describe an extremely simple method of obtaining optically sectioned images with conventional low-power imaging systems in real time. A single spatial frequency grid pattern is projected onto an object. Images taken at three spatial positions of the grid projection are processed to provide 3D images of macroscopic structures.

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.

Most confocal microscopes do not produce images in real time with nonlaser light sources. The tandem scanning confocal microscope does produce such images but, because the pinhole apertures of the Nipkov disk must be placed far apart to reduce cross talk between neighboring pinholes, only 1% or less of the light available for imaging is used. We show that, by using aperture correlation techniques and relaxing the requirement to obtain a pure confocal image directly, one can obtain real-time confocal images with a dramatically increased (25% or even 50%) light budget.

A compact scanning microscope that uses a semiconductor laser both to illuminate a specimen and to detect the signal reflected from it is described. It is demonstrated that the spatial filtering performed by the laser detector ensures confocal operation. Two detection regimes, one employing a laser power monitor and the other using the diode junction voltage as a signal, are compared.