Search

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 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 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.

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.

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 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 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 frequency-modulated continuous-wave technique is used to detect phases of backscattered signals in a single-mode fiber. A distributed interferometric sensor system employing this technique is presented, and interrogation of 28 sensing regions is demonstrated. Spatial resolution 0.7 m and a sensitivity of 3 mrad/ radicalHz are achieved.