Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates.

Multiphoton microscopy is a powerful tool in neuroscience, promising to deliver important data on the spatiotemporal activity within individual neurons as well as in networks of neurons. A major limitation of current technologies is the relatively slow scan rates along the z direction compared to the kHz rates obtainable in the x and y directions. Here, we describe a custom-built microscope system based on an architecture that allows kHz scan rates over hundreds of microns in all three dimensions without introducing aberration.

Agitation-free multiphoton microscopy of oblique planes.

The scanning two-photon fluorescence microscope produces optically sectioned images from the focal plane. It is sometimes desirable to acquire images from other planes of the specimen that are inclined with respect to the focal plane. In this Letter, we discuss the issues concerned with acquiring such images together with the effects of the inclination angle on image resolution and sectioning strength.

Real-time slit scanning microscopy in the meridional plane.

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.

Real-time extended depth of field microscopy.

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.

Aberration-free optical refocusing in high numerical aperture microscopy.

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.

Dynamic axial-position control of a laser-trapped particle by wave-front modification.

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.

Adaptive aberration correction in a confocal microscope.

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.

Active aberration correction for the writing of three-dimensional optical memory devices.

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.