Rimas Juskaitis

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. 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.

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 1.4 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.

Confocal microscopy by aperture correlation

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.

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. We further demonstrate how this high-speed 3D multiphoton imaging system can be used to study neuronal activity at millisecond resolution at the subcellular as well as the population level.

Semiconductor laser confocal microscopy

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.

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. 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.

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. 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.

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. 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.

Enhancement of laser trapping force by spherical aberration correction using a deformable mirror

We have developed a method to enhance axial trapping force in optical tweezers by aberration correction of a laser beam with a membrane deformable mirror. The axial trapping force is strongly dependent on the quality of the laser beam spot, which is deteriorated by aberration due to the refractive index mismatch between a cover glass and water. The aberration correction, therefore, is crucial for stable trapping of a particle and for weak-force measurement with laser trapping. We have evaluated spring constants of the trapping force in the axial direction with and without aberration correction. The enhancement factor of the spring constants by the aberration correction has been achieved as 1.35 for the case of 5-mm sample thickness and as 1.83 for the case of 10-mm sample thickness. The numerical simulation is coincident with the experimental results. [DOI: 10.1143/JJAP.42.L701]

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. To obtain images from oblique planes at high speed, a two-photon system was built wherein a novel optical system is used to provide aberration-free scanning.