Robert H. Cormack

Miniaturized fiber-coupled confocal fluorescence microscope with an electrowetting variable focus lens using no moving parts

We report a miniature, lightweight fiber-coupled confocal fluorescence microscope that incorporates an electrowetting variable focus lens to provide axial scanning for full three-dimensional (3D) imaging. Lateral scanning is accomplished by coupling our device to a laser-scanning confocal microscope through a coherent imaging fiber-bundle. The optical components of the device are combined in a custom 3D-printed adapter with an assembled weight of <2  g that can be mounted onto the head of a mouse. Confocal sectioning provides an axial resolution of ∼12  μm and an axial scan range of ∼80  μm. The lateral field-of-view is 300 μm, and the lateral resolution is 1.8 μm. We determined these parameters by imaging fixed sections of mouse neuronal tissue labeled with green fluorescent protein (GFP) and fluorescent bead samples in agarose gel. To demonstrate viability for imaging intact tissue, we resolved multiple optical sections of ex vivo mouse olfactory nerve fibers expressing yellow fluorescent protein (YFP).

Electrowetting lenses for compensating phase and curvature distortion in arrayed laser systems

We have demonstrated a one-dimensional array of individually addressable electrowetting tunable liquid lenses that compensate for more than one wave of phase distortion across a wavefront. We report a scheme for piston control using tunable liquid lens arrays in volume-bound cavities that alter the optical path length without affecting the wavefront curvature. Liquid lens arrays with separately tunable focus or phase control hold promise for laser communication systems and adaptive optics.

Simultaneous quantitative depth mapping and extended depth of field for 4D microscopy through PSF engineering

An extended depth of field (EDF) microscope that allows for quantitative axial positioning has been constructed. Past work has shown that EDF microscopy allows for features in varying planes to appear sharply focused simultaneously, however an inherent consequence of this is that depth information is lost. Here, a specifically engineered phase plate is used to create a point spread function (PSF) that contains both of the necessary attributes for extended depth of field and quantitative depth mapping. A two-camera solution is used to separate and capture the information for individualized post processing. The result is a microscope that can serve as an essential tool for full 3D, real-time imaging.

Simulation of electrowetting lens and prism arrays for wavefront compensation

A novel application of electrowetting devices has been simulated: wavefront correction using an array of electrowetting lenses and prisms. Five waves of distortion can be corrected with Strehl ratios of 0.9 or higher, utilizing piston, tip-tilt, and curvature corrections from arrays of 19 elements and fill factors as low as 40%. Effective control of piston can be achieved by placing the liquid lens array at the focus of two microlens arrays. Seven waves of piston delay can be generated with variation in focal length between 1.5 and 500 mm.

Noise removal in extended depth of field microscope images through nonlinear signal processing

Extended depth of field (EDF) microscopy, achieved through computational optics, allows for real-time 3D imaging of live cell dynamics. EDF is achieved through a combination of point spread function engineering and digital image processing. A linear Wiener filter has been conventionally used to deconvolve the image, but it suffers from high frequency noise amplification and processing artifacts. A nonlinear processing scheme is proposed which extends the depth of field while minimizing background noise. The nonlinear filter is generated via a training algorithm and an iterative optimizer. Biological microscope images processed with the nonlinear filter show a significant improvement in image quality and signal-to-noise ratio over the conventional linear filter.

Wide-angle nonmechanical beam steering using liquid lenses.

Nonmechanical beam steering is a rapidly growing branch of adaptive optics with applications such as light detection and ranging, imaging, optical communications, and atomic physics. Here, we present an innovative technique for one- and two-dimensional beam steering using multiple tunable liquid lenses. We use an approach in which one lens controls the spot divergence, and one to two decentered lenses act as prisms and steer the beam. Continuous 1D beam steering was demonstrated, achieving steering angles of ±39° using two tunable liquid lenses. The beam scanning angle was further enhanced to ±75° using a fisheye lens. By adding a third tunable liquid lens, we achieved 2D beam steering of ±75°. In this approach, the divergence of the scanning beam is controlled at all steering angles.

Reducing noise in extended depth of field microscope images by optical manipulation of the point spread function

This work describes improved methods and algorithms for implementing extended depth of field (EDF) microscopy through point spread function (PSF) engineering. It utilizes adaptive optics to create a test bed on which to evaluate new phase shapes for EDF. Being able to quickly and cheaply design novel PSFs is essential to overcome limitations of EDF that have prevented the technology from reaching mainstream use. Further improvement is made by reducing the noise normally seen in EDF images. Computational optics principles are used to first encode the noise with an identifiable pattern and a specially-tailored non-linear algorithm then removes the noise. This approach improves a microscopes imaging capabilities in photon-starved applications such as live-cell fluorescence and object tracking.

Real-time extended-depth DIC microscopy

Real-time visualization of live-cell dynamic processes has been realized in differential interference contrast (DIC) microscopy, with an extended-depth-of-focus (EDF) increase of about one order of magnitude. In addition, the diffraction-limited lateral resolution of the microscope is preserved. Experimentally, a custom-designed waveplate inserted in the optical path of a microscope causes feature information, from within the entire 3D specimen volume, to be uniformly encoded into a single CCD image in a way that, after processing, defocus blur artifacts are removed. The result is that extended-depth feature information can be visualized at video rates during live-cell dynamics investigations because there is no longer the need to acquire multi-focus image stacks at each time point. Retrieving the encoded extended-depth information requires specialized digital image processing techniques. This work concentrates on digital filter design for the reconstruction of the waveplate-encoded images. As a measure of filter quality, the signal-to-noise ratio (SNR), the modulation transfer function and the least mean square values are evaluated. Obtaining a high SNR and a lateral resolution comparable to those in conventional single-focus-plane microscopy images at the same time is a challenging goal in EDF microscopy. Filters are created in the frequency domain on the basis of the measured waveplate-encoded point spread functions. Results show that it is possible to produce video-rate, extended-depth-offocus images that have low noise levels and diffraction-limited resolution. This is illustrated by movies of fluorescent beads and of cytoplasmic streaming in live stamen hair cells from the spiderwort plant, Tradescantia, using extendeddepth DIC microscopy.

Real-time quantitative differential interference contrast (DIC) microscopy implemented via novel liquid crystal prisms

A phase shifting differential interference contrast (DIC) microscope, which provides quantitative phase information and is capable of imaging at video rates, has been constructed. Using a combination of phase shifting and bi-directional shear, the microscope captures a series of eight images which are then integrated in Fourier space. In the resultant image the intensity profile linearly maps to the phase differential across the object. The necessary operations are performed by various liquid crystal devices (LCDs) which can operate at high speeds. A set of four liquid crystal prisms shear the beam in both the x and y directions. A liquid crystal bias cell delays the phase between the e- and o-beams providing phase-shifted images. The liquid crystal devices are then synchronized with a CCD camera in order to provide real-time image acquisition. Previous implementation of this microscope utilized Nomarski prisms, a rotation stage and a manually operated Sénarmont compensator to perform the necessary operations and was only capable of fixed sample imaging. In the present work, a series of images were taken using both the new LCD prism based microscope and the previously implemented Sénarmont compensator based system. A comparison between these images shows that the new system achieves equal and in some cases superior results to that of the old system with the added benefit of real-time imaging.

A new expanded point information content design approach for 3D live-cell microscopy at video rates

Through a combination of optical design and algorithm development, a new expanded point information content (EPIC) microscope has been developed that is capable of extending the depth of field while simultaneously super locating the depth position of complex biological objects to within an accuracy of 75 nm. The data is then combined to form 3D animations of live-cell biological specimens. This is accomplished without the need to acquire multi-focal image stacks and is thus well suited for high-speed imaging.