Understanding the limits of remote focusing.

It has previously been demonstrated in both simulation and experiment that well aligned remote focusing microscopes exhibit residual spherical aberration outside the focal plane. In this work, compensation of the residual spherical aberration is provided by the correction collar on the primary objective, controlled by a high precision stepper motor. A Shack-Hartmann wave front sensor is used to demonstrate the magnitude of the spherical aberration generated by the correction collar matches that predicted by an optical model of the objective lens. The limited impact of spherical aberration compensation on the diffraction limited range of the remote focusing system is described through a consideration of both on-axis and off-axis comatic and astigmatic aberrations, which are an inherent feature of remote focusing microscopes.

Sensitivity of remote focusing microscopes to magnification mismatch.

Remote focusing (RF) is a technique that greatly extends the aberration-free axial scan range of an optical microscope. To maximise the diffraction limited depth range in an RF system, the magnification of the relay lenses should be such that the pupil planes of the objectives are accurately mapped on to each other. In this paper we study the tolerance of the RF system to magnification mismatch and quantify the amount of residual spherical aberration present at different focusing depths. We observe that small deviations from ideal magnification results in increased amounts of residual spherical aberration terms leading to a reduction in the diffracted limited range. For high-numerical aperture objectives, the simulation predicts a 50% decrease in the diffracted limited range for 1% magnification mismatch. The simulation has been verified against an experimental RF system with ideal and nonideal magnifications. Experimentally confirmed predictions also provide a valuable empirical method of determining when a system is close to the ideal phase matching condition, based on the sign of the spherical aberration on either side of focus.

Random access parallel microscopy.

We introduce a random-access parallel (RAP) imaging modality that uses a novel design inspired by a Newtonian telescope to image multiple spatially separated samples without moving parts or robotics. This scheme enables near-simultaneous image capture of multiple petri dishes and random-access imaging with sub-millisecond switching times at the full resolution of the camera. This enables the RAP system to capture long-duration records from different samples in parallel, which is not possible using conventional automated microscopes. The system is demonstrated by continuously imaging multiple cardiac monolayer and Caenorhabditis elegans preparations.

Spinning disk-remote focusing microscopy.

Fast confocal imaging was achieved by combining remote focusing with differential spinning disk optical sectioning to rapidly acquire images of live samples at cellular resolution. Axial and lateral full width half maxima less than 5 µm and 490 nm respectively are demonstrated over 130 µm axial range with a 256 × 128 µm field of view. A water-index calibration slide was used to achieve an alignment that minimises image volume distortion. Application to live biological samples was demonstrated by acquiring image volumes over a 24 µm axial range at 1 volume/s, allowing for the detection of calcium-based neuronal activity in Platynereis dumerilii larvae.

Application of the windowed-Fourier-transform-based fringe analysis technique for investigating temperature and concentration fields in fluids.

The present work is concerned with the development and application of a novel fringe analysis technique based on the principles of the windowed-Fourier-transform (WFT) for the determination of temperature and concentration fields from interferometric images for a range of heat and mass transfer applications. Based on the extent of the noise level associated with the experimental data, the technique has been coupled with two different phase unwrapping methods: the Itoh algorithm and the quality guided phase unwrapping technique for phase extraction. In order to generate the experimental data, a range of experiments have been carried out which include cooling of a vertical flat plate in free convection conditions, combustion of mono-propellant flames, and growth of organic as well as inorganic crystals from their aqueous solutions. The flat plate and combustion experiments are modeled as heat transfer applications wherein the interest is to determine the whole-field temperature distribution. Aqueous-solution-based crystal growth experiments are performed to simulate the mass transfer phenomena and the interest is to determine the two-dimensional solute concentration field around the growing crystal. A Mach-Zehnder interferometer has been employed to record the path-integrated quantity of interest (temperature and/or concentration) in the form of interferometric images in the experiments. The potential of the WFT method has also been demonstrated on numerically simulated phase data for varying noise levels, and the accuracy in phase extraction have been quantified in terms of the root mean square errors. Three levels of noise, i.e., 0%, 10%, and 20% have been considered. Results of the present study show that the WFT technique allows an accurate extraction of phase values that can subsequently be converted into two-dimensional temperature and/or concentration distribution fields. Moreover, since WFT is a local processing technique, speckle patterns and the inherent noise in the interferometric data do not affect the resultant phase values. Brief comparisons of the accuracy of the WFT with other standard techniques such as conventional Fourier-filtering methods are also presented.