Sharika Mohanan

Abstract:

Previously, we introduced a microscope design that enabled rapid, random-access well plate imaging [ eLife, 10, e56426 (2021)10.7554/eLife.56426]. Here, we implement this design in a low-cost, compact, and portable prototype (the Exeter Multiscope) and apply it to the problem of capturing the contraction of cardiomyocyte monolayers, which have been plated into nine wells within a 96-well plate. Using a transmissive rather than reflective geometry, each well is sampled using 500 × 500 pixels across a 1.4 × 1.4 mm field of view, acquired in three colours at 3.7 Hz per well. The use of multiple illumination wavelengths provides post-hoc focus selection, further increasing the level of automation. The performance of the Exeter Multiscope is benchmarked against industry standard methods using a commercial microscope with a motorised stage and demonstrates that the Multiscope can acquire data almost 40 times faster. The data from both Multiscope and the commercial systems are processed by a 'pixel variance' algorithm that uses information from the pixel value variability over time to determine the timing and amplitude of tissue contraction. This algorithm is also benchmarked against an existing algorithm that employs an absolute difference measure of tissue contraction.

Abstract:

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.

Abstract:

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.