Study of the effects of spherical aberration and signal levels on a diffraction-based multiplane microscope and its application to evaluate the fluid shear stress around a cell

Abstract

Multifocal/multiplane microscopy (MUM) is a technique to acquire simultaneously several planes at sample and obtain axially extended 4D imaging. This is an important characteristic that allows to track fast single molecules/particles three-dimensionally, in real time and over wide axial ranges (≈ 8 µm). MUM avoids possible ambiguous localisations due to the scanning of the imaged plane to acquire a 3D volume over time. For this thesis, a diffraction multiplane system has been characterised to evaluate the impact of different levels of spherical aberration and signal and applied to measure velocity and shear stress fields due to the flow of a liquid around a cell. The spherical aberration has been quantified via the curves of sharpness that can measure the amount of aberrations in images. This has shown that the measured plane spacing grows as the spherical aberration increases. The influence of spherical aberration on image sharpness as a function of emitter axial position could potentially be used to generate correction factors and improve the accuracy on the recovered positions. In terms of performance, the axial range over which the expected axial positions can be calculated with accuracies of at least 100 nm has been shown to vary linearly with the signal level in the studied range. The signal to noise ratio (SNR) threshold below which the axial range goes to 0 µm has been calculated to be 1.23 ± 0.71. It has also been demonstrated that the axial range can be potentially raised by enlarging the plane spacing. Regarding the precision on the axial positions, this varies exponentially with the signal with a decay constant of 0.51 ± 0.10 per SNR unit. This work has generated two equations to predict the expected axial range and precision, given the system parameters are known. Concerning its applications, MUM has been tested to perform micro-particle image velocimetry (µPIV), a technique able to reconstruct the velocity and shear stress fields imposed by a liquid flowing around a cell. The system has been, first, tested in absence of cells, achieving, within 10 µm from the coverslip glass, an accuracy on the calculated velocity of (0.42 ± 0.32) µm/s. This value is slightly worse than that obtained by using a confocal microscope, which is (0.30 ± 0.13) µm/s. Above 10 µm, instead, MUM performance is considerably inferior than that reached with the confocal microscope. In presence of cells MUM has been used for the first time to capture the perturbations to the expected laminar flow, allowing to measure velocities of 30 µm/s and shear stresses of 3 Pa around the observed cell. The reconstructed fields show characteristics similar to those reported in the literature. However, the observation of unexpected velocity and shear stress values indicate a reduction in accuracy caused by false axial localisations.