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
View/ Open
Date
2019-09Author
Guastamacchia, Michele Gabriele Raffaele
Metadata
Show full item recordAbstract
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.