Aberration retrieval for laser scanning microscopy

Smid, Pieter Henry (2019) Aberration retrieval for laser scanning microscopy. PhD thesis, University of Nottingham.

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This thesis explores different indirect wavefront sensing methods for detection of primary Zernike aberrations in laser scanning microscopes. All the presented aberration retrieval methods rely on analysing intensity distributions in the focal region and within the first dark ring of the Airy spot.

First a home-built reflection confocal microscope with a Zernike modal wavefront sensor is discussed (Chapter 3). Aberrations present in the sample or imaging system are measured indirectly by sequentially applying Zernike modes with a deformable membrane mirror (DMM) while maximising the detected intensity signal at the pinhole of the confocal microscope. When maximum intensity is reached at the pinhole, the Zernike mode(s) imposed by the DMM correct for the wavefront aberrations present in the sample and imaging system. The sensitivity of this modal method for measuring aberrations is discussed as a function of pinhole size for different Zernike modes and the difference between modal wavefront sensing in reflection and fluorescence is considered.

Large aberrations present a challenge for modal wavefront sensors since they can give rise to incorrect measurements due to cross-talk effects between the different Zernike terms. A way to solve this problem is to run through several correction iterations. This thesis proposes a new extension for modal wavefront sensing to tackle large sample induced aberrations more efficiently (Chapter 4). The new method uses an initial wavefront pre-correction which is based on a ray-tracing simulation of the sample. As a result, the number of iteration steps required is significantly reduced because the pre-correction removes the most relevant large aberrations present, thereby increasing the speed of the overall correction process.

Odd aberrations such as coma cannot be detected and corrected for in a reflection microscope because of a double-pass effect where the in-going light path and the return light path pass of different sides of any element present in the system such as the DMM. As a result of this effect, odd aberrations are cancelled out after the second pass through the system and the measurement/correction system is not able to detect the presence of them. Nevertheless, the focal spot at the image plane will suffer from odd aberrations and these will affect the imaging performance of the microscope. A new method is presented in this thesis to break up the double-pass effect and allow odd aberrations to be detected and corrected for in a reflection confocal microscope (Chapter 5). To achieve this the beam is scanned across an edge and the edge response is used to determine the aberrations present as opposed to looking just at the intensity passing through the confocal pinhole. This method is illustrated by looking at coma, a common odd aberration found in optical microscopy. It is shown that the image of the edge (edge response) displays a characteristic distortion which is typical of coma and the amount of coma present in the imaging system can be estimated from the edge response curve.

Finally a novel aberration retrieval method is presented. This method is aimed at retrieving the amplitude of primary Zernike aberrations (astigmatism, coma, spherical aberration) in the pupil (Chapter 6). The primary Zernike aberrations are retrieved by fitting a set of orthogonal circle functions within the central region of the intensity distribution recorded at up to 3 different image planes, typically taken at focus and then either side of focus. Characteristic combinations of aberration sensitive fitting coefficients (so-called aberration indicators) are derived for each primary aberration (astigmatism, coma, spherical aberration) and it is shown that these indicators can be used for aberration retrieval. Importantly for aberration retrieval the indicators are selected so that there is a linear relationship between the aberration amplitudes and their respective indicators up to amplitude values of about 0.13λ. The issue of aberration cross-talk (when several aberrations are present) is also addressed and it is concluded that the new aberration retrieval method is successful as long as the rms wavefront deviation of all primary aberrations remains below 0.1λ. Benefits of this new approach as opposed to techniques such as the Gerchberg-Saxton algorithm are that it is fast, uses less intensity images and is non-iterative.

In summary, this PhD project makes new contributions to the field of aberration retrieval and adaptive optics in scanning microscopy by i) improving the modal aberration correction technique using an initial pre-corrected wavefront to significantly speed up the aberration correction procedure, ii) overcoming a double-pass cancellation issue in a reflection confocal microscope when looking at odd aberrations by using an edge scan to determine the odd aberrations present and iii) proposing a new phase retrieval technique that uses aberration indicators to retrieve the primary aberrations present in the pupil by looking at no more than three intensity images.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Wright, Amanda J.
See, Chung W.
Keywords: Aberration retrieval; laser scanning microscopy
Subjects: Q Science > QH Natural history. Biology > QH201 Microscopy
Faculties/Schools: UK Campuses > Faculty of Engineering
Item ID: 56678
Depositing User: Smid, Pieter
Date Deposited: 08 Apr 2020 08:10
Last Modified: 06 May 2020 09:04
URI: http://eprints.nottingham.ac.uk/id/eprint/56678

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