Over the past few decades, fluorescence microscopy has proven to become the most widely used imaging technique in the field of life sciences. Unfortunately, all classical optical microscopy techniques have one thing in common: their resolution is limited by the diffraction. Thankfully, due to the very strong interest, development of fluorescent microscopy techniques is very intense, with novel solutions surfacing repeatedly. The major breakthrough came with the appearance of super-resolution microscopy techniques, enabling imaging well below the diffraction barrier and opening the new era of nanoscopy. Among the fluorescent super-resolution techniques, Stimulated Emission Depletion (STED) microscopy has been particularly interesting, as it is a purely optical technique which does not require post image processing. STED microscopy has proven to resolve structures down to the molecular resolution. However, super-resolution microscopy is not a cure to all the problems and it also has its limits. What has shown to be particularly challenging, was the super-resolution imaging of thick samples. With increased thickness of biological structures, the aberrations increase and signal-to-noise (SNR) decreases. This becomes even more evident in the super-resolution imaging, as the nanoscopic techniques are especially sensitive to aberrations and low SNR. The aim of this work is to propose and develop a 3D STED microscope that can successfully image thick biological samples with nanoscopic resolution. In order to achieve that, adaptive optics (AO) has been employed for correcting the aberrations, using the indirect wavefront sensing approach. This thesis presents a custom built 3D STED microscope with the AO correction and the resulting images of thick samples with resolution beyond diffraction barrier. The developed STED microscope achieved the resolution of 60nm in lateral and 160nm in axial direction. What is more, it enabled super-resolution imaging of thick, aberrating samples. HeLa, RPE-1 cells and dopaminergic neuron differentiated from human IPS cells were imaged using the microscope. The results shown in this thesis present 3D STED imaging of thick biological samples and, what is particularly worth to highlight, 3D STED imaging at the 80μm depth, where the excitation and depletion beams have to propagate through the thick layer of tissue. 3D STED images at such depth has not been reported up to date.

Lenses in virtual reality headsets make it possible to put displays close to users' eyes and they provide users with wider field of view. However, they also distort the displayed image. Providing we know the lens distortion properties, it is possible to compensate for the distortion by displaying an image that is distorted in the opposite way. This thesis focuses on measurement of the distortion for an arbitrarily complex lens, while also considering chromatic aberration. A system capable of measuring of the distortion using just a camera attached to the virtual reality headset is designed and implemented. It is based on pattern detection and comparison of relative positions of points on the display and their correspondences in the distorted image. The result is a 2D distortion map that can be used to transform images such that they appear undistorted to the virtual reality headset users.

A new type of adaptive optics (AO) based on the principles of digital holography (DH) is proposed and developed for the use in wide-field and confocal retinal imaging. Digital holographic adaptive optics (DHAO) dispenses with the wavefront sensor and wavefront corrector of the conventional AO system. DH is an emergent imaging technology that gives direct numerical access to the phase of the optical field, thus allowing precise control and manipulation of the optical field. Incorporation of DH in an ophthalmic imaging system can lead to versatile imaging capabilities at substantially reduced complexity and cost of the instrument. A typical conventional AO system includes several critical hardware pieces: spatial light modulator, lenslet array, and a second CCD camera in addition to the camera for imaging. The proposed DHAO system replaces these hardware components with numerical processing for wavefront measurement and compensation of aberration through the principles of DH. We first design an image plane DHAO system which is basically simulating the process the conventional AO system and replacing the hardware pieces and complicated control procedures by DH and related numerical processing. In this original DHAO system, CCD is put at the image plane of the pupil plane of the eye lens. The image of the aberration is obtained by a digital hologram or guide star hologram. The full optical field is captured by a second digital hologram. Because CCD is not at the conjugate plane of the sample, a numerical propagation is necessary to find the image of the sample after the numerical aberration compensation at the CCD plane. The theory, simulations and experiments using an eye model have clearly demonstrated the effectiveness of the DHAO. This original DHAO system is described in Chapter 2. Different from the conventional AO system, DHAO is a coherent imaging modality which gives more access to the optical field and allows more freedom in the optical system design. In fact, CCD does not have to be put at the image plane of the CCD. This idea was first explored by testing a Fourier transform DHAO system (FTDHAO). In the FTDHAO, the CCD can directly record the amplitude point spread function (PSF) of the system, making it easier to determine the correct guide star hologram. CCD is also at the image plane of the target. The signal becomes stronger than the image plane DHAO system, especially for the phase aberration sensing. Also, the numerical propagation is not necessary. In the FTDHAO imaging system, the phase aberration at the eye pupil can be retrieved by an inverse Fourier transform (FT) of the guide star hologram and the complex amplitude of the full field optical field at the eye pupil can be obtained by an inverse FT of the full field hologram. The correction takes place at the eye pupil, instead of the CCD plane. Taking FT of the corrected field at the eye pupil, the corrected image can be obtained. The theory, simulations, and experiments on FTDHAO are detailed in chapter 3. The successful demonstration of FTDHAO encourages us to test the feasibility of putting CCD at an arbitrary diffraction plane in the DHAO system. Through theoretical formulation by use of paraxial optical theory, we developed a correction method by correlation for the general optical system to perform the DHAO. In this method, a global quadratic phase term has to be removed before the correction operation. In the formulation, it is quite surprising to find that the defocus term can be eliminated in the correlation operation. The detailed formulations, related simulations, and experimental demonstrations are presented in Chapter 4. To apply the DHAO to the confocal retinal imaging system, we first transformed the conventional line-scanning confocal imaging system into a digital form. That means each line scan is turned into a digital hologram. The complex amplitude of the optical field from each slice of the sample and aberration of the optical system can be retrieved by digital holographic process. In Chapter 5, we report our experiments on this digital line-scanning confocal imaging system. This digital line-scanning confocal image absorbs the merits of the conventional line-scanning confocal imaging system and DH. High-contrast intensity images with low coherent noise, and the optical sectioning capability are made available due to the confocality. Phase profiles of the samples become accessible thanks to DH. The quantitative phase map is even better than that from the wide field DH. We then explore the possibility of applying DHAO to this newly developed digital line-scanning confocal imaging system. Since optical field of each line scan can be achieved by the DH, the aberration contained in this field can be eliminated if we are able to obtain the phase aberration. We have demonstrated that the phase aberration can be obtained by a guide star hologram in the wide field DHAO systems. We then apply this technique to acquire the aberration at the eye pupil, remove this aberration from the optical fields of the line scans and recover the confocal image. To circumvent the effect of phase aberration on the line illumination, a small collimated laser beam is shone on the cylindrical lens. Thus the image is solely blurred by the second passage through the aberrator. This way, we can clearly demonstrate the effect of DHAO on the digital line-scanning confocal image system. Simulations and experiments are presented in chapter 6, which clearly demonstrates the validity of this idea. Since line-scanning confocal imaging system using spatially coherent light sources has proven an effective imaging tool for retinal imaging, the presented digital adaptive optics line-scanning confocal imaging system is quite promising to become a compact digital adaptive optics laser scanning confocal ophthalmoscope.

Clinical wavefront-guided corneal ablation has been now the most technologically advanced method to reduce the dependence of glasses and contact lenses. It has the potential not only to eliminate spherocylindrical errors but also to reduce higher-order aberrations (HOA). Recent statistics show that more than 96% of the patients who received laser in situ keratomileusis (LASIK) treatment reported their satisfaction about the improvement on vision, six months after the surgery. However, there are still patients complaining that their vision performance did not achieve the expectation or was even worse than before surgery. The reasons causing the unexpected post-surgical outcome include undercorrection, overcorrection, induced HOA, and other postoperative diseases, most of which are caused by inaccurate ablation besides other pathological factors. Therefore, to find out the method to optimize the LASIK procedures and provide a higher surgical precision has become increasingly important. A proper method to calculate ablation profile and an effective way to control the laser beam size and shape are key aspects in this research to resolve the problem. Here in this Master of Philosophy degree thesis, the author has performed a meticulous study on the existing methods of ablation profile calculation and investigated the efficiency of wavefront only ablation by a computer simulation applying real patient data. Finally, the concept of a refractive surgery system with dynamical beam shaping function is sketched, which can theoretically overcome the disadvantages of traditional procedures with a finite laser beam size.

A perfect duality exists between the problem of space-based orbit determination from line-of-sight measurements and the problem of designing an interplanetary autonomous navigation system. Mathematically, these two problems are equivalent. Any method solving the first problem can be used to solve the second one and, vice versa. While the first problem estimates the observed unknown object orbit using the known observer orbit, the second problem does exactly the opposite (e.g. the spacecraft observes a known visible planet). However, in an interplanetary navigation problem, in addition to the measurement noise, the following "perturbations" must be considered: 1) light-time effect due to the finite speed of light and large distances between the observer and planets, and 2) light aberration including special relativistic effect. These two effects require corrections of the initial orbit estimation problems. Because of the duality problem of space-based orbit determination, several new techniques of angles-only Initial Orbit Determination (IOD) are here developed which are capable of using multiple observations and provide higher orbit estimation accuracy and also they are not suffering from some of the limitations associated with the classical and some newly developed methods of initial orbit determination. Using multiple observations make these techniques suitable for the coplanar orbit determination problems which are the case for the spacecraft navigation using visible planets as the solar system planets are all almost coplanar. Four new IOD techniques were developed and Laplace method was modified. For the autonomous navigation purpose, Extended Kalman Filter (EKF) is employed. The output of the IOD algorithm is then used as the initial condition to extended Kalman filter. The two "perturbations" caused by light-time effect and stellar aberration including special relativistic effect also need to be taken into consideration and corrections should be implemented into the extended Kalman filter scheme for the autonomous spacecraft navigation problem.

<strong>Purpose:</strong> Although orthokeratology (non-surgical corneal reshaping, Corneal Refractive Therapy, CRT®) has been used for almost a half century, contemporary CRT's outcomes and mechanisms still require investigation. A series of studies was designed to examine different aspects of non-surgical corneal reshaping for myopic and hyperopic corrections, including the efficacy and stability of this procedure, the effect of the lens material characteristics (Dk/t), and the corneal or superficial structural change (e. g. corneal/epithelial thickness) in corneal reshaping. <br /> <strong>Methods:</strong> In the CRT® for myopia (CRT1) study, 20 myopes wore CRT® lenses on one eye and control lenses on the contralateral eye (eye randomized) for one night while sleeping. Corneal topography and refractive error were measured the night prior to lens insertion, immediately after lens removal on the following morning and at 20 and 60 minutes and 3, 6 and 12 hours later. In the CRT® for hyperopia (CRTH) study, 20 ametropes wore CRT®H lenses on one eye for one night while sleeping, the contralateral eye (no lens wear) served as control (eye randomized). Corneal topography, aberrations and refractive error were measured the night prior to lens insertion, immediately after lens removal on the following morning and at 1 and 3, 6, 12 and 28 hours later. In the relatively long term (4 weeks) CRT® for myopia (CRT2) study, 23 myopes wore CRT® lenses overnight and removed their lenses on awakening. Visual Acuity (VA), subjective vision, refractive error, aberrations, and corneal topography were measured at baseline, immediately after lens removal on the first day and 14 hours later, and these measurements were repeated on days 4, 10, and 28. The treatment zone size was demarcated by the change in corneal curvature from negative to positive and vice versa, using tangential difference maps from the corneal topographer. In the study of effects of Dk/t on CRT® for myopia (CRTHDK), 20 myopic subjects were fit with Menicon Z (MZ) lenses (Dk/t=90. 6, Paragon CRT®) on one eye and an Equalens II (EII) CRT® lenses (Dk/t=47. 2) on the contralateral eye (eye randomized). Corneal topography, refractive error and aberrations were measured before lens insertion (baseline), and the following day after overnight lens wear, on lens removal and 1, 3, 6, 12 hours later. In the study of short term effects of CRT® for myopia and hyperopia (STOK), 20 ametropes wore CRT® and CRT®H lenses in a random order on one eye (randomly selected). The lenses were worn for 15, 30 and 60 minutes (randomly ordered, with each period taking place on a different day). Refractive error, aberrations, corneal topography, and corneal/epithelial thickness (using OCT) were measured before and after lens wear. The measurements were performed on the control eyes at 60 minutes only. <br /> <strong>Results:</strong> In the CRT1 study, after one night of CRT® for myopia, the central cornea flattened and the mid-periphery steepened, and myopia reduced. In the CRTH study, after one night of CRT® for hyperopia, the central cornea steepened and the para-central region flattened, myopia was induced or hyperopia was reduced, all aberrations except for the astigmatism increased and signed spherical aberration (SA) shifted from positive to negative. In the CRT2 study, after 4 weeks of CRT® lens wear, in general, the treatment zones stabilized by day 10, vision improved, myopia diminished, total aberration and defocus decreased and higher order aberrations (HOAs) including coma and SA increased. The visual, optical and subjective parameters became stable by day 10. In the CRTHDK study, after one night of CRT® (MZ vs. EII) lens wear, the central corneal curvature and aberration were similar with a slight exception: The mid-peripheral corneal steepening was greater in the EII (lower Dk/t) lens-wearing eyes compared to the MZ (higher Dk/t) eyes. In the STOK study, after brief CRT® and CRT®H lens wear, significant changes occurred from the 15 minutes time point: The corneal shape and optical performance changed in a predictable way; the central cornea swelled less than the mid-periphery after CRT® lens wear, whereas the central cornea swelled more than the para-central region after CRT®H lens wear; the central epithelium was thinner than the mid-periphery after CRT® lens wear and was thicker than the para-central region after CRT®H lens wear. <br /> <strong>Conclusion:</strong> After one night of lens wear, CRT® and CRTH® lenses were effective for myopia and hyperopia correction, respectively. In the 4 week CRT study, the treatment zone size changed during the first 10 days. Its size was associated with VA, refractive error, aberrations, and subjective vision. In the CRTHDK study, after one night of lens wear, changes in corneal shape were slightly different, with more mid-peripheral steepening in the lower Dk lens-wearing eyes compared to the higher Dk lens-wearing eyes. Changes in central corneal shape and optical performance were similar in both eyes. In the STOK study, CRT® lenses for myopia and hyperopia induced significant structural and optical changes in as little as 15 minutes. The cornea, particularly the epithelium, is remarkably moldable, with very rapid steepening and flattening possible in a small amount of time.

<strong>Purpose:</strong> Although orthokeratology (non-surgical corneal reshaping, Corneal Refractive Therapy, CRT®) has been used for almost a half century, contemporary CRT's outcomes and mechanisms still require investigation. A series of studies was designed to examine different aspects of non-surgical corneal reshaping for myopic and hyperopic corrections, including the efficacy and stability of this procedure, the effect of the lens material characteristics (Dk/t), and the corneal or superficial structural change (e. g. corneal/epithelial thickness) in corneal reshaping. <br /> <strong>Methods:</strong> In the CRT® for myopia (CRT1) study, 20 myopes wore CRT® lenses on one eye and control lenses on the contralateral eye (eye randomized) for one night while sleeping. Corneal topography and refractive error were measured the night prior to lens insertion, immediately after lens removal on the following morning and at 20 and 60 minutes and 3, 6 and 12 hours later. In the CRT® for hyperopia (CRTH) study, 20 ametropes wore CRT®H lenses on one eye for one night while sleeping, the contralateral eye (no lens wear) served as control (eye randomized). Corneal topography, aberrations and refractive error were measured the night prior to lens insertion, immediately after lens removal on the following morning and at 1 and 3, 6, 12 and 28 hours later. In the relatively long term (4 weeks) CRT® for myopia (CRT2) study, 23 myopes wore CRT® lenses overnight and removed their lenses on awakening. Visual Acuity (VA), subjective vision, refractive error, aberrations, and corneal topography were measured at baseline, immediately after lens removal on the first day and 14 hours later, and these measurements were repeated on days 4, 10, and 28. The treatment zone size was demarcated by the change in corneal curvature from negative to positive and vice versa, using tangential difference maps from the corneal topographer. In the study of effects of Dk/t on CRT® for myopia (CRTHDK), 20 myopic subjects were fit with Menicon Z (MZ) lenses (Dk/t=90. 6, Paragon CRT®) on one eye and an Equalens II (EII) CRT® lenses (Dk/t=47. 2) on the contralateral eye (eye randomized). Corneal topography, refractive error and aberrations were measured before lens insertion (baseline), and the following day after overnight lens wear, on lens removal and 1, 3, 6, 12 hours later. In the study of short term effects of CRT® for myopia and hyperopia (STOK), 20 ametropes wore CRT® and CRT®H lenses in a random order on one eye (randomly selected). The lenses were worn for 15, 30 and 60 minutes (randomly ordered, with each period taking place on a different day). Refractive error, aberrations, corneal topography, and corneal/epithelial thickness (using OCT) were measured before and after lens wear. The measurements were performed on the control eyes at 60 minutes only. <br /> <strong>Results:</strong> In the CRT1 study, after one night of CRT® for myopia, the central cornea flattened and the mid-periphery steepened, and myopia reduced. In the CRTH study, after one night of CRT® for hyperopia, the central cornea steepened and the para-central region flattened, myopia was induced or hyperopia was reduced, all aberrations except for the astigmatism increased and signed spherical aberration (SA) shifted from positive to negative. In the CRT2 study, after 4 weeks of CRT® lens wear, in general, the treatment zones stabilized by day 10, vision improved, myopia diminished, total aberration and defocus decreased and higher order aberrations (HOAs) including coma and SA increased. The visual, optical and subjective parameters became stable by day 10. In the CRTHDK study, after one night of CRT® (MZ vs. EII) lens wear, the central corneal curvature and aberration were similar with a slight exception: The mid-peripheral corneal steepening was greater in the EII (lower Dk/t) lens-wearing eyes compared to the MZ (higher Dk/t) eyes. In the STOK study, after brief CRT® and CRT®H lens wear, significant changes occurred from the 15 minutes time point: The corneal shape and optical performance changed in a predictable way; the central cornea swelled less than the mid-periphery after CRT® lens wear, whereas the central cornea swelled more than the para-central region after CRT®H lens wear; the central epithelium was thinner than the mid-periphery after CRT® lens wear and was thicker than the para-central region after CRT®H lens wear. <br /> <strong>Conclusion:</strong> After one night of lens wear, CRT® and CRTH® lenses were effective for myopia and hyperopia correction, respectively. In the 4 week CRT study, the treatment zone size changed during the first 10 days. Its size was associated with VA, refractive error, aberrations, and subjective vision. In the CRTHDK study, after one night of lens wear, changes in corneal shape were slightly different, with more mid-peripheral steepening in the lower Dk lens-wearing eyes compared to the higher Dk lens-wearing eyes. Changes in central corneal shape and optical performance were similar in both eyes. In the STOK study, CRT® lenses for myopia and hyperopia induced significant structural and optical changes in as little as 15 minutes. The cornea, particularly the epithelium, is remarkably moldable, with very rapid steepening and flattening possible in a small amount of time.

This research mainly develops a new z-position measurement based on the chromatic aberration effect. An objective-type dark-field illumination scheme is built to produce diascopic chromatic aberration light, and aimed to enhance the signal-to-noise ratio. The xenon lamp is adapted to create white light with continuous spectrum, besides, lens with low Abbe number is needed to extend the degree of chromatic aberration, so lens made of PMMA is as a chromatic aberration component. In the proposed system, the depths of samples in micro-channel is illuminated by the dispersed light and scatter the optical signals, which are captured by a low numerical aperture (N.A.) objective lens. After the simple normalization, the intensity ratio of two selected wavelengths 450 nm (blue light) and 670 nm (red light) from the scattered spectrum becomes a reliable index for the depth information of the detecting objects. By means of establishing the relationship between depth and intensity ratio, every object flowing through diagnosed spot is able to be determined the depth level by cross-referencing the database. By using spectrometer as detector, delicate moving components for light filtering or electrical stage for light scanning can be excluded for high-speed z-position detection. Furthermore, in order to identify the depth level of sample with high flowing rate, avalanche photodiodes are adapted to achieve rapid detection. The experimental results show that the relationship between depth and intensity ratio is a parabola curve, but in this research, the region which tends to behavior linearly is adapted. The proposed system provides a linear detection range of ¡Ó15 £gm for particles with a diameter of 20 £gm. The lens with high Abbe number only obtains ¡Ó10 £gm with linear detection range though, the resolution for size is better than PMMA. The BK7 lens is capable to discriminate the depth change of 2 £gm micro-beads, note that there is no limitation of depth discrimination in this system, because of the measurement is achieved by cross-referencing the linear line. The use of UV-Vis-NIR spectrometer enable this system to analyze the depths of the samples in flow rate 0.5 mm/s. To gain the higher performance, the two avalanche photodiodes are utilized, and the short(CWL=450 nm, ¡Ó20 nm) and long(CWL=650 nm, ¡Ó20 nm) band pass filter are also equipped to represent enhancements of blue and red ray. The effective detection range extends to ¡Ó25 £gm and has high linearity(R square=0.99285) after the optimization of light stop. In high flowing rate detection, this system is able to identify the depth of sample when the flow velocity is 4.167 mm/s, the calculated throughput is 126 particles/s. It also successfully analyzes the depth of flowing human erythrocytes under the flow velocity is 2.778 mm/s, the velocity which the developed system is capable to analyze is about 5-8 folds to the conventional micro-PIV system. With this novel and simple approach, there will be the quantified information from z-direction of flowing body for bio-analysis, and also benefits estimating the performance of micro structure or device in the microfluidic chip, also the analysis of flow field. Except for dynamical detection, this system also be capable to apply in a open and static situation, such as cell or tissue proliferation assay.

No description available.

A detailed study of the aberrations of anamorphic optical systems is presented. This study has been developed with a theoretical structure similar to that of rotationally symmetric optical systems (RSOS) and can be considered a generalization.A general method of deriving the monochromatic primary aberration coefficient expressions for any anamorphic system types with double plane symmetry has been provided.The complete monochromatic primary aberration coefficient expressions for cylindrical anamorphic systems, toroidal anamorphic systems and general anamorphic systems with aspheric departure have been presented, in a form similar to the Seidel aberrations of RSOS.Some anamorphic image system design examples are provided that illustrate the use and value of the theory developed.