| People have never stopped exploring and observing small systems like cells. Since the first optical microscope was invented400years ago, the microscope imaging techniques are constantly innovated. There is an increasing need to extend these techniques into the third dimension, so that dynamic interactions between two or more components can be studied in whole living cells.In this dissertation, we demonstrate a simple, on axis multi-plane imaging technology that delivers real time3D imaging over cellular volumes. Our technique utilizes a quadratically-distorted phase grating (QD grating), in the form of an off axis-Fresnel zone plate that, by introducing a detour phase term, imparts an equal but opposing focal power in the positive and negative diffracted orders, thus providing a ability to image multiple (3-9) object planes simultaneously on a single image plane. The QD grating can be used in simple attachments which are fully compatible with commercial microscopes and standard camera systems, to record wide-field images focused simultaneously on multiple specimen depths. Thus the biggest advantage of this technique is that various light sources could be used, rather than coherent light source limited in most of other3D imaging techniques, which avoids the cell bleaching and/or damaged. Furthermore, simultaneity in measurement is important in applications for studies of rapidly changing objects in cell-biology (e.g. organelle and flagella dynamics), fluid-flow problems and other high-speed,3D tracking applications.However, maximizing the optical efficiency and improving the image quality are crucial in most modern microscope imaging applications. When using QD grating, the first challenge is to maximize photometric efficiency in the usable diffraction orders, by reducing the intensity in the unused higher orders, without compromising QD grating performance or functionality; the second challenge is to diminish chromatic aberration induced in non-zero diffraction orders of QD grating.In order to assess the influence of errors and thus find dominant ones to mitigate a comprehensive error budget, in the form of theoretical analyses, computer simulations and ray-tracing, is established. The analysis presented shows that with achievable accuracies, the plotting error, the residual surface roughness, the thickness of the spectral filters and QD grating substrate, the misplacing of the grating (both lateral and axial) and the delivery of images in the±1diffraction order do not produce significant image defects. And the axial spacing and location of the in-focus images are altered slightly by the use of thick lenses and the aberrations introduced by other optics. The influence on the ability to balance the energy in the multi-focal images arises from the slight change in the NA of the imaging system between diffraction orders of the QD grating. For narrow-spectral bands the etch depth error dominates other errors, but the analysis here was in1D and we expect this effect to be less in2D QD gratings. The most serious error in fluorescence imaging arises from the wavelength-dependent diffraction angle of the±1diffraction orders. In multi-focal imaging this effect may be largely corrected by pre-dispersing the light before it strikes the QD grating.Based on some principles of binary optics, it is demonstrated that grating efficiency could be improved by multi-etch fabrication, using theoretical models of gratings based on an analytical solution to the optimisation of the multi-level phase conditions. Under the conditions of refractive index n=1.46(fused silica) and wavelength of incident light λ=600nm, a set of optimised parameters of grating structure are obtained, which achieve a balanced intensity distribution between diffraction orders and maximum value of grating efficiency. Under these conditions each order contains28.84%,30.3%and30.45%of the flux, when the grating is single (binary-level), double (4-level) and triple (6-level) etched, separately. So we may conclude that double etches is good enough because little efficiency gain would be obtained in3etch levels. And the results are particularly beneficial when using multiple QD gratings, where diffraction losses cumulate. For example,9-plane simultaneously imaging, using dual back-to-back QD gratings would result in higher order losses of25.14%for binary-level grating compared to17.37%for4-level grating. Then some grating fabrication methods are explored, and film deposition technology might be our potential grating fabrication method due to the high accuracy of film thickness. However, for the long term, any process that allows the production of a continuous surface profile (grey level rather than discrete levels), or very-precise alignment of etches on the same side of the substrate (alignment better than about2microns), would offer new opportunities.Because QD gratings are dispersive, the images may be chromatically smeared if the dispersion is not corrected. In most of our former applications, due to the need to limit chromatic distortion, the QD grating-based technique is narrow band, limiting the incident spectral bandwidth, restricting photon flux and hindering application to multiple-fluorophore life science imaging. So we demonstrate an optically and ergonomically-efficient correction in this dissertation-using of a pair of grisms (a grating combined with a prism, both are commercially-available) in multi-plane polychromatic imaging, exploiting the inherent chirp of the QD grating to achieve a near-complete correction of the principal chromatic defects in the3D imaging by simply changing the grism separation in an unfolded, axial, optical system. It is this configuration, in which first-order diffraction of a selected wavelength (mid visible in this case) occurs for an un-deviated beam, that is exploited here to provide a simplified chromatic control system. It is shown that a collimated output beam with easily-varied chromatic shear is achieved by this grism pair. We assess this chromatic shear produced as a function of the grism separation by measuring the angle at which two different laser wavelengths are brought to focus as a function of the grism separation. We then successfully correct the dispersion in3D imaging of a compact polychromatic source using QD grating. For further application, we simulate the performance of the grism correction to the full bandwidth of the fluorophore imaging. The results show that for eGFP and Cy5the dispersion are nearly corrected, though for the broader bandwidth mCherry, there is evidence of some residual chromatic smearing manifest in some low-brightness "wings". Further exploration would be focused on the chromatic correction in9-plane imaging system and a more compact optical alignment to be compatible with commercial microscopes.In conclusion, the highlights of innovation of this work are following:1) Novel error budget methods-1D mathematical models of phase grating fabrication errors have been established, and influence of both fabrication errors and system errors induced by QD grating are assessed.2)1D mathematical models of multi-level (2,4,6) phase grating with balanced intensity distribution between diffraction orders have been established, which illustrate that4.4%efficiency gain is obtained when the QD grating is double etched (4-level) with optimised parameters.3) An innovated technique to correct chromatic smearing in QD grating based3D imaging system, using a simple, linear, grism (grating and prism) pair without compromising image quality. |
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