Thesis
Novel optical developments for axial super-resolution microscopy at the mesoscale
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2024
- Thesis identifier
- T16848
- Person Identifier (Local)
- 201860577
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- A balancing act inherent to all optical microscopy is the amount of a specimen that can be viewed in a single image and the level of spatial detail that can be resolved within it. From the theoretical basis of resolution set up by Abbe, Rayleigh and Sparrow in the 1800 and 1900s, it is understood that to obtain high (sub-cellular) resolution, a high numerical aperture (NA) lens is required. Generally speaking, with conventional optics, a high NA lens also has a high magnification, diminishing the amount of a specimen observed at once to a few hundred square microns. Furthermore, conventional optics are restricted by the physical nature of light; it is not possible to resolve detail below approximately half of the wavelength of the light used to illuminate the specimen. This phenomenon, known as the diffraction limit, is a driving factor in the advancement of super-resolution microscopy in recent years. While optical or chemical phenomena are utilised to breach the diffraction limit of imaging objective lenses in super-resolution microscopy, due to further requirements, high NA lenses are still necessary, meaning that only a handful of cells are imaged simultaneously with the exceptional resolution achieved by these systems. In 2016, McConnell et al. presented a novel bespoke objective lens with the unusual lens prescription of a low magnification and comparatively high NA (4x/0.47). The system, called the Mesolens, was designed with such a prescription to image large cell volumes (6 mm x 6 mm x 3 mm) while retaining sub-cellular resolution in 3D. The Mesolens has allowed for imaging of entire bodies and organs, resolving every fluorescently labelled organelle of interest within the specimen. Several optical developments have been reported to speed up the acquisition rate across the large imaging volume and to bring the 3D resolution of the system towards isotropy. However, until the work discussed in this thesis, the axial resolution of the Mesolens remained at the diffraction limit reported in the initial paper of 7 μm. In this work, I have developed two new illumination modalities that manipulate the excitation light before it reaches the specimen plane of the Mesolens, and both provide nanometre-scale axial resolution. This lays the groundwork for further exciting potential projects. The first technique demonstrated in this work extends the well utilised microscopic technique of Total Internal Reflection Fluorescence (TIRF) from the approximately 100 μm2 FOV available to commercially available high magnification, high NA TIRF objectives to the full field of view (FOV) of the Mesolens in widefield (4.4 mm x 3.0 mm). The modality, which we call MesoTIRF, allows for more than a five-fold improvement in contrast, with excellent signal-background ratio and near isotropic resolution. For practicality and to allow for full characterisation, this system was designed as a prototype initially around an upright microscope system (Chapter 2) before adapting for the Mesolens (Chapter 3) and thorough characterisation and demonstration of the imaging capability of this new illuminator is provided here. The second technique extends another microscopy method to the mesoscale, in this instance standing wave (SW) illumination. This method generates an interference pattern to excite fluorescence from labelled cellular structures and produces high-resolution images of three-dimensional specimen in a two-dimensional image. SW microscopy is generally performed with high magnification, high NA objective lenses: this results in high resolution images but only within a small FOV so the cell numbers are again limited. In Chapter 4, I will present a technique for performing SW imaging at the mesoscale. In the 4.4 mm x 3.0 mm FOV of the Mesolens, we performed topological mapping of more than 16,000 live cells simultaneously, alongside characterisation of the illumination mode and further applications in fixed mammalian cell imaging.
- Advisor / supervisor
- McConnell, Gail
- Resource Type
- DOI
关系
项目
| 缩略图 | 标题 | 上传日期 | 公开度 | 行动 |
|---|---|---|---|---|
|
|
PDF of thesis T16848 | 2024-03-12 | 公开 | 下载 |