Thesis
Optical sectioning techniques for widefield fluorescence mesoscopy with the Mesolens
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2023
- Thesis identifier
- T17415
- Person Identifier (Local)
- 201686804
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Optical sectioning in fluorescence microscopy is the process of removing out-of-focus light from the final image. Several techniques have been developed over the years and the gold standard remains confocal laser scanning microscopy (CLSM). As a point scanning instrument, a confocal microscope acquires data one pixel at a time which results in long acquisition times for large field of view (FOV), high-resolution images. Moreover, the confocal method is not useful when imaging large volume specimens with a low power, low numerical aperture (NA) lens. Hence optical sectioning techniques for widefield fluorescence imaging which use a digital camera to rapidly acquire high-resolution images are highly sought after. Widefield detection requires much less excitation light intensity allowing gentler imaging with less photobleaching and phototoxic effects simultaneously outperforming CLSM in acquisition speed. This thesis reports the development of optical sectioning imaging modalities that have been specified to be compatible with the unique properties of the Mesolens, a novel microscope objective lens that combines millimetre scale FOV, submicron lateral resolution and a long working distance. Firstly, HiLo microscopy was adapted into HiLo mesoscopy. HiLo mesoscopy performs optical sectioning by modulating the in-focus signal with random speckle illumination while leaving out of focus signal uniform. Thus, a weighted map of the image is created that holds information of where the image is in focus and where it is out of focus. HiLo mesoscopy achieved a section thickness of 5.2±0.3 μm at an acquisition speed of 30 seconds per image pair with an additional post-processing time of ~5 minutes compared to CLSM with a section thickness of approximately 6 μm and an acquisition speed of 15 minutes for a three-frame-average final image. Next, a Gaussian light sheet illuminator was constructed using a cylindrical lens to form a light sheet that covered the field of view of the Mesolens. Although the optical section thickness was 30 μm at the center and 40 μm at the edge of the Mesolens FOV, ten times larger than the axial resolution of 7 μm of the Mesolens would require for light sheet microscopy, in optically cleared and thick specimens the out-of-focus background was reduced when compared to widefield illumination when using this simple setup. Compared to CLSM, the section capability of the Gaussian light sheet was inferior, however, the acquisition speed is only limited by the camera framerate and the photodose to the specimen was low, as can be expected from a light sheet illumination setup. Lastly, a structured illumination microscopy (SIM) technique called blind-SIM was implemented to attempt super-resolution (SR) at the mesoscale. Blind-SIM uses random speckle illumination and maximum likelihood estimation algorithms (deconvolution) to estimate both the illumination pattern and the object (fluorophore distribution) to achieve super-resolution. Using the blind-SIM toolbox images of pollen grain and Actin network of MeT5A cells were processed and resulted in images with increased peak signal-to-noise ratio (pSNR) of 64.71 for the pollen grain and 67.04 compared to 15.21 pSNR for a widefield image of the Actin network. Processing time for an image stack comprised of 50 slices with 2691 x 2337 pixels per image was on the order of 10 hours, suggesting a processing time for a single full FOV Mesolens image of approximately 4 days. This thesis achieved proof of concept for the above-mentioned widefield optical sectioning techniques making them a viable alternative to CLSM and opened the path to tackling specific challenges for each technique.
- Advisor / supervisor
- McConnell, Gail
- Resource Type
- DOI
- Date Created
- 2021
Relations
Items
| Thumbnail | Title | Date Uploaded | Visibility | Actions |
|---|---|---|---|---|
|
|
PDF of thesis T17415 | 2025-06-27 | Public | Download |