New optical techniques and hardware for studying live cell dynamics

Student thesis: Doctoral Thesis


Fluorescence optical microscopy has become an integral technique in the life sciences and has opened the door to investigating live biological specimens non-invasively at sub-cellular spatial resolutions with high specificity and temporal resolutions. One of the limiting factors of optical microscopy is that the spatial resolution is dictated by the diffraction limit of light.;This work shows the first use of LEDs to carry out widefield axial super-resolution standing wave microscopy with high temporal resolution. The technique was used to image red blood cell membrane dynamics in real time with no increase in photobleaching or toxicity rates compared to standard widefield imaging. This work also presents 3D computational reconstructions of the data allowing for easier visualisation and the possibility of carrying out further quantitative analysis.;Following on from Chapter 2, is an investigation into the development and application of multi-wavelength standing wave microscopy on live specimens in both emission and excitation modalities. These techniques are henceforth referred to in this thesis as TartanSW. This investigation found that using multiple excitation wavelengths allowed for a reduction in the nodal contribution of the images resulting in obtaining 32.3 % more spatial information about the structure of the specimen. It is also shown that by taking the difference images between each excitation channel the standing wave antinodal planes could be reduced in thickness enabling axial resolutions on the order of 55 nm when imaging live cell experiments.;The multi-emission technique was shown that it could be applied to be applied to imaging biological specimens using both widefield and confocal microscopy. However, the widefield data was not in line with the expected theoretical structure. There is the possibility of using plane ordering though to infer the directionality of a specimen structure and extract height maps though further work to develop computational tools to enable this will have to be implemented.;Finally, this thesis describes the work carried out making use of a new high-brightness 340 nm LED to develop a fast switching 340/380 nm illuminator and demonstrate its application for ratiometric Fura-2 Ca2+ imaging of live cell specimens with sub-5 nM precision that supports full frame video-rate temporal resolutions.
Date of Award8 Jan 2019
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsUniversity of Strathclyde
SupervisorGail McConnell (Supervisor) & David McKee (Supervisor)

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