Fluorescence lifetime imaging microscopy (FLIM) is an imaging modality that is able to provide key insights into subcellular processes. When used to measure Förster resonance energy transfer (FRET), for instance, it can discern protein-protein interactions and conformational changes. This kind of information is highly useful in the drug screening process in order to determine the effectiveness of drug leads and their mechanisms of action. FLIM has yet to be successfully translated to high-content screening (HCS) platforms due to the high throughput and fine temporal and spatial resolution requirements of HCS.
Our prototype HCS FLIM system uses a time-resolving instrument called a streak camera to multiplex the FLIM scanning process, allowing for 100 confocal spots to be simultaneously scanned across a sample. There have been a few major advancements to the prototype. First the fiber array used to connect the fluorescence channels to the streak camera was characterized. Its alternating fiber delay scheme was successful in greatly reducing optical crosstalk between adjacent channels. Next, an optical beam scanner for parallel excitation beams was designed and implemented, greatly improving the possible scan speeds of the system. The streak camera was upgraded to a higher repetition rate sweep, and modifications to system components and reconstruction procedures were made to accommodate the new sweep unit. A single-photon avalanche diode array was also tested as a possible replacement for the streak camera, and was found to offer photon detection efficiency advantages. Finally, improvements were made to the excitation power and optical throughput of the system in order to reduce the required exposure time.
These advances to the prototype system bring it closer to realizing the requirements of HCS FLIM, and provide a clear picture for future improvements and research directions. / Thesis / Doctor of Philosophy (PhD) / Fluorescent proteins are commonly used to tag subcellular targets so that they can easily be distinguished with a fluorescence microscope. While this can help visualize where different organelles and proteins are located in the cell, a great deal more information can be gained by measuring the fluorescence lifetime at each point in the sample, which is highly sensitive to the microenvironment. Fluorescence lifetime imaging microscopy (FLIM) has the potential to be a powerful technique for testing drug leads in the drug discovery process, although current FLIM systems are not able to provide the high throughput speeds and high temporal resolution required for drug screening. This thesis project has succeeded in improving a highly parallel FLIM microscope by reducing inter-channel crosstalk, implementing an optical scanner, improving power and optical throughput, and investigating future time-resolving instruments. This progress has brought the prototype setup closer to being used in a drug screening environment.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/21467 |
| Date | January 2017 |
| Creators | Tsikouras, Anthony |
| Contributors | Fang, Qiyin, Engineering Physics |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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