Pushing the boundaries of microscopy in time and space : a microscope built for highspeed single-molecule imaging and a microscope built for microgravity

Abstract

Cell signalling is a fundamental biological activity by which cells respond and adapt to chemical signals in their environment. All cells have these processes; from single-celled organisms such as yeast to cells to multi-cellular organisms. Optical microscopy is the most common method for observing the activity of living cells, including cell signalling. This thesis describes work pushing microscopy to new heights, both figuratively and literally. Firstly the limits of size and timescale were pushed to observe individual molecules of signal transduction proteins in live cells. Transcription factors are proteins that control gene expression, it is not yet fully known how these correctly find their DNA binding sites in a maze of many throughout the genome. It is thought that clustering may be a mechanism by which this is done, single-molecule fluorescence microscopy and in vivo stoichiometry analysis can elucidate this. Here a single-molecule fluorescence microscope was developed and optimised for highly inclined laminated optical-sheet (HILO) imaging capabilities and was used to image the transcription factors Mig1 in yeast, RelA in U2OS cancer cell lines and the androgen receptor (AR) in prostate cancer cell lines. Mig1-GFP results were compared to those previously obtained to assess the microscope function. HILO microscopy was used on cells before and after stimulation and the images analysed to measure changes in molecular complex size of these transcription factors before and after activation. Secondly the limits of where microscopes can be used were explored. Gravityscope is a new microscope built to operate on board a parabolic flight, which offers microgravity (10-2g) and hypergravity (2g) conditions. Cell signalling has been shown to be severely affected by low gravity across diverse areas from the human immune system, metabolism and in microorganisms such as yeast. Gravityscope was used to image yeast, a model cell signalling experiment: uptake of fluorescent glucose in yeast.