This PhD thesis presents the development of a cost-effective and open (hardware and software) microscopy platform able to perform automated single molecule localisation microscopy (SMLM) for super-resolved high-content analysis (HCA). The work is motivated by the desire to increase access to advanced microscopy techniques like SMLM and HCA, including in lower resourced communities. For SMLM I have utilised and refined easySTORM, a low-cost and robust implementation of dSTORM. For automated microscopy I have developed a novel optical autofocus module that can maintain focus lock during extended easySTORM acquisitions and maintain focus as the microscope images multiple fields of view (FOV) across a multiwell plate. Initially I worked with commercial microscope frames and then extended automated microscopy and easySTORM to a cost-effective, modular, open microscopy platform, “openFrame” that can be adapted to a wide range of microscopy modalities. For this, I combined the openFrame with a low-cost multimode diode laser bank and novel low-cost, cooled CMOS sensor cameras.
I developed two different hardware-based autofocus systems focussing near infrared laser beams from fibre-coupled diode laser sources onto the microscope coverslip with the back reflection being imaged at a secondary autofocus camera. The first autofocus technique presented uses two cylindrical lenses to create a collimated elliptical beam profile with differing confocal parameters (and therefore different range/precision) in orthogonal directions. The second autofocus technique presented utilises a slit to form the elliptical beam profile and then uses a convolutional neural network (CNN) to determine the microscope defocus from the autofocus camera images. The CNN autofocus was then able to operate robustly for months over a range of ±100μm and achieve an accuracy of greater than 200nm. I integrated this autofocus system with an automated multiwell plate easySTORM microscope applied to image focal adhesion structures within melanoma cells and bacteria undergoing phagocytosis in THP1 cells.