Abstract
Many techniques have been developed to better understand the cellular
processes which govern normal function and pathogenesis in humans. The
technique which first allowed scientists to peer into this complex world was
light microscopy. However, the nature of light placed a physical limit to the
resolution achievable by light microscopy to no less than ∼200 nm. But at
the start of the 21st century, techniques which broke this resolution limit
emerged, giving rise to the field of super-resolution microscopy (SRM). SRM
circumvented the resolution limit through selective or stochastic activation
of fluorophores. Initial SRM techniques achieved ∼20 nm resolution, sufficient to resolve some individual large proteins. More recently, a new SRM
technique was introduced which achieved ∼1 nm resolution. A low-power
excitation beam with a central minimum was employed where localisation
of fluorophores was achieved through minimising the recorded photon flux,
giving rise to the name of the method: MINFLUX. We aim to produce an
improved MINFLUX microscope and apply it to living biological samples to
investigate protein distribution and changes in disease—namely Alzheimer’s
disease. Our initial target was the ryanodine receptor, a large protein known
to redistribute in cardiac and neuronal diseases.
Major steps toward the construction of a functional MINFLUX microscope
were achieved. A rudimentary super-resolution image of a synthetic fluorescent sample was generated by our system using a form of MINFLUX. Due to
limitations in time, our system was not able to be used for the visualisation
of proteins such as the ryanodine receptor in human tissue. However, with
the appropriate future direction, our system shows great promise in fulfilling this aim and uncovering pathophysiology for the guidance of disease
management and treatment.