Optical sectioning for 3D super-resolution microscopy

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Microscopy has been an essential tool in advancing our understanding of biology for centuries. Recent developments have improved the ability of microscopes to resolve close objects by a factor of >20 (recognised with the Nobel Prize in 2014), making it possible to image thin samples at the nanometre scale, which is the size of individual protein molecules. It has remained challenging, however, to perform super-resolution imaging of cellular structures in thick samples, such as tissues and organs, because of the large amount of out of focus light from above and below the image plane and the aberrations that are introduced as the light passes through the sample. We have recently built a state of the art super-resolution microscope that removes the aberrations using adaptive mirror technology that was first developed for the Hubble space telescope. We have demonstrated that this 4Pi-SMS microscope can image cells at molecular resolution, but its sensitivity and resolution deteriorate dramatically if the samples are too thick.We now propose to address the problem of out of focus background light in thick samples using another new approach called spatio-temporal focusing. This uses a special laser that produces pulses of light at twice the wavelength (and half of the energy) needed to excite the fluorescent molecules that the microscope detects. By shaping these pulses, we can produce conditions where the only fluorescent molecules to be excited lie in a thin sheet (1-2 micrometres thick) where two photons from the laser activate them simultaneously. This means that we only excite the molecules that we want to detect and reduces the background out of focus light by a factor of ~100. Incorporating spatio-temporal focusing into the 4pi SMS system will make it possible to perform quantitative super-resolution imaging on more complex samples, such as tissues or organoids, which will allow a whole new range of questions to be addressed. As a proof of principle, we will test how well this microscope can image single molecules in the ovaries of the fruitfly, Drosophila, and in mouse intestinal organoids, as these samples are readily available and are more than 50 micrometres thick. More specifically, we plan to investigate the molecular organisation of a conserved set of polarity proteins that make one side of a cell different from the other. This will provide a challenging test for the spatio-temporal focusing 4Pi-SMS, because several of these proteins are localised on the apical side of epithelial cells in both flies and mammals and therefore lie more than 10 micrometres deep in the sample. Visualising these proteins with 20 nanometre precision and being able to count the number of molecules in a complex will allow us to answer major open questions in the field. For example, we plan to investigate how the boundary between the apical and lateral sides of epithelial cells is specified and how the key apical polarity factor, atypical protein kinase C, is recruited to the apical membrane. The 4Pi-SMS microscope allows one to see structures inside tissues that are not visible with other light microscopy methods, and it is therefore hard to predict what new features we may find.