Award details

Optical sectioning for 3D super-resolution microscopy

Reference	BB/P026486/1
Principal Investigator / Supervisor	Professor Daniel St Johnston
Co-Investigators / Co-Supervisors	Dr Edward Allgeyer, Dr George Sirinakis
Institution	University of Cambridge
Department	Gurdon Institute
Funding type	Research
Value (£)	150,787
Status	Completed
Type	Research Grant
Start date	01/09/2017
End date	28/02/2019
Duration	18 months

Abstract

The recent combination of Single Molecule Switching microscopy, adaptive optics and a 4Pi detection geometry, here termed 4Pi-SMS, has enabled imaging with <20 nanometre isotropic resolution throughout the entire volume of whole cells. However, the currently used widefield illumination scheme is not suitable for tissue imaging as: (1) It generates prohibitively high background levels for efficient single molecule detection and localization. (2) It compromises the aberration correction capabilities of the microscope. The purpose of this grant is to drastically improve the optical sectioning capabilities of the 4Pi-SMS microscope and extend its application range to tissue samples by integrating a two-photon spatiotemporal focusing module. Spatiotemporal focusing is a two-photon excitation technique implemented in a widefield format. We will use a previously proven optical geometry where ~100 femtosecond pulses, produced by a Ti:Sapphire laser, are stretched with a blazed grating, and are then increasingly compressed with the help of a telescope as they propagate through the sample, reaching their shortest duration, and hence highest intensity, at the focal plane of the objective. In this way TPE excitation is confined in a thin layer around the focal plane with an expected z-depth of 1.2 micrometres which is orders of magnitude smaller than widefield illumination. To validate the technique, we will use Drosophila egg chambers and mouse intestinal organoids as a test systems, as these are 50 micrometres and >100 micrometres thick respectively. We will focus on imaging endogenously-tagged epithelial polarity proteins to address the stoichiometry of polarity complexes in different regions of the cell and the nature of the boundaries between different polarity domains.

Summary

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.

Impact Summary

This project sets out to extend the range of single molecule switching (SMS) microscopy to thicker samples by using spatiotemporal focusing to illuminate a single sample plane, so that the images are not degraded by out of focus light. If successful, this will allow the super-resolution imaging of samples up to 100 micrometres thick, such as tissues and organoids, with 20nm resolution in all three dimensions. The incorporation of spatiotemporal focussing into the 4Pi-SMS microscope in the Gurdon Institute will provide groups in Cambridge with access to a super resolution system that can image tissue samples with almost molecular resolution. This has the potential to transform many areas of research that involve analysis of cellular architecture and the stoichiometry of protein complexes inside cells. The 4Pi-SMS system for which we are designing the spatiotemporal focusing module is being duplicated in Oxford and EMBL. Incorporation of spatiotemporal focusing into these systems, as well as the original system at Yale, will extend these benefits to a wide range of researchers around the world. We will also encourage other groups using SMS microscopy to exploit this approach by making the detailed designs and the technical specifications of the system freely available through open access publications and the web. The spatiotemporal focusing module developed by this project has the potential to improve other types of imaging by reducing background out of focus light, and we will explore the possibility of developing a commercial version through collaborations with industrial partners.

Committee	Not funded via Committee
Research Topics	Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	Tools and Resources Development Fund (TRDF) [2006-2015]
Funding Scheme	X – not Funded via a specific Funding Scheme

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