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Adaptive Optical Microscopy Systems: Unifying theory, practice and applications

Periodic Reporting for period 3 - AdOMiS (Adaptive Optical Microscopy Systems: Unifying theory, practice and applications)

Reporting period: 2019-09-01 to 2021-02-28

Fluorescence microscopy is a central tool for investigation of molecular structures and dynamics that take place in biological cells and tissues. Coupled with progress in labeling methods that highlight molecules and features of interest, these microscopes permit observation of biological structures and processes with unprecedented sensitivity and resolution. Such microscopes are vital tools in development of our understanding of the functioning of cells, tissues and organisms, with benefits ranging from better understanding of the nature of life to enabling medical advances.

These microscopes have been enabled by the engineering development of diverse optical systems that provide different capabilities for the imaging toolkit. However, all of these methods can suffer from the effects of the variable optical properties of biological specimens. The optical distortions – or aberrations – introduced by these specimens blur the images, reducing image contrast and resolution, which limits the range of applications. Adaptive optics (AO) methods were originally developed for astronomical telescopes, in order to compensate for the blurring effects of turbulence in the atmosphere. More recently, they have been developed to overcome aberration problems in microscopes, recovering the optimal imaging performance. This technology shows great promise to improve virtually all types of research or commercial microscopes, but significant challenges must still be met before adaptive optics can be widely implemented in routine imaging.

The AdOMiS project concerns the development of the next generation of adaptive optics technology for microscopes. Many previous advances have been made through development of bespoke AO solutions to individual imaging tasks. However, the diversity of microscopy methods means that individual solutions are often not translatable to other systems. This project involves the creation of theoretical and practical frameworks that tie together AO concepts and provide a suite of scientific tools with broad application. This systems approach encompasses theoretical modelling, optical engineering and the requirements of biological applications.

The new technology developed in this project will enable deeper imaging inside specimens – microscopes will be able to image more of a specimen, including at depths that are currently not accessible. It will enable faster imaging – through improved efficiency, it will be possible to observe fast biological processes in greater detail. It will also improve super-resolution microscopes – these super-resolution methods reveal details that are too small to detect in a conventional microscope, but their sensitivity to aberrations limits their application; adaptive optics will improve these methods and make them more broadly applicable.
Several microscope systems have been built to act as test-beds for new adaptive optics methods and as systems for demonstration of newly-enabled applications. This includes two confocal and multiphoton laser scanning microscopes, which are the most common high-resolution imaging tools in biological research, and a light sheet microscope. We have also built a new generation of super-resolution microscopes with adaptive optics capability. Other systems have been introduced for the calibration an optimisation of adaptive optics elements. These test-beds have been used to extend beyond phase calibration to polarisation, which is required for several microscope applications.

Significant effort has been in the development of mathematical models of aberration measurement and correction. A framework has been developed to tie together existing diverse methods and to permit side-by-side comparisons of effectiveness in different microscope applications. This has shown which aberration correction methods are more applicable in certain operating regimes than other, which will enable future users to tailor their methods more precisely to applications. Further theoretical development has been undertaken to model aberrations in complex microscope systems, including so-called 4-pi systems that use dual opposing objective lenses. New mathematical representations and control strategies enable the implementation of adaptive optics in these advanced microscopes.

We have also moved the direction of research into other areas of imaging where adaptive optics can provide benefit. An area of major success is in endoscopy, where we have implemented imaging systems through multimode fibres for probing deep within living brain tissue. We have also investigated the complex optical effects in graded index (GRIN) lenses, which are commonly used for endoscopic imaging, including in medical applications.

We have worked with research partners to build up capabilities in biomedical applications, which will allow the full exploitation of our new methods. Through this work, we have access to applications in cell biology, immunology and neuroscience, which will allow demonstration of the full range of capabilities of the novel system and methods.
The AdOMiS project will generate the underlying adaptive optics technology that will underpin a wide range of advanced microscopes. These microscopes will be able to image at increased depths in specimens, at greater efficiency and higher speed. By improving the capabilities in super-resolution microscopes, they will open up new areas of application, which will improve our knowledge of biological function.

One of the key advances is a comprehensive theoretical framework that can bring together diverse current methods of adaptive optics and permit comparison between different technologies. This should provide guidance to developers as to the optimum methods that should be employed for particular applications. We will generate tools that improve the ability to measure aberrations with higher sensitivity and over a greater range of operation than was previously possible.

The project will also produce tools for the generation and control of light, both for correction of aberrations through adaptive optics, but also for wider applications. This includes control of the light’s polarisation, which is important for many imaging applications, and also the control of ultra short light pulses, which are used in many microscopes.

New and unexpected applications have opened up that permit deeper imaging inside tissues, which is particularly useful for neuroscience imaging. A suite of endoscopic and microscopic tools will be available to provide more detail from deep within brain tissue. This will enable neuroscientists to understand more about the nature of operation of brains by observing the connections between neurons and the development of networks in the brain.