BioLaser: Establishing a High-Resolution Laser Ablation Tomography Platform for UK Bioimaging Research

Background summary
Two- and three-dimensional (tomographic) images of biological materials provide insight into the relationships between subcellular structure and function. However, particularly for hard or opaque tissues, instruments that can do this are currently large and extremely expensive. Furthermore, sample preparation and/or imaging is slow, preventing cost effective analysis of the many samples commonly required for biological studies. Thus, there is urgent need for new methods to image and quantitatively describe biological samples at high resolution and speed, allowing analysis of traits currently considered intractable to high-throughput investigation. While the component laser and imaging technologies exist, getting them to work together, and crucially, to achieve high-resolution sub-cellular imaging on biological samples, requires modifications and early concept exploratory and validation investigations. We propose novel modifications to existing, first-generation laser ablation (high-powered, ultrafast laser pulses) technology, enabling new biological insight and understanding into biological problems that were previously intractable using conventional techniques.

Project summary
'BioLaser: Biological Investigations by Laser Ablation Tomography' is a pump-priming project that aligns world-class expertise in laser technology (Institute for Manufacturing, IFM, at the University of Cambridge), plant research, physiology (NIAB, Cambridge) and image analysis (both centres). The approach centers around the use of precision lasering (based on a technique called 'Laser Ablation Tomography', LAT). This will be combined with in-process monitoring to adjust the ablation process according to the composition and topography of the sample ('metrology'), to generate precision ablated biological samples of unprecedented quality for microscopy and 'chemical imaging' platforms that render high-resolution 2D & 3D images, allowing quantitative analysis of cellular and subcellular structures. Using an array of plant tissues, we will evaluate & validate the parameters required to design a precision LAT bioimaging system. This project is structured around for key approaches, all of which must be addressed to successfully develop LAT-based methodologies for high-resolution bioimaging:

1. Expertise in laser-based manufacturing technologies: IMF provides this project with world-class expertise in precision laser research, engineering and manufacturing. Such on-site expertise is the foundation for project success.

2. Precise ablation control: To achieve high-resolution imaging, a uniform plane is required. This needs precise knowledge of the position and power of the laser alongside measurement of the ablated surface after each laser pulse, provided via in-process metrology to ensure high-resolution imaging is truly achievable.

3. Advanced tools for imaging and analysis: The precision machined samples produced will be investigated using a suite of state-of-the-art 2D & 3D imaging and analysis approaches, exploiting imaging expertise at the UofC.

4. Image validation: the tools and approaches to be developed are powerful and precise. However, used without parameterisation and validation, the resulting images could be of limited scientific value. The image data generated will be validated using conventional approaches, providing a feedback loop for parameter optimisation.

This project will lay the foundation for future manufacture of a benchtop prototype, delivered via IFM. Achieving the project aims would result in a step-change over first generation LAT systems that currently work at low magnification. Ultimately, we aim to establish a LAT bioimaging hub based in Cambridge, and to facilitate the production of similar platforms for the UK and beyond. This capacity-building approach will promote the critical-mass in technology uptake necessary for the potential impact of precision LAT bioimaging to be fully realised.

Background: 3D images provide insight into relationships between subcellular structure and function. However, CT or MRI instruments are slow and expensive, and conventional microscopy requires relatively transparent samples. Thus, new methods are needed to image and measure the internal structures of plants with high resolution and speed, allowing analysis of traits currently intractable to high-throughput investigation. Laser ablation removes a thin (100nm) layer of material from the substrate via high frequency laser pulses. The sample surface is imaged and the process repeated, producing a 2D stack used to build a 3D image. While established in materials science, such laser-ablation tomography (LAT) is under-exploited in biology, and has potential to overcome the limitations of other sectioning & imaging methods. In addition, volatilised plasma created during ablation can be chemically analysed, providing additional spatial sample composition information.

Project: BioLaser brings together experts in laser manufacturing (IFM), plant sciences (NIAB) and image analysis (both centres). We will combine an integrated ablation/imaging/chemical analysis system with metrology and enhanced optical delivery to control the morphology of imaged surfaces - a first in plant biology. Additionally, digital holography in conjunction with optical coherence tomography will tackle the challenge posed by heterogeneous materials in biological samples. This is a step-change over first generation LAT systems that currently work at low magnification. Using an array of plant tissues, we will evaluate and validate the parameters required to design a precision LAT bioimaging system, laying the foundation for future manufacture of a benchtop prototype, via IFM. Ultimately, we aim to facilitate the production of LAT-based bioimaging platforms for the UK and beyond, promoting the critical mass in technology uptake necessary for the potential impact of such platforms to be fully realised.

'BioLaser' is a timely application of sophisticated laser ablation techniques and in-process metrology, combined with state-of-the-art optical and chemical analysis systems. The project will test the feasibility of high-precision laser ablation tomography as an alternative to the time-consuming and labour-intensive constraints of biological sample preparation and imaging, thus overcoming phenotyping bottlenecks in order to fully exploit the power of modern genetic analysis approaches. By developing the hardware, software and methodological tools that enable phenotyping at sub-cellular scales, this project offers unique opportunities for UK bioscience. The project team have excellent track records in engagement, exploitation and outreach. NIAB is a respected member of the international plant science community, with close ties to the agricultural industry; the Centre for Industrial Photonics at IFM, which has close links to laser and microscope system manufacturers, has a world-class reputation. The project is multi-disciplinary, bringing together physics, plant biology, computer science and microscopy, and serves as an example of how different fields can work together to create a breakthrough technology.

Within academic disciplines, the work will benefit:
1. Plant biologists interested in affordable systems to measure cellular and subcellular structures via 2D or tomographic images.

2. Plant geneticists who desire to understand the genetic control of plant micromorphology, but are stifled by the lack of high-throughput means of analysis or screening samples.

3. Microscopy and bioimaging technologists will benefit from access to another tool to add to the methods available to visualise and quantify cellular structures. We will design the prototype LAT system such that an array of different microscopes and bioimaging tools can be integrated and tested.

4. Computer scientists and machine vision experts will be able to build on findings from this project that integrate lasers, microscopes and image capture and analysis for quantitative descriptions of biological materials. The images we produce will be available for exploring further analysis methods.

5. Photonic scientists will benefit from discovering how to measure and control laser optics for ablation of heterogeneous biological materials. The innovative combination of digital holography and optical coherence tomography will be of great interest to test in other laboratories, particularly those working with heterogeneous materials.

6. Experts in engineering, metrology and manufacturing sciences will gain insight into the system integration required for the development of the LAT platform, and potentially launch new thinking about scaling down high-end instrumentation into smaller scale, affordable machines for greater access to advanced tools.

The multidisciplinary nature of the project will benefit the participating individuals, with the close proximity of all project partners within Cambridge allowing the frequent personal interaction and coordination between scientists and engineers that is essential to ensure project success. The project also provides a focal point around which scientists across multiple disciplines can coalesce, eg '3CS', a new initiative that brings together NIAB, the University of Cambridge Plant Sciences, and the Sainsbury Laboratory. Benefits will extend to researchers in the UK and worldwide, through established connections by scientists at NIAB and IFM, and the network of interested parties and potential future collaborators to be established during the project.

Findings will be disseminated in peer-reviewed journals and at scientific conferences in the UK and internationally (e.g. European Workshop on Laser Ablation). Results and know-how will be transferred to potential imaging system manufacturers and end-users, and to the wider public through Open Days and the IFM Pathways to Manufacturing programme.