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

Lead Research Organisation: National Inst of Agricultural Botany
Department Name: Genetics and Breeding

Abstract

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.

Technical Summary

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.

Planned Impact

'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.

Publications

10 25 50
 
Description The laser ablation tomography (LAT) platform has been developed and tested on a range of exemplar specimens, each addressing an important biological question that takes advantage of the unique aspects of LAT compared with conventional bioimaging methods.

Hardware: Femto- and pic-second laser systems have been optimised for use on biological specimens that have spatially heterogenous materials (cell walls, water, intercellular air spaces, etc). Affordable yet high quality optics microscopes were purchased and tested.

Software: novel control software was written to integrate the movement and operation of the lasers so that precise ablation of the specimen surface can be obtained while preserving the integrity of subcellular structures and chemical composition for imaging and spatial quantitative measurement of analytes in the sample. This was not a trivial task, yet essential for extracting quantitative data at eh micron level from the images and (in future) the chemical analysis of the ablation plume. The software allows precise matching of the laser and the X,Y, Z position of the target. Control software using C# was designed to create a modular, plug-and-play system that will work seamlessly with different lasers, microscopes and cameras so that a wide range of specimen types and applications can be easily accommodated.
Commercially available software (Amira) for creating the 3D images from the image stack was used, which can also track and measure key features in the images (e.g. vascular elements).

Imaging:
wheat grains: the relative size, number and spatial distribution of A and B-type starch granules were visualised. This work involved collaboration with other BBSRC-funded work at NIAB.
Wheat root: tomographic images of root sections from a root hairless mutant and wildtype were compared with images obtained using conventional SEM. NIAB collaboration
oat stems: stem wall thickness was imaged and quantified. NIAB collaboration
Brachypodium stems: xylem and phloem were highlighted by the Amira software and tracked as vasculature passed from the internode stem through the node, and illustrated the anamastoses. The spatial distribution of fluorescent label from GFP-labelled proteins was visible in the images. Work was done in collaboration with the Sainsbury Lab.
Rice leaves: tissue detail was imaged and compared with images obtained using conventional microscopy. Collaboration with Sheffield Univ.
Exploitation Route We plan to submit follow-on grant proposals to continue development of the LAT platform
Discussions with partners in the manufacturing sector will continue to help design and build a prototype benchtop system in future work
The project has laid the groundwork to make a benchtop LAT available to labs and bioimaging centres where there is no expertise in lasers or resources to buy more expensive platforms
Access to a LAT system opens up new possibilities of measurements that are technically not possible using conventional means, e.g. architects and structural engineers needing to quantify the depth of burn in wood timbers used in building.
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The U.Cam IfM laser research team have had discussions with affiliated manufacturers with whom the envisioned benchtop LAT system can be designed and built. This has given the companies early sight of the developing system. These discussions will continue post-project. The IfM PhD student on the project continues development work on the project in his thesis work post-project. Several technical details remain, and these need to be resolved before a prototype commercial system can be designed. Follow-on work (depending on funding success) will build a prototype system in collaboration with manufacturing partners to test a benchtop LAT system. The current project work emphasised the development of hardware and software that enables a modular plug-and-play approach so that different lasers, microscopes, cameras, etc, can be integrated and controlled with the same control software, making the LAT system flexible for different user needs, types of specimens and applications.
First Year Of Impact 2019
Sector Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description Collaborated with Armstrong Optical, Ltd on testing a Precitec Optronik chromatic optical probe to perform chromatic confocal topography imaging of wheat endosperm to reveal micron-scale subcellular structures 
Organisation Armstrong Optical
Country United Kingdom 
Sector Private 
PI Contribution U. Cambridge Institute for Manufacturing PhD student provided samples, laser ablation platform and interpretation of results
Collaborator Contribution Armstrong optical provided the optical probe and image analysis
Impact The company helped researchers decide if this probe was appropriate for the task, and if the result was acceptable. The preliminary image provided insight into the sample, and helped researchers decide if they would purchase the probe from the collaborating company. Disciplines: Optics, photonics, plant biology
Start Year 2018
 
Description Collaboration on imaging botanical specimens and consultation on bioimaging 
Organisation University of Cambridge
Department The Sainsbury Laboratory
Country United Kingdom 
Sector Academic/University 
PI Contribution Sainsbury Laboratory provided samples and consultation.
Collaborator Contribution NIAB provided consultation and helped interpret results of tomographic imaging of vascular anastomoses through Brachypodium stem internodes. U. Cambridge Institute for Manufacturing provided the laser ablation platform and expertise of the PhD student on the project.
Impact may lead to joint publication. disciplines: photonics, plant biology
Start Year 2019
 
Description collaboration on bioimaging in rice 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution U. Cambridge Institute for manufacturing PhD student did the laser ablation tomography and production of 3D images of rice leaves. NIAB helped with interpretation of results.
Collaborator Contribution U. Sheffield supplied the samples and interpretation of results.
Impact The collaboration provided the student with additional samples of biological interest. disciplines: photonics, plant biology
Start Year 2019
 
Description Discussion meeting on bioimaging at the Cambridge Advanced Imaging Centre 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact University of Cambridge/Institute for Manufacturing PhD student, NIAB researcher and Cambridge Advanced Imaging Centre researcher met to discuss possibilities of collaboration and mutual bioimaging needs and capabilities.
Year(s) Of Engagement Activity 2019
 
Description Establishing a high-resolution Laser Ablation Tomography Platform for UK Bioimaging Research 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact PhD student on the project presented a poster to NIAB researchers and students, describing project and initial results.
Year(s) Of Engagement Activity 2017
 
Description Poster: "Biolaser": System Architecture of a High-Resolution Laser Ablation Tomography Platform 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Master's student Georgios Kokkinos presented a poster on his software control systems at the EPSRC-CDT ultra-precision engineering workshop.
Year(s) Of Engagement Activity 2018