Enhancing spatial and temporal resolution for isotropic volumetric imaging and 3D cell tracking

Lead Research Organisation: Imperial College London
Department Name: Dept of Physics

Abstract

Optical microscopy is ubiquitous in biological sciences with fluorescence microscopy in particular being utilised to map specific labelled proteins and/or structures. While the majority of such research is performed on populations of cells growing on glass slides, increasingly there is an appreciation that more realistic environments are required to obtain relevant data on biological processes, and ultimately this means using live biological models. A range of small, optically accessible, organisms (e.g. zebrafish, nematode worms, etc) provide convenient live samples for studying biological processes in vivo. Such samples are inherently three-dimensional (3-D), zebrafish being <1 mm in diameter when under 16 days old, and therefore require 3-D discrimination to provide unambiguous positional/structural information. Most 3-D microscopy is undertaken with laser scanning microscopes that scan a spot of light through the sample, building up a map of fluorescence intensity point by point. Such scanning microscopes are typically optimised for higher magnifications (i.e. small fields of view), suffer from unequal resolution (transverse better than axial) and require significant financial investment (e.g. often >£150K). An alternative method of acquiring 3-D data is optical projection tomography (OPT), the optical equivalent to X-ray computed tomography, which can be implemented on a standard wide-field imaging microscope and can provide 3-D imaging at a fraction of the cost of point scanning systems. In OPT, wide-field images (either fluorescence or transmitted light) of a rotating sample are acquired at different orientations. These images can be used to reconstruct the 3-D distribution of fluorescence/absorption.
The standard approach to OPT imposes three key constraints: the requirement that at least the front half of the sample must be 'in focus', that the whole sample must stay in the field of view throughout the acquisition to prevent artefacts in the reconstruction process and that the sample must be non-scattering (i.e. transparent). The first two constraints limit the achievable spatial resolution, since they require the numerical aperture (NA) of imaging system to be small. This limit can be overcome by scanning the imaging lens, and therefore the focal plane, through the rotating sample while acquiring the angularly-resolved images. This produces an 'in focus' image of the whole sample that is superimposed on an out of focus "back-ground" signal that can be removed during image processing. We propose to extend this approach to yet higher resolution imaging of selected sub-volumes within the sample by incorporating a lateral scanning microscope stage to allow the motion of a "volume of interest" (VOI) inside a larger specimen (e.g. an organ) to be maintained in focus as the sample rotates. This VOI can then be modelled as a detailed structure within a larger 'unstructured' volume, to permit high resolution reconstruction without artefacts associated with parts of the sample entering/leaving the field of view. This would permit isotropic high resolution 3-D imaging of, e.g. immune cell distribution in specific organs in live zebrafish, which is currently not possible using the standard commercially available instruments.
To address the limits to temporal resolution, we would investigate a novel "orthogonal scanning approach", acquiring sequential images at right-angles with respect to each other, such that the 3-D structure/location of features within the sample could be determined much faster than the rotation period. This could be applied, e.g. to follow cell migration within a live zebrafish. Finally we will extend this system to simultaneously acquire two images at different wavelengths of light using a commercially available spectral image splitter. By analysing these two wavelength channels we will be able to indirectly probe the signalling events that control the immune response and occur within cells.

Technical Summary

We propose to use focal plane scanning OPT, where a high NA imaging lens is scanned axially during the acquisition at each projection angle to acquire high resolution information from all depths of the specimen replicating a parallel projection, to achieve isotropic high resolution volume of interest (VOI) imaging in whole intact samples. Due to the short depth of field (a result of using high NA optics) regions outside the VOI appear significantly blurred and can be accounted for in the reconstruction process using appropriate spatial frequency filtering to suppress artefacts. To realise this system the VOI will have to be tracked during the acquisition as the sample rotates, achieved by appropriate control of lateral and axial motorized translation stages. We will apply this to imaging the spatial distribution of immune cells with respect to the gut epithelium in 6-16 day old zebrafish (available transgenic models) in response to inflammation instigated by a high cholesterol diet (HCD). Moreover we will demonstrate the capability of this OPT system to acquire time-lapse tracking information with a temporal resolution at a fraction of the total acquisition time by employing an orthogonal angle scanning procedure. By acquiring two sequential images at orthogonal angles, the approximate 3-D position of objects (i.e. cells) can be calculated. Therefore over the full acquisition of the OPT data set (i.e. many pairs of orthogonal images) the objects can be tracked. We will use this to extend our investigation to the spatio-temporal dynamics of immune cells in the same HCD zebrafish. Finally we propose to investigate the cellular signalling processes that govern the immune response using a FRET biosensor for the activation of Caspase-1. A spectral image splitter will be incorporated into the system to simultaneously acquire donor and acceptor fluorescence images, which can subsequently be analysed to indicate the relative Caspase-1 activity in this HCD model.

Planned Impact

This project aims to develop a novel high resolution 3-D imaging modality aimed at enabling volume of interest (VOI) imaging on a micron scale and rapid 3-D feature tracking within intact live organisms, such as zebrafish embryos, of mm size. The proposed techniques would be widely applicable to objectives across biology, biomedical research and beyond with potential impact in basic biology research, drug discovery, healthcare, food security and materials science.
Besides the potential impact on academic research, the proposed new imaging capabilities would also benefit drug discovery. Pharmaceutical companies would benefit through more precise assays of the action of their compounds within specific organs in live zebrafish up to 16 dpf, with particular impact in studies of responses in whole organisms and phenotypes associated with cell migration (e.g. metastasis) and recruitment (e.g. inflammation) as well as tumour development. In general, zebrafish can serve as a cost effective and genetically tractable disease model in pharmaceutical development, e.g. providing initial in vivo screens for drug efficacy and toxicity before the expensive mammal testing phase - thereby addressing the "3Rs" agenda - as well as having the potential to measure/observe 'off-target' effects. Thus, this project would highlight new opportunities for instrumentation manufacturers and software developers in the UK and elsewhere - particularly in drug discovery sector but also for general microscopy and imaging applications.
The proposed system could be provided as an OPT 'add-on' to existing commercial microscopes and would provide a more cost effective route to 3-D imaging for general biological/biomedical research compared to advanced laser scanning confocal/multiphoton microscope systems. For drug discovery, zebrafish-based assays would require the development of automated animal handling and imaging/analysis systems and there would be considerable potential to extend the readouts, e.g. to spectroscopic measurements of relevant fluorescent labels. The potential impact on healthcare and society follows from advances in the understanding of fundamental biological processes, disease mechanisms and in more effective discovery and testing of new therapies. A further application would be non-destructive 3-D imaging of intact samples (e.g. histopathology) since high resolution OPT would enable whole complex samples to be studied without sectioning, which could help preserve fragile pathologies. This could have clinical benefit as well as impact on preclinical studies. A further key impact on healthcare could be in regenerative medicine where the development of techniques to promote and control cell and tissue growth for medical therapies could be imaged using high resolution OPT - potentially in a nondestructive manner to enable the development of cell/tissue growth to be monitored. The 3-D imaging capabilities could also be applied to a range of other extended samples including for the characterisation of biomaterials and also for materials science outside biomedicine, e.g. to study or evaluate polymer structures or the outcomes of 3-D (lithographic) "printers". 3-D imaging of intact (live) samples is also important for plant biology and OPT is finding increased application to study crop development and the action of diseases, chemicals and pests that impact food security. This field would particularly benefit from higher resolution 3-D imaging capabilities.
The results of this project would be disseminated through open access publications and presentations at such as BiOS Photonics West and CHI-HCA in the USA and ECBO/Laser Munich in Europe. Imperial has strong links with pharma and these would be exploited to reach companies like AstraZeneca, GSK and GE Healthcare, with whom the Photonics Group has already collaborated extensively. We would also explore opportunities for plant imaging with local plant biologists and companies like Syngenta.
 
Description We extended the performance of the 3-D imaging technique optical projection tomography (OPT) to improve spatial resolution, light collection efficiency and perform 3-D time-lapse cell tracking. Moreover, this was achieved with moderate modifications to a standard widefield fluorescence microscope, an item of equipment available in most biological/biomedical research labs. The overall motivation for this work was to provide a relatively simple and cost-effective microscopy technique to perform quantitative 3-D imaging of mm sized samples, for example biological research in live zebrafish embryos.
We first developed a stable imaging chamber and sample rotation stage that can be mounted onto any scientific widefield microscope, therefore extending the microscope's capability to perform standard OPT (i.e. 3-D imaging). The standard approach to OPT, often described as the optical analogue of X-ray CT, acquires widefield images, or 'projections', of a 3-D sample using an extended depth of field (i.e. the front half of the 3-D sample is imaged 'in focus') typically at 100's of rotation angles, which limits the achievable spatial and temporal resolution.
Improvements in these were achieved by combining focal scanning with a high numerical aperture (NA) objective lens to produce projections that contain both high resolution in focus information from the full extent of the sample together with out of focus light. We initially proposed to do this by directly scanning the microscope objective, but the required distance (~1 mm) resulted in relatively low frame rates (~1 Hz). Instead we opted for remote focal scanning using an electrically tuneable lens (ETL). In this approach the objective lens remained stationary and an additional optical system (incorporating the ETL) was inserted on the output of the microscope to realise significantly faster focal scanning (10s-100s Hz). This provides significantly improved light collection efficiency (x64 times), equating to an efficiency of x9 for a given sample plane, and improved spatial resolution (x3.5 times). In addition the use of focal scanning allows a particular region of interest within the sample to be imaged at higher resolution without suffering from significant reconstruction, which is not possible suing the standard approach. This use of a much higher NA means that the microscope defocuses over a much shorter distance, so objects outside the region of interest appear significantly defocussed and do not produce significant artefacts. This increased the efficiency of the system further, since all the acquisition time was used to image the region of interest and not the full extent of the sample.
We also employed an alternative 'orthogonal' rotational scanning approach using the remote focussing system, where each orthogonal pair of projections were acquired in quick succession followed by a user defined delay (defining the time-lapse resolution). Each pair of orthogonal projections was used to approximately triangulate the position of objects (e.g. a population of immune cells in a zebrafish embryo) at each time-point, and the data set as a whole analysed to track the cell motion. This realised 3-D cell tracking at much better time-lapse resolution and a significantly reduced light exposure compared to acquiring a full OPT acquisition at each time-point.
Exploitation Route A key aspect of the instrumentation developed during this project is the realisation of OPT on a standard widefield microscope frame. Commercially available optically sectioning microscopes used for acquiring 3-D datasets are priced at a level where typically only imaging facilities or institutions can purchase them and not individual researchers. Our relatively minor adaptation to a standard widefield microscope for OPT can provide a 3-D imaging capability to individual research groups through modification of existing equipment. It can them be extended to realise improved resolution and light collection efficiency through focal scanning, as developed during this project. Locally at Imperial College London we have secured funding from a BBSRC Impact Acceleration Account to install an OPT system on a widefield microscope in the imaging facility in the Department of Biological Sciences. This will make OPT easily available to the local biological research community, and we are providing seminars, training and support to help researchers explore if this technique can help with their 3-D imaging needs. To try and realise wider academic impact we are in discussion with a potential industrial partner to make this adaptation commercially available. We will also make our design available on our research group website along with open-source acquisition/reconstruction software in MicroManager, a popular open-source hardware control environment used in microscopy.
There is also potential for using small semi-transparent model organisms in the drug-discovery pipeline. This would require appropriate imaging technology for in vivo assay screening. OPT represents a potential screening tool in combination with automated sample handling, which could be addressed with an industrial partner operating in the pharma sector.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology,Other

URL https://www.imperial.ac.uk/photonics/research/biophotonics/instruments--software/optical-tomography/
 
Description Working with an SME partner, Cairn Research Ltd, initial prototype OPT imaging systems have been constructed. These include a low magnification system for imaging ~1 cm sized field of view and x4 magnification system for imaging ~1 mm sized fields of view, as employed in this project. As we continue to demonstrate the applications of OPT to an increasingly wide audience, we would hope that this will develop into a commercial kit for both academic and non-academic markets.
First Year Of Impact 2018
Sector Other
 
Description Impact Acceleration Award
Amount £13,721 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2015 
End 02/2016
 
Title Micromanager OPT acquisition software 
Description Developed acquisition programme in the using the open-source MicroManager software platform (https://micro-manager.org/wiki/Micro-Manager). 
Type Of Technology Software 
Year Produced 2016 
Open Source License? Yes  
Impact Any MicroManager supported cameras and other ancillary microscope components (e.g. filterwheels, shutters, etc) can be easily incorporated into the OPT acquisition software. 
 
Title OPT sample chamber for widefield microscope 
Description Sample chamber that can be mounted on a standard widefield microscope to perform optical projection tomography on mesoscopic samples (<~1 mm diameter) including in vivo imaging of zebrafish embryos. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2015 
Impact Secured BBSRC Impact Acceleration Account funding to Install an OPT sample chamber in the microscopy facility in the Department of Biological Sciences at Imperial College London. We are also in preliminary discussions with potential industrial partner about making this sample chamber commercially available. 
 
Description 3D optical tomography for ex vivo and in vivo imaging 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact "3D optical tomography for ex vivo and in vivo imaging", Invited oral presentation: SELECTBIO's Bioimaging: From Cells to Molecules
Year(s) Of Engagement Activity 2016
URL https://www.labtube.tv/video/3d-optical-tomography-for-ex-vivo-and-in-vivo-imaging
 
Description BiOS conference presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Region of interest and sub-volume optical projection tomography for high resolution 3D imaging of specific volumes within in vivo specimens on a commercial widefield microscope. Thomas Watson, Natalie Andrews, Laurence Bugeon, Margaret D. Dallman, Paul M. W. French and James McGinty.
Optical Projection Tomography (OPT) is a technique that can measure the absorption and/or fluorescence distribution in 3D samples including ~transparent in vivo model organisms such as C.elegans and D.rerio (zebrafish). In this talk we will present two opportunities presented by focal scanning OPT that overcome some of the limitations inherent in pure projection tomography; namely region-of-interest (RoI-) OPT and sub-volume (SV-) OPT. We will present these novel focal scanning OPT techniques implemented on a commercial widefield microscope for in vivo imaging of zebrafish embryos.
Year(s) Of Engagement Activity 2017
URL https://spie.org/PWB/conferencedetails/three-dimensional-microscopy
 
Description BiOS conference presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Functional imaging of live Zebrafish using fluorescence lifetime optical projection tomography. Natalie Andrews, Samuel Davis, Carys Hay, Marie-Christine Ramel, Laurence Bugeon, James McGinty, Margaret J. Dallman and Paul M. W. French.
Current microscopy techniques are not optimal to image fluorescence in whole live animals. We present fluorescence lifetime optical projection tomography (FLIM OPT) applied to functional imaging in live transgenic zebrafish expressing a Caspase 3 Förster Resonance Energy Transfer (FRET) biosensor. We can detect changes in Caspase 3 activity in embryo Tg(Ubi:Caspase3biosensor) zebrafish after induction of apoptosis by gamma irradiation. Though development of compressive sensing and multiplexed imaging with two imaging arms we have applied OPT and FLIM OPT to adult zebrafish, enabling us to quickly acquire datasets so the fish can be recovered and imaged longitudinally.
Year(s) Of Engagement Activity 2017
URL https://spie.org/PWB/conferencedetails/imaging-manipulation-analysis-biomolecules-cells-tissues
 
Description McGinty, Global Engage Microscopy Congress Invited Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact 3D optical tomography for ex vivo and in vivo imaging
Invited talk, Global Engage Microscopy Congress (2015)
Year(s) Of Engagement Activity 2015
URL http://www.globalengage.co.uk/microscopy.html
 
Description McGinty, OSA Optics in Life Sciences, OMP conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Techniques to improve the spatial and temporal resolution in optical projection tomography: remote focal scanning and time-lapse cell tracking
J. McGinty, L. Chen, S. Kumar, Y. Alexandrov, N. Andrews, D. Kelly, M. J. Dallman, P. M. W. French
OSA Optics in Life Sciences, OMP conference, Vancouver, 2015


Optics & Photonics Congresses (OPCs) are clusters of new and established topical meetings in order to bring together leaders among communities within optics. Congresses are designed to retain the collegial settings of OSA topical meetings and provide richer experiences for networking, information sharing and discussion across the disciplines of optical science and engineering. They also offer opportunities for more special events including plenary sessions, symposia, short courses and joint exhibits.
Year(s) Of Engagement Activity 2015
URL https://www.osapublishing.org/conference.cfm?meetingid=122&yr=2015
 
Description McGinty, Winter School on Neural Engineering Invited Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Optical imaging of 3-D biological samples
Invited talk, Winter School on Neural Engineering in conjunction with the CDT in Neurotechnology and the NETT EU Marie Curie initial training network in Neural Engineering (2015)
Year(s) Of Engagement Activity 2015
URL http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/engineering/centreforneurotechnology/n...
 
Description Open source optical projection tomography 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Website hosting information regarding open-source hardware implementation of optical projection tomography (OPT) on a commercial inverted microscope. This includes CAD designs for the OPT adaptor plate and acquisition software implemented in MicroManager (https://micro-manager.org/). Links to open source analysis and reconstruction software is also provided.

This open source hardware and software will also be described in a publication currently being reviewed (March 2017) entitled "Optical Projection Tomography Integrated Microscope using Open-Source Hardware and Software", Thomas Watson, Natalie Andrews, Samuel Davis, Laurence Bugeon, Margaret D. Dallman and James McGinty
Year(s) Of Engagement Activity 2017
URL https://www.imperial.ac.uk/photonics/research/biophotonics/instruments--software/optical-projection-...
 
Description Presentation and mentoring to the Sensor CDT at Cambridge University 
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 Presentation and mentoring to the Sensor CDT at Cambridge University on Optical Projection Tomography
Year(s) Of Engagement Activity 2016
 
Description Talk to the head of the Japan Science and Technology Agency (JST) and embassy officials 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Optical Tomography and whole body imaging.
Invited presentation to a delegation including the head of the Japan Science and Technology Agency (JST) and embassy officials, Imperial College London (2014).
Year(s) Of Engagement Activity 2014
 
Description Watson, OSA Biomedical Optics conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Remote focal scanning and sub-volume optical projection tomography (poster)
T.J. Watson, N. Andrews, E. Harry, L. Bugeon, M.J. Dallman, P.M.W. French and J. McGinty
OSA Biomedical Optics. Optical Tomography and Spectroscopy: Fluorescence, Raman, polarization spectroscopy and imaging (2016)

Advances in biomedical optics and biophotonics are being translated from the scientific lab bench into clinical medical and surgical applications to impact and improve our health. Fundamental discoveries in optical science and engineering have not only driven new questions in the medical sciences, but have enabled new ways to detect, diagnose, and treat diseases such as cancer and neurological disease.
This new OSA Congress will focus on technological solutions to medical challenges and medical applications, complementing the OSA Congress on Optics in Life Sciences. It will cover a diversity of cutting-edge research and innovative new tools and techniques, and will bring together an international group of leading engineers, optical and medical scientists, and physicians, as well as junior researchers and graduate students, who are engaged in optical methods to advance discovery and application of medical science to clinical practice. With over 400 attendees gathering at a beach-front resort in south Florida, this must-attend meeting affords a unique and exceptional opportunity for intellectually stimulating one-on-one interactions with leaders and colleagues in our field in a casual, inviting environment. We welcome you to join us for this exciting event.
Year(s) Of Engagement Activity 2016
URL http://www.osa.org/en-us/meetings/optics_and_photonics_congresses/biomedical_optics/