Periodic Reporting for period 1 - OPTIMAR (OPTical Imaging of Molecular and signalling Activity in Real-time: application to flatfish metamorphosis)
Reporting period: 2021-01-01 to 2022-12-31
With the world population increasing from the present 7 billion to 10 billion in 2050, aquaculture is the obvious answer to meeting the growing demand for fish. Flatfish are attractive species for aquaculture due to their high market value and consumer demand. However, abnormal development during metamorphosis is often a problem and has hampered the development of successful flatfish aquaculture.
Flatfish metamorphosis is an enigmatic biological process during which a symmetric larva transitions into an asymmetric benthic juvenile, with both eyes on the same side of the head. Although the external morphological changes associated with flatfish metamorphosis are known, how internal structures and functions modify is understudied. Larval metamorphosis has been studied using histological sections and gene expression profiling. However, the first requires sacrificing fish and the latter uses pool of larvae. Hence, there is an increasing demand for high-quality non-invasive 3D imaging tools to directly visualise the mechanisms of metamorphosis in whole live flatfish. This offers a unique opportunity for selective plane illumination microscopy (SPIM) and optical projection tomography (OPT), which are popular in biomedical research for imaging mesoscopic scale (0.5-10mm) model organisms (e.g. zebrafish larvae). However, their full potential for non-model organisms has not been properly explored.
The overall goal of the OPTIMAR project was to develop non-invasive mesoscopic optical imaging tools for real-time 3D imaging, to directly visualise and study the mechanisms of metamorphosis in whole flatfish. OPTIMAR combined state-of-the art-mesoscopic imaging modalities with recent advances in marine sciences, inverse problems and computer science. The developed imaging tools provide a unique means of assessing quality, welfare and flatfish metamorphosis progression, which are valuable indicators of sustainability in aquaculture. Moreover, these tools have shown potential to gain insights into the multitude of developmental transitions occurring in other marine organisms.
Flatfish metamorphosis is an enigmatic biological process during which a symmetric larva transitions into an asymmetric benthic juvenile, with both eyes on the same side of the head. Although the external morphological changes associated with flatfish metamorphosis are known, how internal structures and functions modify is understudied. Larval metamorphosis has been studied using histological sections and gene expression profiling. However, the first requires sacrificing fish and the latter uses pool of larvae. Hence, there is an increasing demand for high-quality non-invasive 3D imaging tools to directly visualise the mechanisms of metamorphosis in whole live flatfish. This offers a unique opportunity for selective plane illumination microscopy (SPIM) and optical projection tomography (OPT), which are popular in biomedical research for imaging mesoscopic scale (0.5-10mm) model organisms (e.g. zebrafish larvae). However, their full potential for non-model organisms has not been properly explored.
The overall goal of the OPTIMAR project was to develop non-invasive mesoscopic optical imaging tools for real-time 3D imaging, to directly visualise and study the mechanisms of metamorphosis in whole flatfish. OPTIMAR combined state-of-the art-mesoscopic imaging modalities with recent advances in marine sciences, inverse problems and computer science. The developed imaging tools provide a unique means of assessing quality, welfare and flatfish metamorphosis progression, which are valuable indicators of sustainability in aquaculture. Moreover, these tools have shown potential to gain insights into the multitude of developmental transitions occurring in other marine organisms.
OPTIMAR consisted of five work packages (WPs).
WP1. Light propagation modelling | Mesoscopic imaging modalities work well for embryos and larvae that are highly transparent. However, for juvenile fish these methods are compromised due to scattering. These can cause stripes and shadows along the illumination direction that will affect the image quality and lead to incorrect quantitative conclusions. The Fellow, Dr. Teresa M Correia, developed accurate light propagation models (forward models) and angularly selective measurements of scattered and fluorescence light, in addition to transmitted light, which enabled quantitatively accurate reconstructions.
WP2. Image reconstruction from undersampled datasets | Imaging dynamic biological processes in 3D with high spatial and temporal resolution can be very challenging. Therefore, Dr. Correia developed image reconstruction strategies, based on the idea of compressed sensing, which generate high quality reconstructions from OPT and SPIM accelerated (undersampled) acquisitions, and thus, enable the visualisation of fast dynamic processes. However, compressed sensing-type algorithms do not achieve computational speeds compatible with real-time applications. Hence, a physics-informed deep learning image reconstruction method was proposed to accelerate scans and achieve real-time reconstructions. The proposed deep learning method successfully generates images from accelerated acquisitions in real time, whereas compressed sensing can take several minutes to reconstruct a complete 3D image.
WP3. Optimal source and detector sampling | The methods developed in WP2 were combined with illumination and detection strategies to: 1) reduce the number of projection images used for the OPT reconstruction; 2) reduce the number of z-stack images required for SPIM imaging. OPT was accelerated by acquiring less than 30 projections instead of several hundreds, whereas SPIM acquisitions were accelerated by 8-fold by using a patterned illuminations strategy.
WP4. Mesoscopic imaging system | An automated mesoscopic imaging system, controlled by computer, with 360-degree rotation, was set up at the host institute. The user-friendly software enables GPU-accelerated 3D reconstructions from 2D projection of transmitted and fluorescent using standard reconstruction methods and advanced deep learning-based reconstructions methods. In addition, it allows users to easily visualise and navigate 3D images.
WP5. Experimental studies using flatfish | Abnormal metamorphosis was induced by treatment with blockers of the thyroid axis. Control and treated flatfish larvae groups at different stages of development were studied using mesoscopic imaging and sequencing techniques. Flatfish sample preparation required testing and optimising tissue clearing and fluorescence labelling protocols. Our results indicate that the proposed mesoscopic imaging tools can be used to gain spatial and temporal insights into the hormone driven tissue and organ remodelling processes that occur during metamorphosis.
OPTIMAR outcomes have been shared on our websites (https://www.ccmar.ualg.pt/ | https://qbioimaging.github.io/) Twitter (@TeresaM2Correia | @CienciasDoMar), LinkedIn and other social media platforms.
WP1. Light propagation modelling | Mesoscopic imaging modalities work well for embryos and larvae that are highly transparent. However, for juvenile fish these methods are compromised due to scattering. These can cause stripes and shadows along the illumination direction that will affect the image quality and lead to incorrect quantitative conclusions. The Fellow, Dr. Teresa M Correia, developed accurate light propagation models (forward models) and angularly selective measurements of scattered and fluorescence light, in addition to transmitted light, which enabled quantitatively accurate reconstructions.
WP2. Image reconstruction from undersampled datasets | Imaging dynamic biological processes in 3D with high spatial and temporal resolution can be very challenging. Therefore, Dr. Correia developed image reconstruction strategies, based on the idea of compressed sensing, which generate high quality reconstructions from OPT and SPIM accelerated (undersampled) acquisitions, and thus, enable the visualisation of fast dynamic processes. However, compressed sensing-type algorithms do not achieve computational speeds compatible with real-time applications. Hence, a physics-informed deep learning image reconstruction method was proposed to accelerate scans and achieve real-time reconstructions. The proposed deep learning method successfully generates images from accelerated acquisitions in real time, whereas compressed sensing can take several minutes to reconstruct a complete 3D image.
WP3. Optimal source and detector sampling | The methods developed in WP2 were combined with illumination and detection strategies to: 1) reduce the number of projection images used for the OPT reconstruction; 2) reduce the number of z-stack images required for SPIM imaging. OPT was accelerated by acquiring less than 30 projections instead of several hundreds, whereas SPIM acquisitions were accelerated by 8-fold by using a patterned illuminations strategy.
WP4. Mesoscopic imaging system | An automated mesoscopic imaging system, controlled by computer, with 360-degree rotation, was set up at the host institute. The user-friendly software enables GPU-accelerated 3D reconstructions from 2D projection of transmitted and fluorescent using standard reconstruction methods and advanced deep learning-based reconstructions methods. In addition, it allows users to easily visualise and navigate 3D images.
WP5. Experimental studies using flatfish | Abnormal metamorphosis was induced by treatment with blockers of the thyroid axis. Control and treated flatfish larvae groups at different stages of development were studied using mesoscopic imaging and sequencing techniques. Flatfish sample preparation required testing and optimising tissue clearing and fluorescence labelling protocols. Our results indicate that the proposed mesoscopic imaging tools can be used to gain spatial and temporal insights into the hormone driven tissue and organ remodelling processes that occur during metamorphosis.
OPTIMAR outcomes have been shared on our websites (https://www.ccmar.ualg.pt/ | https://qbioimaging.github.io/) Twitter (@TeresaM2Correia | @CienciasDoMar), LinkedIn and other social media platforms.
OPTIMAR highlighted the importance of cutting-edge mesoscopic imaging technologies for marine sciences. OPTIMAR proposed unique 3D mesoscopic imaging tools, protocols to perform studies using non-model organisms, and helped bridge the existing biology/optics/mathematics/computer science gap. It proposed several innovative solutions to overcome some of the main limitations of mesoscopic imaging techniques. More specifically, OPTIMAR proposed solutions to address the scattering problem in juvenile fish and potentially other marine organisms, novel illumination and detection strategies to accelerate data acquisition that, together with deep learning-based image reconstruction methods, enable real-time 3D imaging. Thus, OPTIMAR imaging tools provide researchers unprecedented opportunities to study the development of flatfish and other marine organisms, from an embryo to its juvenile form.
The OPTIMAR mesoscopic imaging system and software tools (available on GitHub once published) are extremely useful for researchers and companies working in various areas of marine biology and aquaculture and also particularly valuable to biomedical research.
OPIMAR produced education material and a frugal version of the system for outreach activities, including Science is Wonderful! Digital, to explain the physics behind the 3D microscopes used in OPTIMAR and inspire the next generation of STEM students and professional.
The OPTIMAR mesoscopic imaging system and software tools (available on GitHub once published) are extremely useful for researchers and companies working in various areas of marine biology and aquaculture and also particularly valuable to biomedical research.
OPIMAR produced education material and a frugal version of the system for outreach activities, including Science is Wonderful! Digital, to explain the physics behind the 3D microscopes used in OPTIMAR and inspire the next generation of STEM students and professional.