Work Packages (WPs)

 

WP1: Super-resolution optical imaging for the analysis of cellular processes

Fluorescence super-resolution microscopy has seen a tremendous development over the last two decades, with the emergence of new and powerful techniques which allow for resolving structural details in cells and tissues far beyond the diffraction limit. Stochastic Optical Fluctuation Imaging (SOFI, developed by partner UGO) temporal blinking of fluorescent dyes or quantum dots for enhancing the spatial resolution and contrast of a microscopic image. SOFI not only provides super-resolution, but also, live- cell compatibility. Light-Sheet Microscopy (LSM) is a popular fluorescence microscopy technique that allows for imaging live samples with minimal light load and extreme contrast, but it does not provide super-resolution. The objective of the project of ESR1 based at UGO is to combine SOFI and LSM, which will allow for super-resolution imaging of thick live samples, with unprecedented quality and spatial resolution.

A major limitation of traditional LSM, which uses a Gaussian beam, is its rapid divergence away from focus. Recently, partner USTAN has demonstrated that using an Airy beam allows for high resolution over a much larger field of view of ESR2 based at USTAN is to build a Light-Sheet Microscope with Airy beam excitation for high-contrast deep-tissue imaging.

Complementing the efforts at UGO and USTAN, PQ will focus on advancing the state of the art of stimulated emission depletion microscopy (STED). Typical cell studies demand multilabel staining which is difficult to realise in STED, because this would require multiple depletion laser sources which are costly and impose an undesired light load on the sample. The goal of the ESR3 based at PQ is to explore alternative strategies, based on pulsed excitation together with nanosecond time-resolved detection.

WP2: High-resolution optical imaging of cardiac tissue

Cardiac disease is a most significant cause of mortality worldwide. Partner MPG developed the low-energy anti-fibrillation pacing (LEAP), an innovative concept to control life- threatening electrical turbulence in the heart LEAP, it is crucial to develop high-resolution cardiac imaging systems that will allow understanding the mechanisms of arrhythmia initiation and control. The project of ESR4 based at MPG will develop a system for multimodal imaging, data analysis, and visualisation for Langendorff-perfused intact mouse and rabbit hearts in vitro. The system will permit the real-time simultaneous measurement of membrane voltage, intracellular Ca2+, and mechanical deformation (strain), in a region of interest.

The system to be developed by ESR4 will provide unsurpassed spatial-temporal imaging resolution on organ level in vitro. However, two limitations remain: conventional fluorescent dyes do not permit in vivo long-term studies, and fluorescence imaging is currently limited to visible spectrum and therefore, limited to the surface of the heart.

The project of ESR5 based at MPG will overcome the first limitation by exploiting recent developments of optogenetic sensors, including genetically encoded voltage sensitive fluorescent proteins (VSFPs). The project of ESR5 will develop fiber-optic imaging tools and data processing tools for in vivo optogenetic electrophysiology under minimal invasive conditions, opening the path for in vivo long-term studies. The second limitation will be addressed by project ESR6 based at UGLAS, which will examine methods for increasing the effective collection of emitted fluorescent light from heart tissue during multiphoton excitation.

Partner NCU, who pioneered the development of OCT instruments efforts of MPG and UGLAS by focusing on the newest generation of OCT systems, based on, will complement the wavelength-tunable light sources. The main goal of the project of ESR7 based at NCU is to explore the potential of swept-source OCT to perform organ-scale imaging, in particular, for non-invasive diagnosis of the heart.

WP3: Advanced instrumentation for ophthalmic imaging

The world population is aging, which carries a high risk of eye diseases that significantly affect the quality of life. The projects of ESR8, ESR9 and ESR10 will develop advanced optical instrumentation to improve early diagnosis and follow-up of eye diseases.

A novel approach recently proposed by UPC6, based on hyperspectral imaging technology,which is a non-invasive technology capable of providing spectroscopic data for each pixel of a given image, will be implemented for detection and monitoring of eye diseases affecting the retina. The goal of the project of ESR8 based at UPC is to develop an advanced setup, including digital cameras and light-emitting diodes (LEDs) emitting at several wavelengths.

In spite of the huge amount of research devoted eye characterisation, there is no instrument on the market capable of performing a complete evaluation of vision in a fully autonomous and automatic way. The goal of ESR9 based at IMO is the development and clinical validation of a OCT system that can be incorporated in the instrument developed at UPC, capable of performing a complete evaluation of eye and vision.

OCT can generate images of retinal pathology with higher resolution than any other non- invasive technique. Complementing efforts at UPC, the project of ESR10 based at NCU will develop a new imaging setup that will bring together OCT and Scanning Laser Ophthalmoscope (SLO) with point spread function (PSF) engineering.

The clinical studies of the UPC and NCU prototypes will be done in collaboration with IMO.

WP4: Innovative optical components, methods and software for image analysis

The four projects in this WP involve optical and analysis tools that are specifically designed for optimising the images obtained with the techniques developed in WP1-WP3.

The project of ESR11 based at UPC will focus on identifying cost-effective semiconductor light sources with output power and wavelength optimally tuned for maximum speckle reduction, for the prototypes to be developed for ophthalmic imaging.

Complementing the efforts at UGO, USTAN and PQ, the goal of the project of ESR12 based at CNRS is the realization of nano-structured (NS) substrates for super-resolution fluorescence microscopy based on optical waveguides and photonic crystals. CNRS has shown that NS substrates enable the observation of localized events in cell membranes with highly reduced background noise.

Novel image analysis algorithms will also be developed. Although SOFI is exceptionally simple from a hardware point of view (it only requires rapid imaging with a conventional wide- field fluorescence microscope), image processing is challenging and demanding. Currently, the final super-resolved images require a computational time of minutes, and the raw images suffer from intensity skewing, which unfavourably discriminates dim emitters against bright emitters. The goal of ESR13 based at UGO is to advance the data processing and image deconvolution to make it much faster, and to rectify the brightness skewing problem.

Complementing these efforts, ESR14 based at UPC will develop innovative methods of image analysis to provide new quantitative measures for the classification of complex images. The approach will involve extending the concept of ordinal analysis of time series two-dimensional images and will be applied to both, cardiac and ophthalmic images.