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SUpeR REsolution Adaptive LIght Sheet Microscopy for high resolution volumetric imaging in turbid specimen

Final Report Summary - SURE-ALISM (SUpeR REsolution Adaptive LIght Sheet Microscopy for high resolution volumetric imaging in turbid specimen)

More than a century after the seminal work of S. Ramón y Cajal, the internal clockwork of the nervous system is gradually revealing itself. In particular in the last decades we have witnessed an unprecedented deepening of our understanding of the neuron, the basic building block of the nervous system. At the same time, functional brain imaging techniques such as Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI) and Computed Tomography (CT), give insight into its workings on the macro-scale and can do this with millimeter resolution. Yet knowledge of the macroscopic structure can only hint at the true complexity that hides within the intricate connective network patterns formed between the neurons. Each neuron can connect with thousands of other neurons throughout the brain, and it is precisely this connectivity that gives rise to complex behavior. To truly understand a complex organ such as the brain, it is fundamental to be able to study its dynamics in vivo in its natural, three-dimensional environment.

Unlike nuclear contrast techniques, fluorescence microscopy combines high resolution with high specificity while it is minimally invasive for living biological tissue. In recent years, light sheet fluorescence microscopy has revolutionized the field of developmental biology. Illuminating with a thin sheet of light allows high contrast imaging of life specimens with minimal sample exposure, photo-bleaching, and damage. Wide-field detection with a second objective, orthogonal to the light sheet, allows for rapid three-dimensional imaging. These characteristics make light sheet microscopy ideally suited for studying the development of biological specimen and the evolution of neurodegenerative diseases. Yet, even with light sheet microscopy, the resolution and contrast is limited when looking beyond the outer layers of brain tissue. Inhomogeneities in refractive index blur the image, while scattering and unwanted fluorescence introduce a hazy background that quickly overwhelms the image.

The objectives of this project are to study the inherent effect of surrounding biological tissue on image quality to gain a deeper understanding of its nature, and to discover novel ways to overcome these limitations. This project thus aims to cast a high definition view into challenging biological samples.

We set out by studying how the blur varies in three-dimensional space within a biological sample of approximately a 1x1x1 millimetre cube. We developed algorithms to estimate the blur size and, in a second step, efficient L1-norm regularized deconvolution to improve the sharpness throughout the volume. Although we certainly saw an improvement in image quality, it was limited to the parts of the sample closest to the detection objective. Next we started to build a light sheet microscope with adaptive optics capabilities. Adaptive optics, originally developed to overcome the atmospheric turbulence that hinders astronomical observations, measures and inverts the optical aberrations introduced by the atmosphere. We applied the same technology to correct for sample-induced aberrations. In principle this should provide sharp images throughout the sample. However, we found that in many biological samples the features of interest are not only blurred, but also hidden by a strong auto-fluorescent background. Although the fluorescence of endogenous molecules tends to be relatively weak, in large volumes its combined effect may dominate that of labelled structures. We therefore redirected our efforts to enable high contrast in the presence of a strong, even dominant , background. Our search has been fruitful. We found a new contrast mechanism for light sheet microscopy that can clearly distinguish labelled structures from auto-fluorescence and even laser scattering. When testing the novel method on sample phantoms, we demonstrated a 400-fold contrast improvement. Further tests with cells and bacteria have shown bio-compatibility. We now continue work with intact, live, biological specimen. Our work is currently submitted for publication to a well recognized international peer-reviewed journal.

Our findings have the most immediate impact in biomedical research. Beyond any doubt, researchers in the life sciences and in biomedicine in particular are constantly working at the limits of what is technically possible today. Optical microscopy can give a direct view into the biological machinery and it functioning or its malfunctioning. By pushing the limits of the current technology, our findings light a path towards a deeper understanding of the biological processes and their malfunctions.