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Smart illumination for 3D-imaging of biological tissues
SMART ILLUMINATION FOR 3D-IMAGING OF BIOLOGICAL TISSUES
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Loic LE GOFF *, Lorry MAZZELLA, Sofia CECCHINI, Nicolas LEVI-VALENSI, Marc ALLAIN, Anne SENTENAC, Frédéric GALLAND,
Aix Marseille Univ, CNRS, Centrale Med, Institut Fresnel, 13013, Marseille, France * legoff @ fresnel. fr
https:// doi. org / 10.1051 / photon / 202513436
Three dimensional imaging of living tissues with widefield fluorescence optical microscopy is limited by out-of-focus blur, light dose, and acquisition speed. We discuss recent camera-based strategies that use light more intelligently to improve resolution, sectioning and reduce toxicity when imaging thick biological structures.
The microscope as a scientific instrument for biological discovery emerged in the 17th century. When Robert Hooke examined a thin slice of cork under a microscope, he saw tiny compartments and named them ' cells ', thereby helping to establish the idea that living tissues are made up of repeating units. For a long time afterwards, microscopes continued to reveal shapes— membranes, nuclei and filaments— without necessarily showing what those structures were doing.
Over the last two decades, the use of fluorescence has transformed biological imaging. Fluorescence is the process by which a molecule called fluorophore absorbs light at one wavelength, which excites its electronic state, and then de-excites by emitting light at a different wavelength. Using optical filters that only transmit the emitted light allows labelled structures to stand out with high contrast. This revolution was accelerated by the development of fluorescent proteins( FPs), which are genetically encoded tags that can be fused to proteins of interest inside living cells and organisms [ 1 ]. Through FPs, a wide variety of tools are available nowadays, including calcium or voltage reporters for neural activity, cytoskeletal markers for force and shape investigation, and stress or programmed cell death probes. Once the necessary genome edits have been introduced, entire organisms can be engineered to express these probes in selected tissues or throughout the body, enabling us to observe cellular behaviour in vivo rather than on an isolated glass slide.
Imaging cells within their host organism poses new technical challenges for the microscopist. It requires 3D imaging with high spatial and temporal resolution deep inside whole organisms, such as Drosophila embryos, zebrafish larvae or organoids. There are three practical hurdles to overcome. Firstly, out-of-focus fluorescence creates a blurred background that obscures details especially for deep observations. Secondly, building a 3D stack slice by slice increases the light dose, bleaching fluorophores and disrupting physiology. Thirdly, acquiring many planes is time-consuming; the dynamics we are interested in can exceed the capabilities of our scanners and cameras.
This article explores strategies that use structured and smart illumination to mitigate these constraints. The goal is simple: keep samples alive, capture events as they happen, and reveal structures that were previously hidden— by using light more intelligently.
3D-MICROSCOPY Microscopes are inherently asymmetric instruments. Light propagates along a single, privileged direction— the optical axis. Imaging instruments naturally create contrasted images in the transverse( xy) plane,
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