đź“° Image processing allows unmatched resolution for fluorescent microscopes

đź“° Image processing allows unmatched resolution for fluorescent microscopes

Researchers of Sophia-Antipolis Computer, Signal and Systems Laboratory (I3S – CNRS / University of the Costa Blava) i Villefranche-sur-mer Developmental Biology Laboratory (LBDV – CNRS / Sorbonne University) won the Award for best paper finalist at the conference IEEE International Biomedical Imaging Symposium (ISBI) 2022, for its proposed method that improves the lateral and axial resolution of images acquired with a microscope of fluorescence (Fluorescence is a light emission caused by the excitation of a molecule …) per total internal reflex (In geometric optics, a ray of light that crosses a separation between two means of indexes …) (TIRF).

For all optical microscopes (including fluorescent microscopes), the highest possible spatial resolution is governed by certain fundamental physical laws related to the propagation of light. light (Light is the set of electromagnetic waves visible to the eye …). There is a resolution limit (called the diffraction limit) that cannot be exceeded by physical means (for example, changing the lens setting or aperture). digital (Digital information is information …) of the optical system). In the settingsacquisition (In general, the acquisition is the action of obtaining information or acquiring a …) standard, the limit of diffraction (Diffraction is the behavior of waves when they encounter an obstacle that is not theirs …)that is, the size of the biological entities we are able to distinguish is approximately equal to 200 nanometers (10-6 mm) in the lateral plane (xy plane) and at 500 nm in the axial axis (z direction). Because many biological entities of interest are smaller than these values, the implementation of appropriate superresolution techniques can greatly improve the visualization of subcellular entities and allow understanding of the biological functions that occur at the molecular level.

Recently, a microscope Multi-Angle – TIRF (MA-TIRF) was prototyped (one of three in France) in collaboration with me ‘high school (An institute is a permanent organization created for a specific purpose. It is …) of Biology (Biology, commonly called “bio”, is the science of life ….) Valrose (iBV – CNRS / Inserm / University of the CĂ´te d’Azur). The microscope includes a dedicated reconstruction algorithm that achieves unprecedented axial resolution, far beyond the diffraction limit and still unmatched for commercial imaging systems.imagery (The image is mainly the creation and trade of physical images that …) living cells.

However, the lateral resolution of this system remained limited by diffraction. In this work, the researchers combined MA-TIRF reconstruction with a lateral overresolution technique called Covariance-based res0 superresolution microscopy with intensity estimation (COL0RME) that exploits the behavior statistical (A statistic is, at first glance, a number calculated on a sample ….) independent of standard fluorescent emitters.

(a) A stack image of images acquired by the microscope.
(b) Super resolved side plane images estimated by the COL0RME method.
(key volume (Volume, in physical or mathematical sciences, is a quantity that measures extent …) 3D estimated by the 3D method MA-TIRF COL0RME.

The researchers start from a temporary stack of images acquired by the microscope from different angles, such as those in line (a) of the image above, and then calculate super-resolved images laterally, such as those in line (b). using the COL0RME. method and finally estimate the super-resolved 3D image presented in line (c). He data (In information technology (IT), data is an elementary description, often …) The image above shows microtubules of bovine aortic endothelial cells, stained with Alexa-Fluor 488.

Comparing the 3D volume estimated by the pre-existing reconstruction of MA-TIRF (bottom right of the following image) and the one estimated by the proposed 3D method MA-TIRF COL0RME (top left), it is possible to observe a very good improvement of the resolution lateral all (The all-inclusive as the whole of what exists is often interpreted as the world or …) maintaining a good axial resolution.

Comparison between standard MA-TIRF and 3D MA-TIRF COL0RME methods. Color quantifies the depth of theshows (Generally speaking, a sample is a small amount of material, information or …).

1 – Vasiliki Stergiopoulou, Luca Calatroni, Henrique de Morais Goulart, SĂ©bastien Schaub and Laure Blanc-FĂ©raud. “COL0RME: Super-resolution microscopy based on the location of sparse flickering / fluctuating fluorophores and intensity estimation.” Biological Imaging, vol. 2, e1, 2022.
2 – Doctoral student in the Laboratory Computer science (IT – contraction of information and automation – is the domain …)Sophia-Antipolis Signals and Systems (I3S – CNRS / University of the CĂ´te d’Azur)
3 – Responsible for research (Scientific research first designates all the actions carried out with the aim of …) CNRS (The National Center for Scientific Research, better known by its acronym CNRS, is the largest …) at the Sophia-Antipolis Computer, Signal and Systems Laboratory (I3S – CNRS / University of the CĂ´te d’Azur)
4 – Engineer (“The basic job of an engineer is to solve problems in nature …) CNRS researcher at the Laboratory of Developmental Biology in Villefranche-sur-mer (LBDV – CNRS / Sorbonne University)
5 – CNRS Research Director at the Sophia-Antipolis Computer, Signal and Systems Laboratory (I3S – CNRS / University of the CĂ´te d’Azur)

Vasiliki Stergiopoulou, Luca Calatroni, Sébastien Schaub and Laure Blanc-Féraud3D image super-resolution due to fluorophore fluctuations and reconstruction with MA-TIRF microscopy (3D-COL0RME). ” 19th IEEE International Symposium on Biomedical Imaging (ISBI), 2022.

– Vasiliki Stergiopoulou – PhD student at I3S – Vasiliki.STERGIOPOULOU at univ-cotedazur.fr
– Luca Calatroni – CNRS researcher at I3S – calatroni at i3s.unice.fr
– SĂ©bastien Schaub – CNRS research engineer at LBDV – sebastien.schaub at imev-mer.fr
– Laure Blanc-FĂ©raud – CNRS Research Director at I3S – laure.blanc-feraud at cnrs.fr

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