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Light sheet microscopy

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vECi : A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUE CLEARING AND 3-DIMENSIONAL IMAGING

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

BUDGET & TIMING

EXPERIMENTAL APPROACH

CONTEXT

  • Homogenizing the refraction indice is a crucial step for clearing a sample before observing it with lightsheet microscopy.
  • Many technics exist for clearing samples but with disadvantages: they are often time-consuming or use toxic reactants.
  • A potential way for the optimization of time-consuming clearing method is to play on physical parameters such as pressure.

EXPECTEDOUTCOME

AIMS

  • To optimize the ECi clearing method to be faster and more efficient.
  • To increase the penetration of labeling molecule in the sample.

ABSTRACT

COLLAB.

REFS.

Sup'Biotech Interactive Scientific Poster ©

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

CONTEXT

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

The brain a complex organ

LightSheet microscope

Tissue clearing is needed

Ueda, H. R., Dodt, H.-U., Osten, P., Economo, M. N., Chandrashekar, J., & Keller, P. J. (2020). Whole-Brain Profiling of Cells and Circuits in Mammals by Tissue Clearing and Light-Sheet Microscopy. Neuron, 106(3), 369–387. https://doi.org/10.1016/j.neuron.2020.03.004

Figure 1. Physical and Chemical Principles of Tissue Clearing

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

CONTEXT

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

Klingberg, A., Hasenberg, A., Ludwig-Portugall, I., Medyukhina, A., Männ, L., Brenzel, A., … Gunzer, M. (2017). Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy. Journal of the American Society of Nephrology, 28(2), 452–459. https://doi.org/10.1681/asn.2016020232

Chung, K., & Deisseroth, K. (2013). CLARITY for mapping the nervous system. Nature Methods, 10(6), 508–513. https://doi.org/10.1038/nmeth.2481

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

CONTEXT

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

Variation of a physical parameter for a better efficiency

  • Immunohistochemistry: decreasing the pressure helps to dehydrate the sample.
  • ACT-PRESTO: applying a positive pressure increase the penetration of macromolecules in a sample (Eunsoo, 2016)

 Lee, E., Choi, J., Jo, Y. et al. ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging. Sci Rep 6, 18631 (2016)

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

CONTEXT

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

Expected outcome

LightSheet microscope

Firgure 3.Light-sheet fluorescence brain imaging of tyrosine hydroxylase (TH) expression in representative vehicle-dosed control mouse

Roostalu, U., Salinas, C. B. G., Thorbek, D. D., Skytte, J. L., Fabricius, K., Barkholt, P., … Hecksher-Sørensen, J. (2019). Quantitative whole-brain 3D imaging of tyrosine hydroxylase-labeled neuron architecture in the mouse MPTP model of Parkinson’s disease. Disease Models & Mechanisms, 12(11), dmm042200. https://doi.org/10.1242/dmm.042200

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

AIMS

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

Objective: To optimize the ECI clearing method for lightsheet microscopy observation by shortening the dehydration phase and increasing the efficiency of both dehydration and labelling step. Hypothesis: By playing on the pressure applied to the sample and the solution, the molecules will reach the center of the sample more easily and the water will be removed faster from the sample. Specific aims: To create a vacuum environement using a pump that will force ethanol to penetrate in the sample and make the water evaporate faster for the dehydration step. To increase the penetration of labeling molecules in the core of the sample to obtain a better acquisition of the center during microscopy observations.

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

EXPERIMENTAL APPROACH

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

  • The classical ECi method

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

EXPERIMENTAL APPROACH

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

  • The continuous filtration system

Step 2: Injection

Step 1: Filtration

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

EXPERIMENTAL APPROACH

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

  • The vECi method

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

EXPERIMENTAL APPROACH

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

  • From days to hours
  • Dehydration in classical ECi method requires several days for clearing a sample of the size of a mouse brain
Continuous filtration system:
  • Force ethanol to penetrate the sample
  • Decrease the pressure to helps water evaporate
► Reduce dehydration time to only few hours

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

BUDGET & TIMING

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

GANTT CHART

5 major steps:

  • Performing ECi with a classic protocle
  • Performing VECi
  • Optimizing VECi
  • Verfiying experimental results (Spectroscopy & Microscopy)
  • Comparing both methods

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

BUDGET & TIMING

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

BUDGET

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

EXPECTED OUTCOME

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

ABSTRACT

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

COLLABORATIONS & ACKNOWLEDGEMENTS

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

Alexandre CAUMON

Claire ANGIBAUD

Nessrine TALHA

Hugo BRUANT

vECi: A RAPID AND NON-TOXIC METHOD FOR BRAIN TISSUES CLEARING AND 3-DIMENSIONAL IMAGING

BIBLIOGRAPHY

ANGIBAUD Claire, BRUANT Hugo, CAUMON Alexandre, TALHA Nessrine

  • Chung, K., & Deisseroth, K. (2013). CLARITY for mapping the nervous system. Nature Methods, 10(6), 508–513. https://doi.org/10.1038/nmeth.2481
  • Klingberg, A., Hasenberg, A., Ludwig-Portugall, I., Medyukhina, A., Männ, L., Brenzel, A., … Gunzer, M. (2017). Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy. Journal of the American Society of Nephrology, 28(2), 452–459. https://doi.org/10.1681/asn.2016020232
  • Lee, E., Choi, J., Jo, Y. et al. ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging. Sci Rep 6, 18631 (2016). https://doi.org/10.1038/srep18631
  • Roostalu, U., Salinas, C. B. G., Thorbek, D. D., Skytte, J. L., Fabricius, K., Barkholt, P., … Hecksher-Sørensen, J. (2019). Quantitative whole-brain 3D imaging of tyrosine hydroxylase-labeled neuron architecture in the mouse MPTP model of Parkinson’s disease. Disease Models & Mechanisms, 12(11). https://doi.org/10.1242/dmm.042200
  • Susaki, E. A., Tainaka, K., Perrin, D., Kishino, F., Tawara, T., Watanabe, T. M., … Ueda, H. R. (2014). Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis. Cell, 157(3), 726–739. https://doi.org/10.1016/j.cell.2014.03.042
  • Ueda, H. R., Ertürk, A., Chung, K., Gradinaru, V., Chédotal, A., Tomancak, P., & Keller, P. J. (2020). Tissue clearing and its applications in neuroscience. Nature Reviews Neuroscience, 21(2), 61–79. https://doi.org/10.1038/s41583-019-0250-1
  • Vigouroux, R. J., Belle, M., & Chédotal, A. (2017). Neuroscience in the third dimension: shedding new light on the brain with tissue clearing. Molecular Brain, 10(1). https://doi.org/10.1186/s13041-017-0314-y