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Applicazioni di microscopia ad illuminazione strutturata per studio di fenomeni veloci

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This doctoral research presents applications of structured illumination microscopy (SIM) for studying rapid biological phenomena. Conducted at the University of Milano-Bicocca, the study utilizes fluorescence mechanisms and neural network analysis to enhance imaging techniques aimed at understanding calcium dynamics in neurons. Key methodologies include the combination of epifluorescence and two-photon microscopy to capture millisecond-scale fluorescence variations. The work involves intricate imaging of neuronal structures, such as dendrites and axons, providing insights into membrane potential and ionic channel behavior.

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Applicazioni di microscopia ad illuminazione strutturata per studio di fenomeni veloci

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  1. Universitàdeglistudi di Milano – Bicocca Dipartimento di fisicaG.Occhialini Applicazioni di microscopia ad illuminazionestrutturata per studio di fenomeniveloci Relazionedi dottoratoA.A.:2012 Ciclo XXVII Dottorando: Paolo Pozzi Tutore: Giuseppe Chirico Laboratory of Advanced Biological Spectroscopy

  2. Summary • Fluorescence • “Standard” Microscopy • Structured Illumination • Neural Network Analysis • Calcium Imaging

  3. Fluorescence S2 fluorophore S1 Linear phenomenon

  4. Epifluorescence Microscope CCD Emission filter Lamp Dichroic mirror Excitation filter Microscope Field of View Objective Sample

  5. Two Photon Fluorescence S2 fluorophore S1 Non - Linear phenomenon

  6. Two Photon Microscope Phototube Emission filter Galvanometric mirrors Dichroic mirror Laser Source Microscope Field of View Objective Sample

  7. Scanning Imaging Very slow!!! 10-15 fps max I t

  8. Phase Shaping Standard microscope: FFT Structured Illumination: FFT

  9. Structured Illumination Microscope CCD Emission filter Laser Source Dichroic mirror Microscope Field of View SLM Objective Sample

  10. What do we Watch? Millisecond scale fluorescence variations in multiple locations: Blood flow cross-correlation Neural network monitoring

  11. Neurons Dendrites Ionic Channels Axon

  12. Action Potential Potassium Channels Open Potential Sodium Channels Open Resting potential Time s

  13. Voltage Sensitive Dyes • Membrane Marker • Extremely sensible to Stark Effect • Two photon fluorescence change S2 S1

  14. The experiment Mossy Fiber (Input) Granule Cells (Elaboration) Purkinje Cell (Output)

  15. Imaging

  16. Data Acquisition WIP 5

  17. Calcium Imaging 5

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