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Università degli studi di Milano – Bicocca Dipartimento di fisica G.Occhialini. Applicazioni di microscopia ad illuminazione strutturata per studio di fenomeni veloci. Relazione di dottorato A.A.:2012 Ciclo XXVII. Dottorando : Paolo Pozzi Tutore : Giuseppe Chirico.

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Presentation Transcript
slide1

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

summary
Summary
  • Fluorescence
  • “Standard” Microscopy
  • Structured Illumination
  • Neural Network Analysis
  • Calcium Imaging
fluorescence
Fluorescence

S2

fluorophore

S1

Linear phenomenon

slide4

Epifluorescence Microscope

CCD

Emission filter

Lamp

Dichroic mirror

Excitation filter

Microscope Field of View

Objective

Sample

two photon fluorescence
Two Photon Fluorescence

S2

fluorophore

S1

Non - Linear phenomenon

slide6

Two Photon Microscope

Phototube

Emission filter

Galvanometric mirrors

Dichroic mirror

Laser Source

Microscope Field of View

Objective

Sample

slide7

Scanning Imaging

Very slow!!!

10-15 fps max

I

t

phase shaping
Phase Shaping

Standard microscope:

FFT

Structured Illumination:

FFT

slide9

Structured Illumination

Microscope

CCD

Emission filter

Laser Source

Dichroic mirror

Microscope Field of View

SLM

Objective

Sample

what do we watch
What do we Watch?

Millisecond scale fluorescence variations in multiple locations:

Blood flow cross-correlation

Neural network monitoring

neurons
Neurons

Dendrites

Ionic Channels

Axon

action potential
Action Potential

Potassium Channels Open

Potential

Sodium Channels Open

Resting potential

Time

s

voltage sensitive dyes
Voltage Sensitive Dyes
  • Membrane Marker
  • Extremely sensible to Stark Effect
  • Two photon fluorescence change

S2

S1

the experiment
The experiment

Mossy Fiber (Input)

Granule Cells (Elaboration)

Purkinje Cell (Output)