Applicazioni di microscopia ad illuminazione strutturata per

studio di fenomeni velociRelazione di dottorato A.A.:2012 Ciclo XXVII

Dottorando: Paolo PozziTutore: Giuseppe Chirico

Laboratory of Advanced Biological Spectroscopy

Università degli studi di Milano – BicoccaDipartimento di fisica G.Occhialini

Summary

• Fluorescence

• “Standard” Microscopy

• Structured Illumination

• Neural Network Analysis

• Calcium Imaging

Fluorescence

fluorophore

Linear phenomenon

S1

S2

CCD

ObjectiveEx

cita

tion

filte

r

Emission filterDichroic mirror

Sample

Microscope Field of View

Lamp

Epifluorescence Microscope

Two Photon Fluorescence

fluorophore

Non - Linear phenomenon

S1

S2

Phototube

Objective

Emission filterDichroic mirror

Sample

Microscope Field of View

Two Photon Microscope

Laser Source

Galvanometric mirrors

Scanning Imaging

Very slow!!!

10-15 fps max

I

t

Phase Shaping

𝐸=𝐴 (𝑥 , 𝑦 )𝑒𝑖 𝜑 (𝑥 , 𝑦 )𝑡

FFT

FFT

Standard microscope:

Structured Illumination:

Objective

Emission filterDichroic mirror

Sample

Microscope Field of View

Structured IlluminationMicroscope

Laser Source

CCD

SLM

What do we Watch?Millisecond scale fluorescence variations in multiple locations:

Blood flow cross-correlationNeural network monitoring

Neurons

𝐾 +¿ ¿

𝐶𝑙−𝑁𝑎+¿ ¿

𝐾 +¿ ¿

𝐶𝑙−𝑁𝑎+¿ ¿

𝐴−

Ionic ChannelsDendrites

Axon

Action PotentialPo

tenti

al

−70𝑚𝑉

0𝑚𝑉

−60𝑚𝑉

+30𝑚𝑉

Resting potential

Sodium Channels Open

Potassium Channels Open

Times

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

S2

S1

The experiment

Mossy Fiber (Input)

Purkinje Cell (Output)

Granule Cells (Elaboration)

Imaging

Data Acquisition

WIP

5

Calcium Imaging

5