applicazioni di microscopia ad illuminazione strutturata per studio di fenomeni veloci
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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