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Biophysical Methods of Neurobiology (Dieter Braun)
Biophysical Methods in Neuroscience
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Biophysical Methods of Neurobiology (Dieter Braun)
The Fluorescence ProcessStage 1 : ExcitationA photon of energy hνEX creates an excitedelectronic singlet state (S1'). In chemilumi-nescence, S1’ is populated by a chemicalreaction. Stage 2 : Excited-State LifetimeFor finite time (typ. 1–10 ns), the fluorophoreundergoes conformational changes and issubject to interactions with its molecular envi-ronment. (a) the energy of S1' is partially dissipated,yielding a relaxed singlet excited state (S1). (b) not all excited molecules return to theground state (S0) by fluorescence emission:possibilties are collisional quenching, Fluores-cence Resonance Energy Transfer (FRET) andintersystem crossing.Stage 3 : Fluorescence EmissionA photon of energy hνEM is emitted, returningthe fluorophore to ground state S0. Due toenergy dissipation (S1’ to S1) the energy ofthe emission photon is lower (longer wave-length). Difference is called the Stokes shift,making it possible to detect emission photonsagainst the huge background of excitationphotons.
Fluorescein
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Biophysical Methods of Neurobiology (Dieter Braun)
Fluorescence Microscope
http://www.olympusmicro.com/
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Biophysical Methods of Neurobiology (Dieter Braun)
GFP - a fluorescent ProteinGreen fluorescent protein is also present in the jellyfish A. victoria,and absorbs blue light (488 nm) emitted by aequorin before re-emit-ting it as green light (512 nm)
4nm
2.5 nm
Chromophore after reaction of serine 65 - tyrosine 66 - glycine 67
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Biophysical Methods of Neurobiology (Dieter Braun)
GFP - a fluorescent ProteinGreen fluorescent protein is also present in the jellyfish A. victoria,and absorbs blue light (488 nm) emitted by aequorin before re-emit-ting it as green light (512 nm)
4nm
2.5 nm
Staining of cells which activate a specific protein
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Biophysical Methods of Neurobiology (Dieter Braun)
Imaging of Neuronal Activity
Calcium Indicators to detect Neuronal activity
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Biophysical Methods of Neurobiology (Dieter Braun)
Fura-2 Ca-Dye
RatiometricallyExcitation at 365nm vs 380nm
=> ∆[Ca]free
Chromophore Ca-Binder
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Biophysical Methods of Neurobiology (Dieter Braun)
Calibration in Neurons
Ca-stores (S) (b) Ca-dye (B)
κDBCa[ ]∆Ca[ ]∆
-------------------B[ ]Kd
Ca[ ]before Kd–( ) Ca[ ]peak Kd–( )----------------------------------------------------------------------------------= =κS
SCa[ ]∆Ca[ ]∆
-------------------=
Binding Ratio Binding Ratio
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Biophysical Methods of Neurobiology (Dieter Braun)
Calibration in Neurons
Ca-stores (S) (b) Ca-dye (B)
κDBCa[ ]∆Ca[ ]∆
-------------------B[ ]Kd
Ca[ ]before Kd–( ) Ca[ ]peak Kd–( )----------------------------------------------------------------------------------= =κS
SCa[ ]∆Ca[ ]∆
-------------------=
Binding Ratio
d Ca[ ]dt
--------------- d SCa[ ]dt
------------------ d BCa[ ]dt
-------------------+ +jin jout–
V-------------------=Total:
d Ca[ ]dt
--------------- 1 κS κB+ +( ) Ca[ ]totalδ t( )∆ γ Ca[ ] Ca[ ]before–( )–=
=> Ca[ ]∆ Ae t τ⁄–= τ1 κS κB+ +
γ----------------------------= A
Ca[ ]total∆1 κS κB+ +----------------------------=with ,
Binding Ratio
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Biophysical Methods of Neurobiology (Dieter Braun)
Calibration in Neurons
Ca-stores (S) (b) Ca-dye (B)
Measure [Ca] transients to infer A,τ depending on [B] , kBInfer kS and [Ca] without dye
d Ca[ ]dt
--------------- d SCa[ ]dt
------------------ d BCa[ ]dt
-------------------+ +jin jout–
V-------------------=Total:
d Ca[ ]dt
--------------- 1 κS κB+ +( ) Ca[ ]totalδ t( )∆ γ Ca[ ] Ca[ ]before–( )–=
=> Ca[ ]∆ Ae t τ⁄–= τ1 κS κB+ +
γ----------------------------= A
Ca[ ]total∆1 κS κB+ +----------------------------=with ,
κDBCa[ ]∆Ca[ ]∆
-------------------B[ ]Kd
Ca[ ]before Kd–( ) Ca[ ]peak Kd–( )----------------------------------------------------------------------------------= =κS
SCa[ ]∆Ca[ ]∆
-------------------=
Binding Ratio Binding Ratio
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Biophysical Methods of Neurobiology (Dieter Braun)
Calibration in Neurons
Ca[ ]∆ Ae t τ⁄–= τ1 κS κB+ +
γ----------------------------= A
Ca[ ]total∆1 κS κB+ +----------------------------=with ,
Results interpolated for no Ca-Dye:- 150-300nM Ca influx- Kinetics <100ms- 0.5-1% of Ca of an Action Potential is free=> Measurement of [Ca] in many physiological situations
Helmchen F., Imoto K. & Sakmann B. Ca2+ buffering and action potential-evokedCa2+ signaling in dendrites of pyramidal neurons. Biophys. J. 70, 1069-1081 (1996)
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Biophysical Methods of Neurobiology (Dieter Braun)
Clark, W., 1934. "Infrared photography," J. Biol. Photogr. Ass.2 (3):119-129
Seeing through in infrared
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Biophysical Methods of Neurobiology (Dieter Braun)
Clark, W., 1934. "Infrared photography," J. Biol. Photogr. Ass.2 (3):119-129
Seeing through in infrared
Ti-S Laser:- 700-1100nm- 100-400fs- €120.000
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Biophysical Methods of Neurobiology (Dieter Braun)
2-Photon Fluorescence Imaging
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Biophysical Methods of Neurobiology (Dieter Braun)
2-Photon Fluorescence Imaging
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Biophysical Methods of Neurobiology (Dieter Braun)
Ca-Imaging in Dendrites
Active Backpropagationof Action Potentials
Only in some Branches
Voltage sensitiveCa-Channels
NMDA Receptors
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Biophysical Methods of Neurobiology (Dieter Braun)
Measurements with Ca-Dyes
Quantal Analysisof single spines
Correlate Action Potentialswith Ca-Influx
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Biophysical Methods of Neurobiology (Dieter Braun)
Fluorescent Voltage-Sensitive DyesHigh fields inside Membranes
4nm
100mV / 4nm = 250kV/cmField Strength:
In
Out
E
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Biophysical Methods of Neurobiology (Dieter Braun)
Fluorescent Voltage-Sensitive DyesHigh fields inside Membranes
4nm
100mV / 4nm = 250kV/cmField Strength:
In
Out
E
Fluorescent Dyes: Charge relocation
E
+
-
+
Excitationblue shift
Emissionblue shift
+
Solvatochroism
Messungen von Aktionspotentialen in Dendriten von kultivierten und gentechnisch veränderten Hippocampusneuronen mit spannungssensi-tiven Farbstoffen, Dissertation Bernd Kuhn, 2001, http://tumb1.biblio.tu-muenchen.de/publ/diss/ph/2001/kuhn.pdf
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Biophysical Methods of Neurobiology (Dieter Braun)
Two-Dimensional Spectrum
Excitation λ
Em
issi
on
Change after100mV Depol.
Spectral Shift
Fluorescent Voltage-Sensitive Dyes
Fluorescent Dyes: Charge relocation
E
+
-
+
Excitationblue shift
Emissionblue shift
+
Solvatochroism
Messungen von Aktionspotentialen in Dendriten von kultivierten und gentechnisch veränderten Hippocampusneuronen mit spannungssensi-tiven Farbstoffen, Dissertation Bernd Kuhn, 2001, http://tumb1.biblio.tu-muenchen.de/publ/diss/ph/2001/kuhn.pdf
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Biophysical Methods of Neurobiology (Dieter Braun)
AP Backpropagation in Dendrites (cultured neurons)
Fluorescent Voltage-Sensitive Dyes
Bro
aden
ing
In Vivo detection of Whisker Sensing
Interaction of sensory responses with spontaneous depolarization in layer 2/3 barrel cortex.Proc Natl Acad Sci U S A. 2003 Nov 11; 100(23): 13638-43
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Biophysical Methods of Neurobiology (Dieter Braun)
Second Harmonic Generation (SHG)
ExcitationSHG TPF
P χ 1( )E1 χ 2( )E2 χ 3( )E3 …+ + +=
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Biophysical Methods of Neurobiology (Dieter Braun)
Second Harmonic Generation (SHG)
ExcitationSHG TPF
High-Resolution Nonlinear Optical Imaging of Live Cells by Second Harmonic GenerationBiophys. J. 1999 77: 3341-3349.
P χ 1( )E1 χ 2( )E2 χ 3( )E3 …+ + +=
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Biophysical Methods of Neurobiology (Dieter Braun)
Second Harmonic Generation (SHG)
Voltage-Sensitive SHG- tilting of the SHG-Dye- static E-Field contribution
P χ 1( )E1 χ 2( )E2 χ 3( )E3 …+ + +=
χ 2( ) χsurface2( ) χ 3( )EDC+=
influenced by Membrane Potential ?! PolarizationMechanisms of membrane potential sensing with second-har-monic generation microscopy, Journal of Biomedical Optics -- July 2003 -- Volume 8, Issue 3, pp. 428-431
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Biophysical Methods of Neurobiology (Dieter Braun)
Second Harmonic Generation (SHG)
Also: intrinsic SHG for Histology
Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy.Proc Natl Acad Sci U S A. 2003 Jun 10; 100(12): 7081-6.
Voltage-Sensitive SHG- tilting of the SHG-Dye- static E-Field contribution
P χ 1( )E1 χ 2( )E2 χ 3( )E3 …+ + +=
χ 2( ) χsurface2( ) χ 3( )EDC+=
influenced by Membrane Potential ?! PolarizationMechanisms of membrane potential sensing with second-har-monic generation microscopy, Journal of Biomedical Optics -- July 2003 -- Volume 8, Issue 3, pp. 428-431
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Biophysical Methods of Neurobiology (Dieter Braun)
Gene expression of a few cells
RNA Chip
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Biophysical Methods of Neurobiology (Dieter Braun)
Functional Magnetic
Resolution: 1-3mm @ 3T fMRI: blood oxygenation level
Resonance Imaging (fMRI)
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Biophysical Methods of Neurobiology (Dieter Braun)
Positron Emission Tomography (PET)
1ns time window
3 mm
blood flowwith 15O-label
(rCBF)
dopaminergiccells withF18-dopa
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Biophysical Methods of Neurobiology (Dieter Braun)
Coupling Cells to Planar Electrodes
- Metal electrodes- Isolated electrodes
Cell
CleftSi02p-Si
VM
VE
VS
rJ
VJ(x,y)
Electrode / Sensor(no electrochemical reactions)
Basic Circuit
Search for (a) well-defined system(b) Neuronal Interfaces
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Biophysical Methods of Neurobiology (Dieter Braun)
High-pass filter characteristics 2D
Cell
CleftSi02p-Si
VM
VE
VS
rJ
VJ(x,y)
Electrode / Sensor
Current in Node:
Current through cell
Resulting PDE:
In frequency space with transfer functions:
Also:
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Biophysical Methods of Neurobiology (Dieter Braun)
High-pass filter characteristics 2DFrequency Space
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Biophysical Methods of Neurobiology (Dieter Braun)
High-pass filter characteristics 2DFrequency Space Time Space
0DPoint
ContactModel
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Biophysical Methods of Neurobiology (Dieter Braun)
Imaging of Seal ResistanceLock-In Technique
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Biophysical Methods of Neurobiology (Dieter Braun)
Imaging of Seal ResistanceLock-In Technique
Cascade of transfer functions:
Chip: Volt.-Sens. Dye: Photomultiplier Tube:s
Total:
VSTIM Fluorescence
Cell
CleftSi02p-Si
VM
VE
VS
rJ
VJ(x,y)
Electrode / Sensor
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Biophysical Methods of Neurobiology (Dieter Braun)
Imaging of Seal ResistanceFrequency based Imagings
Cell
CleftSi02p-Si
VM
VE
VS
rJ
VJ(x,y)
Electrode / Sensor
Cascade of transfer functions:
Chip: Volt.-Sens. Dye: Photomultiplier Tube:s
Total:
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Biophysical Methods of Neurobiology (Dieter Braun)
Imaging of Seal Resistance
Cascade of transfer functions:
Total:
Transient Imaging
Cell
CleftSi02p-Si
VM
VE
VS
rJ
VJ(x,y)
Electrode / Sensor
Fourrier-Based Calculation:
Transform VSTIM from time space -> frequency spaceCalculate Expectation VPM in frequency spaceBacktransform VPM from frequency space -> time spaces
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Biophysical Methods of Neurobiology (Dieter Braun)
Transistors
www.biochem.mpg.de/mnphys
Electrochemical clean and CMOS compatible neurointerface
MeasuringVJ-VB
Retzius cell from leech
Rat hipocampus neurons
Averageover 64 signals
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Biophysical Methods of Neurobiology (Dieter Braun)
Measuring of Cell-Surface DistanceIRM: RICM:
Reduction of
Interference Reflection Reflection Interference
d
stray light with λ/4 plates
Contrast MicroscopyMicroscopy
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Biophysical Methods of Neurobiology (Dieter Braun)
Evanescent field
Measuring of Cell-Surface Distance
http://www.olym-pusmicro.com/primer/techniques/fluorescence/tirf/olympusaptirf.html
High NAImplementation
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Biophysical Methods of Neurobiology (Dieter Braun)
TIRF
Evanescent field
Measuring of Cell-Surface Distance
TIRAFTotal InternalReflection Fluorescence
Total Internal ReflectionAquous Fluorescence
FluorescentmarkerExcitation
http://www.olym-pusmicro.com/primer/techniques/fluorescence/tirf/olympusaptirf.html
BuffermarkerExcitation
cell cell
High NAImplementation
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Biophysical Methods of Neurobiology (Dieter Braun)
FLIC:
Measuring of Cell-Surface DistanceFluorescence InterferenceContrast Microscopy
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Biophysical Methods of Neurobiology (Dieter Braun)
Measuring of Cell-Surface DistanceFLIC: Fluorescence Interference Contrast Microscopy
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Biophysical Methods of Neurobiology (Dieter Braun)
Fluorescence Interference Microscopy
rkup
rkdown
Transfer of electrical field due tointerference and multireflection
Situation in fluorescentlayer (i.e. membrane)
Polarizations at Surface
Probability of Excitation or of Emission accordingto Fermi’s Golden rule and dipole far field operator proportional to:
Transitiondipole of
fluorescentdye
Incomingor outgoingelectrical
field
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Biophysical Methods of Neurobiology (Dieter Braun)
Fluorescence Interference MicroscopyPex pEex
2
λex
∫NAex
∫Cyl∫
Layer∫=Integrated Probability of Excitation
Pem pEem2
λem
∫NAem
∫Cyl∫
Layer∫=Integrated Probability of Emission
Expected relative fluorescence Ifluorescence PexPem=
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Biophysical Methods of Neurobiology (Dieter Braun)
Fluorescence Interference MicroscopyNumerical implementation
- First program with fixed layer system, C(no complex numbers, no objects)
- Second program in Java: complex object, more sophisticated numerical methods, easy buffering
Used to check the numerics of first: found some errors
- Third program in LabView:easy to play around and to debug
(a) “Numerical recipes in C” helps a lot for speed
pEex2
λex
∫NAex
∫Cyl∫
Layer∫pE 2
Cyl∫
Layer∫
NAex
∫λex
∫
>105-fold slower
(b) Integration order is crucial
with garbage collection, free layer objects, multiple windows GUI.
Notes
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Biophysical Methods of Neurobiology (Dieter Braun)
’Optical’ Stimulation
FM1-43 uptake
Ca Detection
“Photoconductive”
Remodeling of Synaptic Actin Induced by Photoconductive Stimulation; Cell, Vol 107, 605-616, 30 November 2001
Stimulation
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Biophysical Methods of Neurobiology (Dieter Braun)
’Optical’ Stimulation
FM1-43 uptake
Ca Detection
“Photoconductive”
Remodeling of Synaptic Actin Induced by Photoconductive Stimulation; Cell, Vol 107, 605-616, 30 November 2001
Stimulation
Dark
Bright
low Si-field effect capacitance
high Si-field effect capacitanceVoltage across SiO2
Voltage across Si
=> break-through of SiO2
Probably:
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Biophysical Methods of Neurobiology (Dieter Braun)
Planar Patch-Clamp
Whole cell patch clamp recording performed on a planar glass chip., Biophys J. 2002 Jun;82(6):3056-62.
Automating Patch-Clamp on a Chip
Long holes essential for Gigaseal
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Biophysical Methods of Neurobiology (Dieter Braun)
Temperature FluorescenceImaging of Temperature Laser
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Biophysical Methods of Neurobiology (Dieter Braun)
Temperature FluorescenceImaging of Temperature
Locked Temperature Oscillations
Laser
d=5µm
Laser
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Biophysical Methods of Neurobiology (Dieter Braun)
Two-State Model under Temperature Oscillationswith Temperature
Sensitivity of Fluorescence Signal
Kinetics in Phase Space
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Biophysical Methods of Neurobiology (Dieter Braun)
Two-State Model under Temperature Oscillationswith Temperature
Sensitivity of Fluorescence Signal
Measurements near single molecule level:DNA Hairpins Transfer h in Amplitude and Phase with Fit
Kinetics in Phase Space
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Biophysical Methods of Neurobiology (Dieter Braun)
Kinetics in Phase SpaceImaging Fluorescence Lock-In
Excitation IEx
Modulation E
Phase lag α
Dye(e.g. Temp.)
I q E( ) IEx× td∫=
Slow Imaging
Modulation E(e.g. Temp.)
Excitation IEx
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Biophysical Methods of Neurobiology (Dieter Braun)
Kinetics in Phase SpaceImaging Fluorescence Lock-In
Excitation IEx
Modulation E
Phase lag α
Dye(e.g. Temp.)
Aeiϕ 4π---
I0° I180°–I0° I180° 2Iback–+--------------------------------------------- i
I270° I90°–I270° I90° 2Iback–+------------------------------------------------+=
IEx IExΘ ωt α–( )sin[ ] ωt α–( )sin∆ IEx0( )+=
q q0 A ωt ϕ+( ) 1+sin[ ]=
IαAπ IExq0∆
2------------------------- α ϕ+( )cos 2 IExq0∆ 2πq0IEx
0( )+ +=
I q E( ) IEx× td∫=
Slow Imaging
Co-Modulation as follows
Leads to images with slow detector:
Infer Amplitude and Phase of q:
Iback 2πq0IEx0( ) Iconst+=
Modulation E(e.g. Temp.)
Excitation IEx
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Biophysical Methods of Neurobiology (Dieter Braun)
Kinetics in Phase SpaceImaging Fluorescence Lock-In
Excitation IEx
Modulation E
Phase lag α
Dye(e.g. Temp.)
Aeiϕ 4π---
I0° I180°–I0° I180° 2Iback–+--------------------------------------------- i
I270° I90°–I270° I90° 2Iback–+------------------------------------------------+=
IEx IExΘ ωt α–( )sin[ ] ωt α–( )sin∆ IEx0( )+=
q q0 A ωt ϕ+( ) 1+sin[ ]=
IαAπ IExq0∆
2------------------------- α ϕ+( )cos 2 IExq0∆ 2πq0IEx
0( )+ +=
I q E( ) IEx× td∫=
Slow Imaging
Co-Modulation as follows
Leads to images with slow detector:
Infer Amplitude and Phase of q:
DNA Hairpin under Temperature Oscillation
10 µm 10 µm170Hz 170Hz
Amplitude Phase
Iback 2πq0IEx0( ) Iconst+=
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Biophysical Methods of Neurobiology (Dieter Braun)
Kinetics in Phase Space
10µm
Imaging of Kinetics at Each pixel