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Biophysical Methods of Neurobiology (Dieter Braun)

Biophysical Methods in Neuroscience

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

Biophysical Methods of Neurobiology (Dieter Braun)

Fluorescence Microscope

http://www.olympusmicro.com/

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

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

Biophysical Methods of Neurobiology (Dieter Braun)

Imaging of Neuronal Activity

Calcium Indicators to detect Neuronal activity

Biophysical Methods of Neurobiology (Dieter Braun)

Fura-2 Ca-Dye

RatiometricallyExcitation at 365nm vs 380nm

=> ∆[Ca]free

Chromophore Ca-Binder

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

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

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

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)

Biophysical Methods of Neurobiology (Dieter Braun)

Clark, W., 1934. "Infrared photography," J. Biol. Photogr. Ass.2 (3):119-129

Seeing through in infrared

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

Biophysical Methods of Neurobiology (Dieter Braun)

2-Photon Fluorescence Imaging

Biophysical Methods of Neurobiology (Dieter Braun)

2-Photon Fluorescence Imaging

Biophysical Methods of Neurobiology (Dieter Braun)

Ca-Imaging in Dendrites

Active Backpropagationof Action Potentials

Only in some Branches

Voltage sensitiveCa-Channels

NMDA Receptors

Biophysical Methods of Neurobiology (Dieter Braun)

Measurements with Ca-Dyes

Quantal Analysisof single spines

Correlate Action Potentialswith Ca-Influx

Biophysical Methods of Neurobiology (Dieter Braun)

Fluorescent Voltage-Sensitive DyesHigh fields inside Membranes

4nm

100mV / 4nm = 250kV/cmField Strength:

In

Out

E

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

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

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

Biophysical Methods of Neurobiology (Dieter Braun)

Second Harmonic Generation (SHG)

ExcitationSHG TPF

P χ 1( )E1 χ 2( )E2 χ 3( )E3 …+ + +=

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 …+ + +=

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

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

Biophysical Methods of Neurobiology (Dieter Braun)

Gene expression of a few cells

RNA Chip

Biophysical Methods of Neurobiology (Dieter Braun)

Functional Magnetic

Resolution: 1-3mm @ 3T fMRI: blood oxygenation level

Resonance Imaging (fMRI)

Biophysical Methods of Neurobiology (Dieter Braun)

Positron Emission Tomography (PET)

1ns time window

3 mm

blood flowwith 15O-label

(rCBF)

dopaminergiccells withF18-dopa

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

Vorlesung Biophysik Braun - Methoden in der NeuroBiologie

High-pass filter characteristics 2D

Cell

CleftSi02p-Si

VM

VE

VS

rJ

VJ(x,y)

Electrode / Sensor

Core-Coat Conductor

Iohmic i,VJ∇R

---------- dx⋅=

Net Current:Iohmic

VJ∇R

----------∇ dxdy⋅=

Normalization of all currents to Area:

IohmicVJ∇rJ

----------∇=

R [Ohm/cm²] -> rJ [Ohm]

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:

Biophysical Methods of Neurobiology (Dieter Braun)

High-pass filter characteristics 2DFrequency Space

Biophysical Methods of Neurobiology (Dieter Braun)

High-pass filter characteristics 2DFrequency Space Time Space

0DPoint

ContactModel

Biophysical Methods of Neurobiology (Dieter Braun)

Imaging of Seal ResistanceLock-In Technique

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

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:

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

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

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

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

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

Biophysical Methods of Neurobiology (Dieter Braun)

FLIC:

Measuring of Cell-Surface DistanceFluorescence InterferenceContrast Microscopy

Biophysical Methods of Neurobiology (Dieter Braun)

Measuring of Cell-Surface DistanceFLIC: Fluorescence Interference Contrast Microscopy

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

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=

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

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

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:

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

Biophysical Methods of Neurobiology (Dieter Braun)

Temperature FluorescenceImaging of Temperature Laser

Biophysical Methods of Neurobiology (Dieter Braun)

Temperature FluorescenceImaging of Temperature

Locked Temperature Oscillations

Laser

d=5µm

Laser

Biophysical Methods of Neurobiology (Dieter Braun)

Two-State Model under Temperature Oscillationswith Temperature

Sensitivity of Fluorescence Signal

Kinetics in Phase Space

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

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

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

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+=

Biophysical Methods of Neurobiology (Dieter Braun)

Kinetics in Phase Space

10µm

Imaging of Kinetics at Each pixel