two-photon optogenetics by wave front shaping of ultra...

Two-photon optogenetics by wave front shaping of ultra fast pulses

Valentina Emiliani

Wave-front engineering microscopy groupNeurophysiology and New Microscopies Laboratory

Paris Descartes University, Paris France

Microscopie Non-linéaire en Science du Vivant, 2012

Neurophotonics:

V. Gradinaru et al. J Neurosci. (2007)

Optical stimulation • Photolysis• Optogenetics

Sub cellular (~30 nm) In vivo

Development and use of advanced optical methods for Neurobiology

STED microscope Micro-endoscope

confocal

STED

patterned endomicroscopy

Imaging and functional imaging

A fundamental task in neuroscience research is to establish a map of theneural connections within the brain

INTRODUCTION

Young Frankenstein, M. Brook 1974

not invasive spatial and temporal resolutionflexible reversible

• Light stimulation• Electrode stimulation

Some experimental challenges:mechanical damageslimited spatial resolution difficulty in inhibiting neurons

An alternative way to stimulate neurons is to use light

The use of light is not invasive permits to precisely control the location the time and the strength of stimulation, is flexible and reversible

Richard Fork

(R. Fork, Science, 1971)

INTRODUCTION

Zhang et al. (2006)

Chlamydomonas reinhardtii (algae) Natronomonas pharaonis (archaeobacteria)

Channelrhodopsin ChR2: excitation Halorhodopsin NpHR: inhibition

Zhang et al. (2007)

Optogenetics: Light gated channels and pumps

The demonstration of functional expression of Channelrhodopsin-2 in mammalian (Nagel etal. 2003) and neuronal cells (Boyden et al. 2005) marked the beginning of optogenetics

V. Gradinaru et al. J Neurosci. 2007

Examples: excitation

• Blue light stimulation of the right secondary motor cortex in transgenic mice expressing ChR2 (Thy1::ChR2-EYFP)

F. Zhang, et al.Nature (2007)

Examples: inhibition

• Transgenic C. elegans expressing NpHR in muscles

Key biological questions have been already addressed with simple illumination methods based on wide-field blue light illumination

To individuate subsets of genetically identical connected cells, orestablish the role of specific spatiotemporal excitatory patterns in guidinganimal behavior, more sophisticated illumination methods are required

INTRODUCTION:

Two-photon excitation

Two-photon excitation

Improvement in axial resolution because of non linearity of the two photon effect

Improvement in penetration depth because of the use of longer wavelength

fIS

2

PE2 ⋅τ∝

B. Amos Cambridge

Light gated channels (ChR2)• Low conductivity (~80 fS)

(Feldbauer et al PNAS, 2009)• Low density of channels

(Nagel et al, FEBS Lett.,1995)

Two-photon photoactivation

conventional two-photon excitation volume is too small:

• Laser scanning

Not enough channels to generate an AP

• First demonstration for AP generation in cultured neurons

• High two-photon cross section (~250 GM at 920 nm)

(Rickgauer and Tank PNAS, 2009)

Limited temporal (~30ms for single cell excitation) and axial resolution

First demonstration of 2P ChR2 activation in cultured neurons

Laser scanning

conventional two-photon excitation volume is too small:

• Parallel approach

Two-photon photoactivation

Light gated channels (ChR2)• Low conductivity (~80 fS)

(Feldbauer et al PNAS, 2009)• Low density of channels

(Nagel et al, FEBS Lett.,1995)

Parallel approach: Gaussian beam

• Poor Axial resolution

z

λλ 2

2

sNAK

∝⋅

=resolution axial10

0 µm Focal plane

y

xy

s

Back aperture

Ideally:

• efficiency• multi-scale excitation• ms temporal resolution • μm axial resolution

•Limited to circular shape

OUTLINE

Optical techniques:

Digital holography

Generalized phase contrast method

Temporal focusing

Results:

2P optogenetics in brain slices

Propagation of shaped beams through scattering media

Phase modulation

An efficient way of controlling light distribution is to modify its optical wave front by pure phase modulation (not losses of power)

INTRODUCTION: phase modulation

Multi spot generation

Spot shaping

Liquid crystal based modulators (SLM)Incoming

Light

Reflective

surface

Glass with

electrodes

LC molecules

ΔV1

ΔV2

ΔV3polarization

φ1

φ2

φ3

www.optics-solutions.co.uk

www.doc.com

www.nanomaker.ru

n

INTRODUCTION: phase modulation

Phase-only modulation is obtained by modifying the thickness of the diffractive elements

U(x1,y1)=A (x1,y1)e iΦ(x1,y

1)

))y,x(U(F)y,x(U

))y,x(U(F)y,x(U

001

11

1100−=

=

U(x0,y0)=A (x0,y0)e iΦ(x0,y

0)

Objective back aperture Focal plane

2π

0

Liquid crystal based modulator

Gerchberg and Saxton algorithm [Optik (1972)]

Digital holography: Gerchberg and Saxton algorithm

| G(x1,y1) | = |U(x1,y1) |

2. Inverse Fourier transform

Target illumination

FT‐1

G(x1,y1)

g(x0,y0)= |U(x0,y0)|.eiΦ(x0,y0 )

g1(x0,y0) = |U(x0,y0)|.eiΦ’ (x1,y1 )

| U(x0,y0) |2 =

G’(x1,y1)=|U(x1,y1) | .eiΦ(x1,y1)

FT

g'(x0,y0)

[ ]2

, 00002

11),(),('' ∑ −=

yxyxUyxgE Evaluation step

Random phaseΦ(x0,y0)

1. Building starting function

3. Retain phaseReplace amplitude

Φ(x1,y1)

4. Fourier transform

4a. Retain phase,Replace amplitude with target

Φ’(x1,y1)

4b. STOP

Output phase

D. Oron et al. Progress in Brain Research (2012)

Digital holography: Gerchberg and Saxton algorithm [Optik (1972)]

Φ(x1,y1)

10 μm

20 μm

RESULTS:

C. Lutz et al. Nature Methods (2008)

M. Zahid et al. PlosOne (2010)

E. Papagiakoumou, et al., Optics Exp.(2008) E. Papagiakoumou, et al., Optics Exp. (2009)

x

zy

20μm

S. Yang, et al., J Neuronal Engineering, (2011)F. Anselmi et al. PNAS (2011)

SLM

L1

L2

Beam expander

Laser (405 nm)

Front view

Side view

Objective conjugate plane

Wave front analyzer

Digital holography: The set up

Summary: Digital holography

Digital holography

•Phase modulation: minimize intensity losses•Parallel approach: temporal resolution fixed by the dwell time•Intrinsic optical sectioning: s∝Δz•Possibility for 3D patterning•in vivo

Extension of phase contrast method (Frederik Zernike 1930, Nobel prize 1953)

The generalized phase contrast method (GPC)

Phase map transformed into an intensity map

( )( ) ( ) ( )( )dxdyyxidxdyyxi ∫∫∫∫−

= ,exp,exp1

φφ

Expansion of the phase contrast method (Zernike 1932) to [0, 2π] phase variation:

( ) 2,cos12 yxI φ−≈ [ ] [ ]4;0;0 →ππθ =

J. Glückstad, Optic. Comm (1996)

Digital holography: Gerchberg and Saxton algorithm [Optik (1972)]

E. Papagiakoumou et al. Nature Methods (2010)

λexc=780 nmFluorescence Laser pattern

Two photon-GPC: Results

Phase mask

Laser pattern

DH

GPC

Focal planeSLM plane=Fourier plane

Focal planeSLM plane=conjugate plane

0.2 0.4 0.6 0.8 1.0 1.20.0

0.2

0.4

0.6

0.8

1.0

1.2 DH GPC DMD

2P e

xcita

tion

effic

ienc

y

x/ ΔX

25.0A

A

tot

SPOT =

20.0

15.0

05.0

10.0

} 05.0

25.0

0.2 0.4 0.6 0.8 1.0 1.20.0

0.2

0.4

0.6

0.8

1.0

1.2 DH GPC DMD

2P e

xcita

tion

effic

ienc

y

x/ ΔX

25.0A

A

tot

SPOT =

20.0

15.0

05.0

10.0

} 05.0

25.0

25.0A

A

tot

SPOT =

20.0

15.0

05.0

10.0

} 05.0

25.0

• DH, diffraction efficiency limited by SLM pixel size: (sin X/X)4

• GPC, given by the interferometer characteristics:

F = Aπ/(Aπ+A0)

Diffraction efficiency and excitation field: DH and GPC

A0

A π


• Alignment of phase dot (Wave front analyzer)

Practical challenges to set up GPC+TF:

0 1 2 3 4 5 60.00.20.40.60.81.01.2

Nor

m. I

nteg

rate

d In

tens

ity

Spot

1

1

23

41

23

45

1

2

1

23

e

• SLM=FOV; A spot ~ 1/4 FOV

• Iris:

• Tilt to compensate for grating tilt


F = Aπ/(Aπ+A0)=constant

Practical challenges to set up GPC+TF:

Gaussian beam

10 μm

Axial propagation?

GPC10

0μm

Back aperture

Holographic beam

z

y

10 μm

Back aperture

z

y

Focal plane

Back aperture

Axial resolution, b ∝ s 2 Axial resolution, b ∝ s

z

y

d

D. Oron, E. Tal, Y. Silberberg, Optics Express (2005)

Axial propagation: Temporal focusing

Spatial focusing(a)

z, t

y

S2P

z, t

S2P

τ

Temporal focusing(b)

z, t

y

z, t

τ

Vaziri&Emiliani Curr. Opinion in Neurobiology (2012)

Originally used for wide field two-photon microscopy

fIS

2

PE2 ⋅τ∝

D. Oron ,et al ., Progress in Brain Research (2012)

zR~τc

It works for ultra short pulses (depth of 3μm with τ =10fs)

α

To relax this condition:tilted angle

To optimize off- axis dispersion:blazed grating

Temporal focusing: the principle

5.0

Rline z

z1I−

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎟⎠

⎞⎜⎜⎝

⎛+≈

1

Rintpo z

z1I−

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎟⎠

⎞⎜⎜⎝

⎛+≈

Sample

Dichroicmirror

Objective

Beam expander

SLM

L1

Diffraction grating

Beam dump

Patch pipette

L

Ti: Sa Laser

CCD

AttenuatorIntensity

Phase

Intensity

Phase

Intensity

Phase

Axial resolution: Digital holography and Temporal focusing

E. Papagiakoumou et al. Optics Express (2008)

Sample

Dichroicmirror

Objective

Beam expander

SLM

L1

Diffraction grating

Beam dump

Patch pipette

PCF

L2

L

Ti: Sa Laser

CCD

AttenuatorIntensity

Phase

Intensity

Phase

Intensity

Phase

3D wave front shaping: GPC and Temporal focusing


Gaussian beam Holographic beam+TF

z

y

Axial resolution with temporal focusing: Results

10 μm

100μ

m

-20 -10 0 10 20

Holography Line scanningGPC

z-axis position (µm)

Inte

nsity

(a.u

.)

3μm

Focal plane

z

y

GPC+TF

Grating=830 l/mmObj =60x NA 0.9f=500mm


OUTLINE

Optical techniques:

Digital holography

Generalized phase contrast method

Temporal focusing

Results:

2P optogenetics in brain slices

Propagation of shaped beams through scattering media

Phase modulation

RESULTS: 2P Photoactivation of HEK 293 cells

50 pA

50 ms

Lateral resolution

ChR2-H134R-GFP transfected cultured HEK λEXc=850 nm; excitation density=0.45 mW / μm2 ; 10ms pulse

26 µm

-30 -20 -10 0 10 20 300

1

Nor

mal

ized

inte

grat

ed c

urre

nt

z-axis position (µm)

Axial resolution

n=4/7TheoryExp


Thy1-ChR2-YFP transgenic mice Excitation =0.3-0.5 mW /μm2; depth 60-70μm , 10ms

4 mV

10 ms20 µm

15 µm

10 µm

7 µm

5 µm

Action potential threshold around 10 µm

E. Papagiakoumou et al., Nat. Methods (2010)

10 Hz (5/8)

15 µm spot

10 µm spot

7 µm spot

5 µm spot20 mV

100 ms

Firing frequency increases with spot size

Action potential trains generation

50 ms

40 mV

20 Hz (3/8)

30 Hz (2/8)

20 mV

25 ms

10 µm

RESULTS: 2P Photoactivation in brain slices

25 mV

50 ms

RESULTS. Multiple processes, multiple cells

λEXC=920 nm; Excitation =0.6 mW /μm2; pulse=10ms

50 ms20 mV

A

B 10 µm

λEXC=920 nm, Excitation =0.3-0.5 mW /μm2; pulse=10ms

• Phase modulation of optical wave fronts permit a 3D sculpting of the excitation volume:

Lateral light patterning:digital holographygeneralized phase contrast methods

Axial resolution: temporal focusing

• TF: to control axial resolution AND to maintain shaped patterns in scattering media

CONCLUSIONS

CONCLUSIONS:

Which is the optimal illumination method for optogenetics?

Parallel

Digital holographyGeneralized

phase contrast

Temporal focusingScanning

Microendoendoscopy

Young Frankenstein, M. Brook 1974

D. Oron ,et al ., Progress in Brain Research (2012)c

parallel

CONCLUSIONS: comparison

D. Oron et al PBR (2012)More FlexibilityIncreased FOV3D

Speckles Complexity (algorithm)

CONCLUSIONS: comparison

Wave front engineering microscopygroup

D. Oron (Weizmann Institut)J. Glueckstadt(DTU Fotonik)E. Isacoff(Berkeley Univ.)Cha Min Tang, S. Yang(Maryland School of Medicine)A. Packer(UCL, London)

J. BradeleyS. CharpakD. OgdenD. DiGregorio(Paris Descartes Univ)

Financing:

External Collaborations:

Osnath AssayagAurelien BegueVincent De SarsBenoit Forget Marc GuillonOscar HernandezErwan LedouxMarcel LauterbachEirini PapagiakoumouEmiliano RonzittiVivien SzaboDelphine VardanegaCathie Ventalon

Former membersC. LutzMorad ZahidFrancesca Anselmi

THANK YOU!!

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