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Wolfgang Langbein – Imaging Absorbers with small quantum yield

Imaging Absorbers with small quantum yield

School of Physics and Astronomy, Cardiff University

Wolfgang Langbein


Introduction

• All materials absorb light above the “band-gap” or homo-lumo

separation

• Absorption is used in transmission

microscopy of stained samples

(Histology)

• Most materials do not fluoresce significantly

• Most sensitive and optical sectioning microscopy techniques use

fluorescence (resonant absorption & emission, low background)

• Which other techniques can be used to measure absorption of

non-fluorescent materials sensitively with 3D resolution ?


Fluorophores and the normal molecule

Spin-orbit coupling

mixes singlet & triplet

Flu

orescen

ce

S0

S1

photo

n a

bso

rpti

on

S2

T1

Phosphorescence

Vibrational thermalization

with environment (heating)

Strong vibrational coupling allows

fast vibrational relaxation to the ground state:

Low quantum efficiency of flourescence

Energy results in local heating

Fluorophore “Normal” Molecule

Long (>ns) vibrational relaxation time

from excited to ground state: Photon

emission is dominant relaxation process

S0

S1

S2


Detecting Absorption without Fluorescence

Ab

sorb

ance

Gai

nMeasure change of absorption and

refractive index before vibrational

relaxation

(withing the first picoseconds after

excitation)

Measure heating of surrounding

(nanoseconds after excitation)

Photothermal Microscopy:

Use Probe beam to measure thermal

lensing by temperatue dependent refractive

index n(T)

Photoacoustic Microscopy:

Use microphone to detect pressure waves

created by thermal expansion after pulsed

excitation (non-imaging detection)

Resonant Pump-probe or

Four-wave mixing spectroscopy

pump probe

Fluorescence


Photothermal Microscopy

Science 297, 1160 (2002)

5-80nm Gold NP

1MHz modulation

1-20mW heating at 532nm

Probing at 633nm 2mW

Clean substrates

300nm

polystyrene

beads

80nm

gold beads

10nm

gold beads


Photothermal Heterodyne Imaging

Uses different differential reflectivity

Detected down to 2nm gold particles

Heating and probing powers 3mW, 70mW


Photothermal imaging: Limited specificity

• Photothermal Imaging probes the non-resonant refractive index

changes

• Sensitive to anything that absorps the pump light

• Background in cellular environment


Resonant Pump-Probe Microscopy

doi:10.1038/nature08438

crystal violet chromophore

Haemoglobin in Blood (red)

Two-photon excited at 800nm (20mW)

Probe at 600nm (3mW)


Four-Wave Mixing Imaging of Colloidal

Nanoparticles

Francesco Masia1,2, Iwan Moreels3, Zeger Hens3, Peter Watson1,

Wolfgang Langbein2, and Paola Borri1,2

1. Cardiff University School of Biosciences, UK

2. Cardiff University School of Physics and Astronomy UK

3. Department of Inorganic and Physical Chemistry, Gent University, Belgium


Optical Labels for Bioimaging

The optimal label for optical

bioimaging should be:

small

photostable

biocompatible (nontoxic)

generate an intense optical

signal

Organic dyes and

fluorescent proteins:

small

photobleaching

associated photo-toxicity


Optical Labels for Bioimaging

Invitrogen

Colloidal Quantum Dots

large size

blinking

cytotoxic (Cd or As atoms)

large two-photon absorption

cross-section

Colloidal Gold Nanoparticles:

small (established probes for

electron microscopy)

photostable

biocompatible

large absorption cross section at

the surface plasmon resonance

small fluorescence quantum

yield

Au


Resonant Four-Wave Mixing

Third-order polarization:

*

123EEEE

FWM⋅⋅∝

E1E3 E2

d=20nm

Optical cross-sections (a.u.)

σabs

σsca

σext

Au

500 525 550 575 600 625 650 675

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Absorption (µM

-1cm

-1)

Wavelength (nm)

QD ensemble

Photoluminescence Int. (arb. unit)

CdSe

ZnS

τ23

τ12

varying τ12 → dephasing

varying τ23 → dynamics of the absorption/scattering properties (transient grating)

fixing τ12 and τ23 for maximum FWM and scanning focus position → FWM Imaging


Resonant Four-Wave Mixing

Not relying on (hence not limited by) fluorescence emission

Multiphoton microscopy (intrinsic 3D resolution)

Increased spatial resolution beyond the one-photon diffraction limit due to

the third-order nonlinearity

Coherent signal: Interferometric detection free from incoherent (eg

autofluorescence, Raman) backgrounds

Specific to the absorption resonance of colloidal nanoparticles

Transient changes in extinction (absorption/scattering): “lifetime imaging”

*

123EEEE

FWM⋅⋅∝

E1E3 E2


FWM Microscopy Set-up

• 150-fs pulses at 76 MHz rep. rate

• Oil immersion microscope objectives (MO) of 1.25 NA

• Nanometric positioning of the sample (~10nm resolution)

• Heterodyne detection (interference with reference beam)

• Balanced detection to reject common-mode laser noise

• Transmitted and FWM fields are distinguished by selecting the proper beating frequency

• Polarization selection used to improve signal-to-background ratio

Reference: ν0

Lock-in

x

zy

MO1 MO2

SampleBPD

_

BS3ν0+ ν2

ν0+ ν3

PolAOM2

AOM3

Pol

BS1P2

P3

AOM1P1

ν0+ ν1

BS2


FWM Imaging CdSe/ZnS CQDs

a)

0

1

intensity

590nm

b)

-1 0 1

-1

0

1

y (µm)

x (µm)

-2 0 2

-5

0

5

y (µm)

z (µm)

500 550 600 6500

5

10

15

FWM PL

0.5 µm

Wavelength (nm)

Absorption (cm

-1)

0

2

4

Intensity (a.u.)

a)

1

0

b)

0

8.7E-12

1.7E-11

2.6E-11

3.5E-11

4.4E-11

5.2E-11

6.1E-11

7E -11

7.9E-11

8.7E-11

9.6E-11

1E -10

1.1E-10

1.2E-10

1.3E-10

1.4E-10

1.5E-10

1.6E-10

1.7E-10

1.7E-10

1.8E-10

1.9E-10

2E -10

2.1E-10

2.2E-10

2.3E-10

2.4E-10

2.4E-10

2.5E-10

2.6E-10

2.7E-10

2.8E-10

2.9E-10

3E -10

3.1E-10

3.1E-10

3.2E-10

3.3E-10

3.4E-10

3.5E-10

-10 -5 0 5 10

-10

-5

0

5

10y (µm)

x (µm)-10 -5 0 5 10

x (µm)

Transmission FWM

Proof-of-principle:

CQDs in PMMA spin coated on a coverslip

Three dimensional high spatial resolution:

140nm lateral, 590nm axial

FWM is spectrally selective

to the excitonic ground state absorption

F. Masia et al. Appl. Phys. Lett. 93, 021114 (2008)

Estimated sensitivity limit HzCQDs10


FWM Imaging with PbS CQDs

2µm

0

1

PbS CQDs in a polystyrene matrix

Absorption peak at 1.2µµµµm

Feasibility of near infrared imaging demonstrated with PbS CQDs

The use of a heterodyne interferometric detection overcomes the problem of the

high detector noise in this wavelength range.

Upper limits of the spatial resolution:

400nm (lateral) and 1.6µµµµm (axial)

In collaboration with I. Moreels and Z. Hens

-4 -2 0 2 4-4

-2

0

2

4

y (µm)

z (µm)

1.6µm

-0.5

0.0

0.5

1 0 -1

y (µm)

x (µm)

400nm

Estimated sensitivity limit HzCQDs100

FWM

Masia et al. Phys. Rev. B 82, 155302 (2010)


Single gold NP detection with FWM

20nm GNPs 1

0250nm

0

4 E- 11

8 E- 11

1 .2E - 10

1 .6E - 10

2 E- 10

2 .4E - 10

2 .8E - 10

3 .2E - 10

3 .6E - 10

4 E- 10

4 .4E - 10

4 .8E - 10

5 .2E - 10

5 .6E - 10

6 E- 10

6 .4E - 10

6 .8E - 10

7 .2E - 10

7 .6E - 10

8 E- 10

8 .4E - 10

8 .8E - 10

9 .2E - 10

9 .6E - 10

1 E- 9

1 .04E - 9

1 .08E - 9

1 .12E - 9

1 .16E - 9

1 .2E - 9

1 .24E - 9

1 .28E - 9

1 .32E - 9

1 .36E - 9

1 .4E - 9

1 .44E - 9

1 .48E - 9

1 .52E - 9

1 .56E - 9

1 .6E - 9

We detected single GNPs with diameter down to 10nm at powers

corresponding to negligible average

photothermal heating (~1K)

λ=550nm

E1,E2 in time overlap

E3: 0.5ps delay

10 20 30 40 50 60

1

10

100

FWM Amplitude (a.u.)

d(nm)

d3

Size dependence

absFWMdE σ∝∝ 3

FWM is spectrally selective: decrease of two orders of magnitude

for off-resonance excitation at 670nm


Origin of FWM in metallic NPs

• Excitation of a Surface Plasmon by E1 and E2

• Fast energy transfer (~10fs) to single electron excitation

• Electron-electron scattering thermalizes (~100fs) the electron gas which becomes hot

• E3 probes the variation in the extinction properties at the Surface Plasmon Resonance

• FWM signal is generated ∝ E1 E2 *E3

• Thermalization of the electron gas with the lattice (~1ps)

• Subsequent thermalization with the surrounding medium (~100ps)

τ23E1E2E3

FWM∝ E1E2*E3

t

τ12=0ps

e-e-

e-

e-e-

e-

e-e-

e-

e-e-

e-

e-e-

e-

ωSP

ω

hot

T


Thermalization dynamics

-0.5 0.0 1 10 100 1000

0.0

0.5

1.0

t2=390ps

20 nm GNP

Itot=18.5 kW/cm

2FWM Amplitude (a.u.)

τ23 (ps)

t1=2ps

electron-lattice

NP-surrounding

el-el scattering

Distinction between amplitude and phase modulation (real and imaginary part of

dielectric function) also possible


Spatial Resolution

-0.5 0.0 0.5

-0.5

0.0

0.5

1.0

z (µm)

x (µm)

140nm

470nm

800680950470Axial

190160230140Lateral

2P (2λ)Confocal1PFWM

Intensity Point Spread Function obtained by imaging a single 20nm GNP

High 3D spatial resolution beyond the one-

photon diffraction limit is achieved due to the

non-linear nature of FWM signal

F. Masia et al. Optics Letters 34, 1816 (2009)

Resolution (nm)


FWM Imaging of cells with GNPs

Golgi structures of fixed HepG2 cells co-immunostained with antibody-GNPs

and antibody-Alexa488

FWM

0

15µm

z

c d

2µm

2µm

x

y

Fluorescence

Phase Contrast FWM

10nm GNP

I=65kW/cm2 (0.5pJ/pulse)

SNR ~ 200

5nm GNP

I=110kW/cm2 (0.9pJ/pulse)

SNR ~ 200

Pixel rate of 10Hz

F. Masia et al. Optics Letters 34, 1816 (2009)


Fast Cell Imaging

A pixel rate of 5kHz is achieved

Images of a 10nm GNP-labelled

Golgi structure for different z

positions (0.5µm step)

SNR~140 for I=74kW/cm2

(0.6pJ/pulse)

1

0

0

4E -11

8E -11

1.2E-10

1.6E-10

2E -10

2.4E-10

2.8E-10

3.2E-10

3.6E-10

4E -10

4.4E-10

4.8E-10

5.2E-10

5.6E-10

6E -10

6.4E-10

6.8E-10

7.2E-10

7.6E-10

8E -10

8.4E-10

8.8E-10

9.2E-10

9.6E-10

1E -9

1.0 4E -9

1.0 8E -9

1.1 2E -9

1.1 6E -9

1.2E-9

1.2 4E -9

1.2 8E -9

1.3 2E -9

1.3 6E -9

1.4E-9

1.4 4E -9

1.4 8E -9

1.5 2E -9

1.5 6E -9

1.6E-9

1µµµµm

Projection of Stack-1.avi


Lifetime FWM Imaging

Local thermal conductivity can be measured

Lifetime FWM Imaging can be obtained by changing the delay of P3

Labels with different dynamics of the grating (i.e. GNPs and CQDs) can be distinguished

1

10-2

0

4 .0 00E- 11

8 .0 00E- 11

1 .2 00E- 10

1 .6 00E- 10

2 .0 00E- 10

2 .4 00E- 10

2 .8 00E- 10

3 .2 00E- 10

3 .6 00E- 10

4 .0 00E- 10

4 .4 00E- 10

4 .8 00E- 10

5 .2 00E- 10

5 .6 00E- 10

6 .0 00E- 10

6 .4 00E- 10

6 .8 00E- 10

7 .2 00E- 10

7 .6 00E- 10

8 .0 00E- 10

8 .4 00E- 10

8 .8 00E- 10

9 .2 00E- 10

9 .6 00E- 10

1 .0 00E- 9

1 .0 40E- 9

1 .0 80E- 9

1 .1 20E- 9

1 .1 60E- 9

1 .2 00E- 9

1 .2 40E- 9

1 .2 80E- 9

1 .3 20E- 9

1 .3 60E- 9

1 .4 00E- 9

1 .4 40E- 9

1 .4 80E- 9

1 .5 20E- 9

1 .5 60E- 9

1 .6 00E- 9

τ=0ps τ=0.5ps

τ=2ps τ=10ps

1 µµµµm


Excitation Intensity Effects

The temperature increase in the vicinity of the NP is a key issue for the applicability of the technique to living cells

We investigated the dependence on the excitation power:

• Loss of the signal after high intensity irradiation

• No change in the nonlinear property at these excitation intensities observed in dropcast sample

Estimation of temperature increase (8I0):

Max increase in electron T ~ 3300K

Max increase in lattice T ~ 800K

Average steady state GNP T increase ~ 9K

Disruption of the antibody bonding at high power

-1

0

1 I0=85kW/cm

2

y (µm)

-2 -1 0 1 2

-1

0

1

x (µm)

y (µm)

I0

-1

0

1 2I0

y (µm)

-1

0

1 4I0

y (µm)

-1

0

1 8I0

y (µm)

I

I0=32kW/cm2

0 100 2000

1

single NP

20nm

Ptot (kW/cm

2)

EFWM/(E1E2E3) (arb.u.)


Summary

Four-Wave Mixing imaging with colloidal nanoparticles:

Beyond fluorescence, Background-free coherent detection, High 3D resolution,

Specific (resonant), Transient.

Single gold NPs are detected:

• Thermalisation Dynamics:

-0.5 0.0 0.5

-0.5

0.0

0.5

1.0z (µm)

x (µm)

140nm

470nm

1

0

0

4E-11

8E-11

1.2E- 10

1.6E- 10

2E-10

2.4E- 10

2.8E- 10

3.2E- 10

3.6E- 10

4E-10

4.4E- 10

4.8E- 10

5.2E- 10

5.6E- 10

6E-10

6.4E- 10

6.8E- 10

7.2E- 10

7.6E- 10

8E-10

8.4E- 10

8.8E- 10

9.2E- 10

9.6E- 10

1E-9

1.04E-9

1.08E-9

1.12E-9

1.16E-9

1.2E- 9

1.24E-9

1.28E-9

1.32E-9

1.36E-9

1.4E- 9

1.44E-9

1.48E-9

1.52E-9

1.56E-9

1.6E- 9

20nm

-0.5 0.0 1 10 100 1000

0.0

0.5

1.0

t2=390ps

20 nm GNP

Itot=18.5 kW/cm

2

FWM Amplitude (a.u.)

τ23 (ps)

t1=2ps

z2µmx

y

Imaging of subcellular structures at high

pixel rates and low excitation powers,

compatible with live cell imaging

Measured spatial resolution:

lateral, 140nm; axial, 470nm


Quantum Optoelectronics & Biophotonics Group

Postdoc position on CARS Nanoscopy open -

contact Wolfgang Langbein or Paola Borri for details

Cardiff University, Wales, UK

//langsrv.astro.cf.ac.uk/

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