TeodoraŞcheul

Dr. Iréne Wang, Dr. Jean-Claude Vial

LIPhy, Grenoble, France

STED microscopywith single light source

Summary

I. Introduction to STED microscopyII. STED with one laser source

1. Two-photon single wavelength STED2. Two colours for excitation and depletion -

one source– Experimental set-ups

– Results

– Conclusions and future work

STED microscopy - principle

Switching off the fluorescence ability of the molecules in the periphery off the focus shrinks the fluorescing area

Stefan W. Hell et al., Opt. Lett., 1994, 19, 780-782

S0

S1

exc

Excitation Luminescence

+Excitation STED Luminescence

Zero intensity point

fluoStimulated

emission

Energy levels and optical transitions involved in STED

200 nm

50 nm

STED microscopy – one laser source?

Ti-Sapphire femtosecondlaser Single wavelength

STED

Microchip Nd -YAG laser

Two colours for excitation and

depletion

I. Two-photon excitation and stimulated emission

depletion by a single wavelength

500 550 600 650 700 750 800 850 900 950 10000

20

40

60

80

100

120

140

160

180

200

220

Two p

ho

ton ab

sorp

tion

cross section

(GM

un

it)

Flu

ores

cenc

e (a

rb u

nit

)

Wavelength (nm)

Excitation and Stimulation

DCM

0

1

2

2hν excitation ~ I2 (fs pulse)

STED ~ I (ps pulse)

Two-photon absorption and emission spectra of DCM dye

500 550 600 650 700 750 800 850 900 950 10000

20

40

60

80

100

120

140

160

180

200

220

Two p

ho

ton ab

sorp

tion

cross section

(GM

un

it)

Flu

ores

cenc

e (a

rb u

nit

)

Wavelength (nm)

Excitation and Stimulation

DCM

0

1

2

S0

S1

exc fluo STED

n0

n0vib

n1

n1vib

S0

S1

exc fluo STED

n0

n0vib

n1

n1vib

• Laser source: mode-locked Ti:Sapphire laser (Chameleon CoherentTM, Ultra II): pulse duration 140 fs, repetition rate of 80 MHz.

• The duration of the stretched pulse: ~40 ps

Excitation

Ti:Sa

Camera

Ti:Sa

140 fsStretcher

STED40 ps

Sample

Mono-chromator

PMT

TCSPC board

λ/4 plate

λ/2

plate

Delay Line

P1

P2Excitation

Ti:Sa

Camera

Ti:Sa

140 fsStretcher

STED40 ps

Sample

Mono-chromator

PMT

TCSPC board

λ/4 plate

λ/2

plate

Delay Line

P1

P2

Experimental setup: proof of principle in solution

DCM solution

Scheul et al. Opt. Express, 2011, 19, 18036–18048

a b c

d e fExcitation dd Excitation+STEDeSTED ff

a b c

d e fExcitation dd Excitation+STEDeSTED ff

*Average powers: 25 mW for excitation beam, 80 mW for STED beam

The fluorescence is quenched when the two beams overlap

0 2 4 6 8 10 12 14 160,0

0,2

0,4

0,6

0,8

1,0

0.86*exp(-0.40 ISTED

)+0.14

0.91*exp(-0.79 ISTED

)+0.09

Nor

mal

ized

fluo

resc

ence

sig

nal

ISTED

(GW/cm2)

λ = 680 nm λ = 700 nm

Fluorescence depletion efficiency increases with STED intensity

Fluorescent decays

CCD Images

a b c

d e fExcitation dd Excitation+STEDeSTED ff

a b c

d e fExcitation dd Excitation+STEDeSTED ff

Fluorescent decays

CCD Images

Experimental resultsExcitation STED Exc+STED Scheul et al. Opt. Express,

2011, 19, 18036–18048

Conclusions of Part 1

• We have demonstrated the possibility to quench two-photon excited fluorescence by stimulated emission with a single wavelength

• Standard two-photon microscope into STED microscope Outlook

• We are building Single wavelength STED microscope by implementing the vortex phase plate

• Test different dyes: Pyridines and Styryl dye families, for live cell imaging - large Stokes-shift red fluorescent proteins

+

Vortex phase plate(RPC photonics)

→

Gaussian beam

Doughnut shaped beam

+

Vortex phase plate(RPC photonics)

→

Gaussian beam

Doughnut shaped beam

II. STED with a two color microchip laser• Goal: STED microscopy with a single laser source that delivers two wavelengths

simultaneously.

• Advantages:- Two beams are intrinsically aligned and synchronized. - Low cost

• Disadvantages:- Restricted dye choice (large Stokes shift and UV absorbing dyes)- The need of achromatic optics for the two wavelengths simultaneously

MicrochipNd-YAG

1064 nm

Second harmonic generation

1064 nm

532 nm Sum frequency generation

1064 nm532 nm

355 nm

• Q-switched Nd-YAG laser • Repetition rate of 8 kHz to 150 kHz). • Pulse duration ~1 ns

Experimental setup: confocal towards STED microscope

Segmented waveplate:

λ/2 for 532 nm λ at 355 nm

Reuss et al. 2010 / Vol. 18, No. 2 / OPTICS EXPRESS.

Segm.waveplateSegm.waveplate

Fast axis

Confocal image of fluorescent beads

STED

Experimental – results

355 nm 532 nm

300 400 500 6000,0

0,2

0,4

0,6

0,8

1,0

Abs

/Em

(ar

b.un

its)

Wavelength (nm)

Abs Hoescht Em Hoescht

300 400 500 600 7000,0

0,2

0,4

0,6

0,8

1,0

Abs

/Em

(ar

b.un

its)

Wavelength (nm)

Abs ATTO 390 Em ATTO 390

355 nm 532 nm

Exc (355 nm)Exc (355 nm)+ STED (532 nm)

Conclusions of Part 2

• We have build a confocal microscope with a bicolor laser source that we believe it can be turned in a STED microscope by adding a chromatic birefringentdevice for the beamshaping

• Several dyes proved to be adequate to excitation at 355 nm and stimulated emission at 532 nm

Outlook • Future work will be done on fluorescent beads and

fixed stained cells.

Acknowledgements

Irène Wang Jean-Claude Vial Ciro d’Amico

LIPhy - MOTIV

Thank you!