Short pulses in optical microscopyIvan Scheblykin, Chemical Physics, LU

Outline:

Introduction to traditional optical microscopy based on single photon absorption:

Fluorescence wide-field and conforcal microscopyIntroduction to single molecule imaging

2-photon absorption2-photon confocal fluorescence microscopy

3-photon absorption, second harmonic generation

Microscopy which is not limited by light diffraction

Why do we see objects ?

Changing of the properties of light coming to the sample:Light absorptionLight scatteringChanging of light polarization…

An object emits light itself:LuminescenceSecond-harmonic generation…..

Many different ways to create contrast in optical microscopy

object

Transmission imageAbsorption and scattering

object

Transmission imageAbsorption and scattering

object

100€

Excitation light

Blocking filter

object

object

Transmission imageAbsorption and scattering

object

100€

Excitation light

Fluorescence

Blocking filter

Sample is stained by a fluorescent dye

White boardMicroscope scheme

Numerical Apreture

Spherical angle S

Light collection efficiency

S/4

NA/n = 1 , 50%

NA/n = 0.6, 10%

NAD

22.1

10 microns

Wide-field fluorescence microscopeConfocal fluorescence microscope

3D imaging, z-scan

Single molecule spectroscopy

Can we see one single chromophore ?

Not in absorption, because cross section is too small

= 10-16 cm2 ,

10-8 cm = 0.1 nm

However, we can detect fluorescence light emitted by the molecule!

5

SampleFor SMS

Single molecule imaging

Chemical Physics, Single Molecule Spectroscopy group, LU

Other ways to create contrast

Non-linear processes induced by strong laser light

Observation of fluorescence excitated by

2-photon absorption3-photon absorption

third harmonic signalObservation of second harmonic signal

....

4)3()2()1(

EEEEEEP

PED

Absorption,scattering

Two-photon absorption

Theory - Maria Göppert-Mayer, 1929Experimental observation – 1961Using in microscopy – Denk, Strickler, Webb, Science 1990

Probability of excitaion (W) (Intensity)2

W ( I [ptonots/cm2/s] )2

Absorbed photonFluorescence

Virtual level

i

f

One and Two-photon absorption cross sections

Transition dipole moment moment

Estimation of 2 (WB)

Two-photon excitation versus one-photon excitation

543 nm excitation

1046 nm excitation

Dye solution, safranin O

Resolution of 2-photon microscopy

XY, Z,

1/z4 excitation probability dependenceAnd 1/z2 dependence of total fluorescence(WB)

100 105 1010 1015 1020

1015

1016

1017

1018

1019

1020

1021

1022

1023

1024

1025

1026

1027

1028

Inte

nsi

ty fo

r 2

-ph

oto

n e

xcita

tion

Intensity for 1-photon excitation, photons/second/cm2

)()( 2211 IkIk pp

I2

I1

The same fluorescence signal from the sample

Better Light collection efficiency..

Multi-photon excitation confines fluorescence excitation to a small volume at the focus of the objective. Photon flux is insufficient in out-of-focus planes to excite fluorescence. No confocal pinhole is needed. All fluorescence (even scattered photons) constitutes useful signal.

Photobleaching and photodamage are limited to the zone of 2P excitation and do not occur above or beyond the focus.

Larger penetration depth. IR photons travel deeper into tissue with less scattering and absorption comparing to visible photons. Scattering 1/4 !

In practice - approximaterly 2 times larger penetration depth.

Much smaller background from impurity fluorescence when IR laser is used in comparison with VIS or UV light.

2 photon excitation spectra are usually very broad. Therefore, one laser source can be used for many different dyes having different fluorescence wavelengths. No chromatic aberration problems.

Some advantages of 2-photon excitation versus one-excitation in confocal microscopy

Even scattered fluorescence photons are usefull in 2-photon regime

All the dyes are excited by the same laser!

No effect of chtomatic aberration(White board)

Other ways to create contrast

Non-linear processes induced by strong laser light

Observation of fluorescence excitated by

2-photon absorption3-photon absorption

third harmonic signal

Observation of

second harmonic signal

....

4)3()2()1(

EEEEEEP

PED

Absorption,scattering

SHG microscopy is generally used to observe non-centrosymmetric structuresSHG is forbidden where there is an inversion symmetry, and this constraint makes it a sensitive tool for the study of interfaces and surfaces

One can get a signal even without using any dyes to stain the sample

nsorieneatiooveraveragedN )2(

Number of molecules

SHG is cohherent processes: Intensity N2

Fluorescence is noncohherent processes: Intensity N

Cross-section of SHG on a molecules is very small, but collective response from many molecules can compensate it !

Third harmonic generation image,No dye staining was applied

Optical microscopy beyond diffraction limit

?????

Diffraction limit – distribution of light intensity

However, if the process is nonlinear function of intensity, then the localization is not limited by the wavelength

Excited state depletion

Excitationpulse

S0

S1

Fluorescence

Excitation pulse

Excited state depletion

Excitationpulse

S0

S1

Fluorescence

Stimulated emission

Stimulated emission

Excitation pulse

STED pulse

Excited state depletion

Excitationpulse

S0

S1

Stimulated emission

Stimulated emission

Excitation pulse

STED pulse

Fluorescence is completely suppressed by stimulated emission process.

Kinternal relaxation >KSM >> Kfluorescence

Suturation condition for STED pulse: KSM=Kfluorescence ; Isaturation absorption ~ 1 ns-1

Photons in STED pulse has lower energy to avoid excitation.

Pulse duration should much shorter then S1 lifetime = 1/Kfluores

Imax>> Isaturation

~

Fluorescence

Excitation spot

f(x) - Spatial distribution of the STED pulse

Saturation parameter:

= I max/ Isaturation

x

f(x) = sin2(2/)

x= /100, when =1000

x