Post on 19-Dec-2015
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(
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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