Short pulses in optical microscopy
Ivan Scheblykin, Chemical Physics, LU
Outline:
Introduction to traditional optical microscopy
based on single photon absorption:
Fluorescence wide-field and conforcal microscopy
Introduction to single molecule imaging
2-photon absorption
2-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 absorption
Light scattering
Changing of light polarization
…
An object emits light itself:
Luminescence
Second-harmonic generation
…..
Many different ways to create contrast in optical microscopy
Transmission image
Absorption and scattering
object
Transmission image
Absorption and scattering
Excitation light
100€
object
Blocking filter
object
Sample is stained by a fluorescent dye
Transmission image
Absorption and scattering
Excitation light
100€
object
Fluorescence
Blocking filter
object
White board
Microscope scheme
Numerical Apreture
Spherical angle S
Light collection efficiency

S/4
NA/n = 1 ,
50%
NA/n = 0.6, 10%
1.22  
D
NA
Confocal fluorescence microscope
Wide-field fluorescence microscope
10 microns
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!
Sample
For SMS
5
Single molecule imaging
Chemical Physics, Single Molecule Spectroscopy group, LU
Other ways to create contrast
Non-linear processes induced by strong laser light
 

D  E  4P




(1)
( 2)
( 3)
P   E   EE   EEE  ....
Observation of
fluorescence excitated by
Observation of second
harmonic signal
Absorption,
scattering
2-photon absorption
3-photon absorption
third harmonic signal
Two-photon absorption
Theory - Maria Göppert-Mayer, 1929
Experimental observation – 1961
Using in microscopy – Denk, Strickler, Webb, Science 1990
Probability of excitaion (W)  (Intensity)2
W  ( I [ptonots/cm2/s] )2
f
Absorbed photon
i
Fluorescence
Virtual level
One and Two-photon absorption cross sections
Transition dipole moment moment
Estimation of 2
(WB)
Two-photon excitation versus one-photon excitation
Dye solution, safranin O
543 nm excitation
1046 nm excitation
Resolution of 2-photon microscopy
XY, Z,
1/z4 excitation probability dependence
And 1/z2 dependence of total fluorescence (WB)
k1 p ( I1 )  k 2 p ( I 2 )
The same fluorescence signal from the sample
28
10
27
10
26
I2
Intensity for 2-photon excitation
10
25
10
24
10
23
10
22
10
21
10
20
10
19
10
18
10
17
10
16
10
15
10
0
10
5
10
10
10
15
Intensity for 1-photon excitation, photons/second/cm
I1
20
10
10
2
Some advantages of 2-photon excitation versus one-excitation in confocal microscopy
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.
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
 

D  E  4P




(1)
( 2)
( 3)
P   E   EE   EEE  ....
Observation of
fluorescence excitated by
Absorption,
scattering
Observation of
second harmonic signal
2-photon absorption
3-photon absorption
third harmonic signal
SHG microscopy is generally used to observe non-centrosymmetric structures
SHG 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
 ( 2)  N 
averaged
over orieneations
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
Excitation pulse
S1
Excitation
pulse
Fluorescence
S0
Excited state depletion
STED pulse
Excitation pulse
S1
Excitation
pulse
Stimulated emission
Fluorescence
S0
Stimulated emission
Excited state depletion
STED pulse
Excitation pulse
Stimulated emission
S1
Photons in STED pulse has lower
energy to avoid excitation.
Excitation
pulse
Stimulated emission
Pulse duration should much shorter then
S1 lifetime = 1/Kfluores
Kinternal relaxation >KSM >> Kfluorescence
Fluorescence is completely suppressed
by stimulated emission process.
S0
Suturation condition for STED pulse: KSM=Kfluorescence ; Isaturation absorption ~ 1 ns-1
Imax>> Isaturation
Fluorescence
f(x) - Spatial distribution of the STED
pulse
x 

 
Saturation parameter:
 = I max/ Isaturation
f(x) = sin2(2/)
x= /100, when =1000
Excitation
spot
x
~