Nonlinear Optical Microscopy

X
Y
Non-linear?
Actual response can be written as

y = c
1
x+ c
3
x
3
(this is called a cubic distortion)

Assuming the input is a periodic signal x = cos (et)

y=c
1
cos(et)+c
3
[cos (et)]
3


Trigonometric identity tells us

[cos (et)]
3
= (3/4) cos(et) + (1/4) cos(3et)

The output is thus given by

y=[a1+(3/4)c
1
] cos(et)-(1/4)c
3
cos(3et)

Thus a small cubic nonlinearity gives rise to a modified response at w
but also generates a new signal at 3w

Nonlinear response
1. Applied field distorts the cloud and displaces the electron

2. Separation of charges gives rise to a dipole moment

3. Dipole moment per unit volume is called the polarisation
P = c
1
E ; P is the polarization

c
1
is called the linear susceptibility

This describes linear propagation giving rise to speed of
propagation through the medium (real part) absorption in the
medium (imaginary part)

It can be shown that

C
1
= n - 1

where n is the refractive index of the medium

Linear polarization
Nonlinear polarization
A more realistic equation for polarisation is

P = _
(1)
E + _
(2)
E
2
+ _
(3)
E
3
+ .

where _
(2)
, _
(3)
etc are the second and third order nonlinear
susceptibilities

Normally,

_
(3)
E
3
<< _
(2)
E
2
<< _
(1)
E

Unless, E is very very big.


Symmetry arguments can be used to show that for
isotropic materials even order susceptibilities are zero

Typical Nonlinear Optical
Phenomena
Second Order Processes
Second Harmonic Generation
Sum-Frequency Generation
Third Order Processes
Multi-Photon Absorption*
Stimulated Raman Scattering
Optical Kerr Effect
White Light Generation
Interaction of Light with Matter
...
3 ) 3 ( 2 ) 2 ( 1 ) 1 (
+ + + = E E E P _ _ _
P = induced polarization,
_
(n)
= n
th
order non-linear susceptibility
E = electric field


Linear Processes
Simple Absorption/Reflection
Rayleigh Scattering

_
(3)
<< _
(2)
<< _
(1)
(5-7 orders of magnitude per term)

Second Order Processes
Second Harmonic Generation*
Sum-Frequency Generation
Third Order Processes
Multi-Photon Absorption*
Stimulated Raman Scattering
Optical Kerr Effect
White Light Generation
One and two photon absorption physics
Requires high power:
Absorption only
In focal plane
Greatly Reduces out of plane bleaching
Simultaneous absorption
Virtual State:
Very short lifetime ~10
-17
s
Goeppart-Mayer, ~1936
e.g. fluorescein
One Photon 2 photon
Absorption
probability
Absorption
Coefficient
units
c (50,000)
o (10
-16
cm
2
)
o (10
-50
cm
4
s)
10
-50
cm
4
s=
1 GM (Goppert-Mayer)
Power (photon)
dependence
p P
2
(gives rise to sectioning)
Laser Temporal
dependence
none
1/t
o p o
p
2

/t
One and 2-photon absorption characteristics
Cannot use cw lasers (Ar+)
Xu and Webb, 1996
Slope of 2 at
All wavelengths:
2-photon process
Fluorescein and rhodamine
Power Dependence
2-photon excitation of fluorescein: 3D confinement
Absorption, Fluorescence only
in middle at focal point
Compare 1 and 2-p
Absorption
1-p excites throughout
Radial PSF Axial PSF
Comparable Lateral and Axial
Resolution to confocal

C
r
o
s
s

s
e
c
t
i
o
n

G
M

Max 820 nm
not 1050 nm
Two-photon Absorption Spectrum

1 0
S S
Nominally forbidden in 2-p
2 0
S S
Nominally forbidden in 1-p:
Allowed and stronger in 2-p
Rhodamine Photophysics
10
-12
s
1000 nm TPE
500 nm OPE
800 nm TPE
400 nm OPE
10
-9
s
S
0
S
1
S
2
800 nmstronger than 1000 nmband
Reverse of 1-photon
For all xanthenes:
Fluorescein,
rhodamines

All max ~830 nm
Not ~1000 nm
1 and 2-photon bands
Same emission spectrum
for 1-p, 2-p excitation

Relaxation is independent of
Mode of excitation
Same emission spectrum
For different 2-p wavelengths:
750 and 800 nm
Just like 1-photon emission
Xu and Webb, 1996
Emission Spectrum
1) Emission spectrum is the same as 1-p

2) Emission quantum yield is the same

3) Fluorescence lifetime is the same

4) Spectral positions nominally scale for the same transition:
2-p is twice 1-p wavelength for

5) Selection rules are often different, especially for xanthenes
(fluorescein, rhodamine and derivatives)

Some Generalities about Multi-
photon absorption
Non-decanned Detection
White, Biophys J, 1998
Confocal (1-p)<2-p descanned< 2-p direct

2-p direct collects ballistic and scattered photons
X-Z
projection
Non-descanned Detection
Increases Sensitivity
White, Biophys J, 1998
1-p
2-p
Improved Imaging Depth Due to
Reduced Scattering
All images are descanned

Registration Issues

Focus of White light vs Laser often different by 10-20 microns

Overlapping visible and near-infrared lasers difficult for uncaging

Second Harmonic Generation Alignment is different than Laser

Problems can arise from high peak power
giving rise to unwanted non-linear effects

- Plasma formation leading to cell destruction (makes
holes)

- Accidental 3 photon absorption of proteins and nucleic
acids (700-800 nm) (abnormal cell division)

~ 10 mW at 1.4 NA is good limit at sample
(Scales for lower NA)
Piston, Biophys J. 2000
488 nm 1-photon
Slope=1.2
Bleaching of fluorescein dextran in droplets
710 nm 2-photon
Slope=1.9 (low power)
Piston,2000
NADH=3.65
Coumarin=5.1
Indo-1=3.5
Highly nonlinear:
Higher order processes
Excitation to higher states
Non-linear bleaching (ctd)
For same transition 2-p
Does not bleach more
Than 1-p!
Applications
Autofluorescence of endogenous species in tissues
Need multi-photon excitation, non-descanned detection
For enough sensitivity: small cross sections and quantum yields
Autofluorescence in Tumors
Mitochondria:
NADH, Flavins

NAD not fluorescent
NADH emission to
Monitor respiration
NADH good diagnostic
Of cell metabolism
Small cross section
Quantum yield ~10%
Small delta ~0.1 GM
High concentration

Need non-descanned
Detection to be viable
Imaging Muscle (NADH)
With TPE Fluorescence

Low cross section but
High concentration
Balaban et al
Strata corneum

Keratinocytes

Dermal layer
(elastin, collagen)
fibers
Human Skin Two-photon imaging
So et al
Ann. Rev. BME
2000
More versatile than dyes (but weaker)
MPM enabling, very weak in confocal
Multiphoton bleaching
Need 3D treatment, both radial, axial PSF
Two-photon cross section measurement
Xu and Webb, 1996
Measure by fluorescence intensity, need quantum yield
(same as 1 photon)
Measure wavelength
Measure pulse width
Measure
power
Measure
Fluor.
2
2
1 2
] [

t
t o
hc
NA
P n
a

~
Control power