2. The Perrin-Jablonski Diagram
The life history of an excited state electron in a luminescent probe
Internal
conversion 10-12s
S total spin quantum number
M multiplicity
M= (2S+1)
S singlet state (S=0) M=1
T triplet state (S=1) M=3 S2 S1
Inter-system
Crossing 10-10s T1
Absorption
10-15s
Fluorescence
10-9s
Radiationless
Decay <10-9s
Phosphorescence
10-3s S0
Key points:
Emission has lower energy compared to absorption (one photon excitation)
Triplet emission is lower in energy compared to singlet emission
Most emission/quenching/FRET/chemical reactions occur from the lowest vibrational level of [S]1

3. Epifluorescence microscope
Fluorescence microscopy
No optical sectioning!!
The integral of intensity point spread function for conventional widefield microscope is constant as a function of depth, producing no optical sectioning capabilities.
Epifluorescence microscope
specimen
1PE
How we can achieve sectioning
effect in a fluorescence microscopy?

4. Laser scanning confocal microscopy
Background
Focal
plane
volume
Detector
Emission
filter
pinhole
Beam
expander FS
Dichroic
mirror
Laser
Microscope
objective z x

5. Multiphoton excitation fluorescence microscopy
4/28/2017
Multiphoton excitation fluorescence microscopy
Curso Microscopia

6. The history of two photon excitation and how this phenomenon was finally applied to fluorescence microscopy:
The two-photon excitation process is a quantum phenomenon that was theoretically predicted by Maria Göppert-Mayer in her thesis dissertation during 1931 (Annalen der Physik, Göppert-Mayer, 1931) .
This effect was first observed in 1961 after lasers sources were developed (first two photon excitation experiment used CaF2:Eu2+, Kaiser & Garret, 1961, Phys Rev. Letter 7: ).
Maria Göppert-Mayer received the Nobel Prize in Physics in 1963 for proposing the nuclear shell model of the atomic nucleus, becoming one of only two women to receive a Nobel Prize in Physics (the other being Marie Curie).
In 1976 Berns reported about a probable two-photons effect as a result of focusing an intense pulsed laser beam onto chromosomes of living cells (Berns MW: A possible two-photon effect in vitro using a focused laser beam. Biophys J, 1976, 16: ).
1978 – the first nonlinear scanning optical microscope with depth resolution was described (based in second harmonic generation - the possibility of performing two photon excitation microscopy was outlined; Sheppard CJ, Kompfner R: Resonant scanning optical microscope.Appl Opt, 1978, 17: ).
Biological applications were hampered by the high peak intensities required for priming two photon excitation fluorescence. This obstacle was overcome with the advent of ultrashort and fast pulsed lasers in the 80s.
Winfried Denk, James Strickler and Watt Webb introduced the two photon excitation scanning fluorescent microscope. They demonstrated the applicability of two photon excitation microscopy in biological systems (Denk W., Strickler J.H. and Webb W.W. (1990) Science 248:73-76).
Maria Göppert-Mayer

7. Simultaneous absorption of two photons
Two photon excitation principle
One-photon
two-photon S1 1p 2p
IR light
wavelength
emission S0
E= hc/
emission
Intensity
non linear
process
Simultaneous absorption of two photons

8. High photon density is required!!!
E= hc/
1/abs= 1/1 + 1/2
Since 2PE excitation requires at least two statistically "independent“ photons for each excitation process, its rate depends on the square power of the instantaneous intensity.
High photon density is required!!!

9. The strong focusing capability of a microscope objective allows to generate a volume containing high photon density.
The 2PE process can be generated with IR laser sources, i.e. either continuous wave (CW) or pulsed laser sources.
However there are fundamental advantages about using pulsed laser sources:
1) Even though the instantaneous light intensity is extremely high in pulsed lasers the average power received by the sample is not excesive.
2) The repetition time and pulse width are important parameters to deal with fluorescence phenomena

10. approximation is given by:
The most popular relationship about 2PE is related to the practical situation of a train of beam pulses (ultrashort and fast pulsed laser) focused through a high numerical aperture (NA) objective, with a duration τp and fp repetition rate.
Under controlled conditions, the probability, na, that a certain fluorophore simultaneously absorbs two photons during a single pulse, in the paraxial
approximation is given by:
Two photon absorption cross section (2) are measured in GM (Göppert-Mayer).
1 GM = cm4 s per photon.
for a δ2 of approximately 10 GM,
focusing through an objective of NA >1,
an average incident laser power of ≈ 1–50 mW,
operating at a wavelength ranging from 680 to 1100 nm
with 80–150 fs pulsewidth and 80–100 MHz repetition rate,
one should get fluorescence without saturation
saturation occurs when na=1
saturation declines resolution and lower the
quality of the fluorescence intensity images!
p pulse duration
fp repetition rate
pave average incident power,
2 absortion cross section
h Planck’s constant
c speed of the light
 wavelenght
NA numerical aperture.

11. As example, for fluorescein that possesses a δ2 ≅38 GM at 780 nm, using NA = 1.4, a repetition rate of 100 MHz and pulse width of 100 fs
na≈ 5930 (Pave)2.
Assuming Pave of 1, 10, 20 and 50 mW
na is
, , 1.86, respectively
Saturation start above 10 mW

12. What is so special about the use of ultrashort and fast pulsed lasers?
Pulse repeat
10-8 s
Pulse width
10-13 s
10-9 s
Fluorescence decay
during the pulse time (10-13 s of duration
and a typical lifetime in the 10-9 s range) the molecule has insufficient time to relax to the ground state. This can be considered a prerequisite for absorption of another photon pair
A 106-fold improvement over continuous illumination (CW) is achieved by using 100 fsec 100MHz repetition rate!

13. Other considerations:
The related rate of photon emission per molecule, at a non saturation excitation level, in absence of photobleaching, is given by na multiplied by the repetition rate of the pulses (na x fp)
For fluorescein at non saturating conditions is around
5·107 photons s-1.
It is worth noting that when considering the effective fluorescence emission one should consider a further factor given by the so-called
quantum efficiency (or quantum yield) of the fluorescent molecules.
It is worth noting that the fluorophore emission
spectrum results independent of the excitation
mode from 1PE to MPE like the quantum efficiency!!!!!

14. How to choose the excitation wavelength?
The quantum-mechanical selection rules for two-photon absorption
differ from those for one photon-absorption.
No quantitative predictions about the behavior of two-photon
absorption can be made from the one photon absorption spectrum.
Empirical rule (rule-of-thumb):
The absorption band for one photon excitation is centered at
wavelength l then the appropriate wavelength for a two-photon
excitation process is 2l (3l for three photon).
The two-photon absorption spectra of some dyes are available in the literature
For example see Xu and Webb (1996) J Opt. Soc. Am. B 13,

15. Do it yourself!!!
Notice that one single wavelenght in 2PE mode excite many fluorophores that in one photon case requires more than one excitation wavelenght!
Typical case: Rhodamine and fluorescein are excited with good efficiency using 780 nm

17. From
Svoboda and Yasuda
Neuron, 50:
2006

18. Intrinsic fluorophores

20. Two photon excitation effect has inherent spatial resolution!!!
Great! We can excite a molecule by using two simultaneous photons and get fluorescence as happens with one photon excitation.
But what is the relationship with sectioning effect here????
Two photon excitation effect has inherent spatial resolution!!!
The intensity distribution within the focal region of an
objective having numerical aperture NA = n sin (α) is
given, in the paraxial regime, by
where J0 is the 0th order Bessel function, ρ is a radial coordinate
in the pupil plane, n is the refractive index of the
medium between the lens and the specimen, (α) is the
semi-angle of aperture of the lens:
axial
radial

21. The intensity of fluorescence distribution within the focal region has a I(u, v) behaviour (linear) for the 1PE case
In case of 2PE one has to consider a double wavelength and a square behaviour, i.e. I2(u/2, v/2); quadratic
Consequently the 2PE emission intensity distribution is axially confined!!
In fact, considering the integral over ν, keeping u constant, its behaviour is constant along z for one-photon and has a half-bell shape for 2PE. This behavior is responsible of the 3D discrimination property of 2PE, i.e. of the optical sectioning properties of the 2PE microscope.
Focal volume

22. Now, a very interesting aspect is that the excitation power falls off as the square of the distance from the lens focal point.
In practice this means that the square relationship between the excitation power and the fluorescence intensity I2(u/2, v/2) brings about the fact that 2PE falls off as the fourth power of distance from the focal point of the objective.
This fact implies that those molecules away from the focal region of the objective lens do not contribute to the image formation process and are not affected by photobleaching or phototoxicity.
It is immediately evident that in this case the optical sectioning effect is obtained in a physically different way with respect to the confocal case (no pinholes). 1P 2P

23. inherent spatial resolution!
Rhodamine
Fluorescein
543 nm
780 nm
780 nm
380 nm
80% of the total fluorescence
intensity is confined to within
± 1mm of the focal plane
(femtoliter volume).
Objective NA 1.25
wavelength 780 nm

24. From Rubart M, Circ. Research 2004, 95:1154

25. From Diaspro et al, Biomedical Engineering online, 2006
Up to here we have another method that allows sectioning effect using fluorescence as a contrast as confocal microscopy
MPE = intrinsically 3D
From Diaspro et al, Biomedical Engineering online, 2006
This article is available from:

26. Rayleigh Criterion for the resolution of two adjacent spots:
Plim = 0.61 lo / NAobj
Examples: (lo = 550 nm)
Mag f(mm) n a NA Plim (mm) (NAcond=NAobj)
high dry 10x
40x
oil 100x
63x
MPE used infrared light Plim increase
(the resolution decreases)
BUT
The fact that the background signal coming from adjacent planes tends to zero produces a sort of compensation for the reduced spatial resolution due to the utilization of a wavelength that is twice with respect to the 1PE case.

28. Resolution (high NA objectives ~1.4)
600 nm axial z
300 nm radial x y
In practice very similar to a laser confocal microscope!

29. Superior than confocal (max 200 micrometers)
High penetration
Scattering effect is proportional to the inverse 4th power of the excitation wavelentgh
Thus the longer wavelengths used in 2PE, or in general in MPE, will be scattered less than the ultraviolet-visible wavelengths used for conventional excitation
Allows to reach fluorescence targets in depth within thick samples (up to 700 m).
It has been shown that two-photon fluorescence images can be obtained throughout almost the entire grey matter of the mouse neocortex by using optically amplified femtosecond pulses.
Superior than confocal (max 200 micrometers)

30. How to distinguish among one, two or three photon absorption
Sample: human skin
wavelength: 960 nm
Fluorophore: NADPH
From Master et al, (1997) Biophys. J 72:
For two photon absorption:
cuadratic dependence of the power of the incident light with the fluorescence emission intensity
For one and three photon absorption
the dependence of the power of
the incident light with the fluorescence
emission intensity is linear and cubic
respectively.

31. Practical use of UV excited probes!!
Intrinsic fluorophores such as NADH, FADH can be easily measured
From Diaspro et al, Biomedical Engineering online, 2006
This article is available from:

32. 1p 2p LAURDAN emission Intensity wavelength
360 nm
440 nm
780 nm
emission 2p 1p
Intensity
wavelength
The wavelength of the excitation light and the Rayleigh and Raman scattering
associated with it, is considerably different from that of the fluorescence emission
great increase in the signal to noise ratio for detection!!

33. Objectives
Important: Objectives should transmit infrared light!!
Recent developments in objective lenses improve the quality of the multiphoton excitation images

34. Modes of detection Descanned detection is superior for thick samples
From Diaspro et al, Biomedical Engineering online, 2006
This article is available from:

35. Laser Sources

36. Advantages Sectioning effect without pinholes
 Low photobleaching and photodamage rate
 Separation of excitation and emission
 No Raman from the solvent
 Deep penetration in tissues
 Single excitation wavelength for many dyes
 No expensive UV optics (for UV excited fluorophores)

37. Disadvantages
 Is only suitable for fluorescence images
(reflected light images is not currently available)
 The technique is not suitable for imaging highly
pigmented cells and tissues which absorb near
infrared light.
 Laser source is expensive (~ u$s )

38. Multicolor experiment, single wavelength. Avoid chromatic aberrations
From Diaspro et al, Biomedical Engineering online, 2006
This article is available from:

39. Applications in live animals
the penetration in tissues is high (up to 800 m)

40. two photon excitation – 3D reconstruction
Skin SC from live mouse
two photon excitation – 3D reconstruction
SC lipid
membranes
hair

41. Excise skin from human – 2 hours after surgery
two photon excitation – stack - autofluorescence

42. Applications Deep tissue imaging (Brain, skin, etc)
2PE can prime photochemical reaction within subfemtoliter volumes inside solutions, cells and tissues (photolabile ”caged” compounds)
Imaging of living specimen for longer period of time
Live Animal imaging (intrinsic fluorophores)

43. = Photon + molecule complex
SHG
Linear spectroscopy:
Fluorescence
Absorption
Scattering
Linear spectroscopy:
Induced Dipole
“Dressed state”
= Photon + molecule complex
The interaction of a weak electric field (excitation light) with the media will induce a linear response of the polarization

44. Non Linear spectroscopy
SHG
Non Linear spectroscopy
For intense electrical field non linear properties of the polarization become apparent:
Fluorescence
Multiphoton
Absorption w1 w1
w1+ 1 w1
2w1
The interaction of the strong electric field (excitation light) with the media will induce a non linear response of the polarization
Non centrosymmetric molecules can generate second harmonic
Second harmonic gives structural information
Biological application => Collagen fibers, muscle fibers
Second harmonic

45. Muscle fibers Laser @920nm SHG Size 81x81µm Size 27x27µm
The signal is exc/2
Sectioning capability
No out of focus signal
The signal is sensitive to the organization of the tissue

46.
SHG - Skin dermis