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

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?

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

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

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 7:229-231). 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, 16:973-977). 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, 17:2879-2885). 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. 1990 - 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 1906 - 1972

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

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!!!

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

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 = 10-50 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.

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 5.93 10-3, 5.93 10-1, 1.86, 2.965 respectively Saturation start above 10 mW

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 pulses @ 100MHz repetition rate!

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!!!!!

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, 481-491.

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

From Svoboda and Yasuda Neuron, 50:823-839 2006

Intrinsic fluorophores

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

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

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

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

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

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: http://www.biomedical-engineering-online.com/content/5/1/36

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 16 1.00 15 0.25 1.10 40x 4 1.00 40 0.65 0.42 oil 100x 1.6 1.52 61 1.33 0.204 63x 2.5 1.52 67.5 1.40 0.196 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.

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

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)

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: 2405-2412 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.

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: http://www.biomedical-engineering-online.com/content/5/1/36

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!!

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

Modes of detection Descanned detection is superior for thick samples From Diaspro et al, Biomedical Engineering online, 2006 This article is available from: http://www.biomedical-engineering-online.com/content/5/1/36

Laser Sources

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)

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 200.000)

Multicolor experiment, single wavelength. Avoid chromatic aberrations From Diaspro et al, Biomedical Engineering online, 2006 This article is available from: http://www.biomedical-engineering-online.com/content/5/1/36

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

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

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

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)

= 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

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

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

Laser @820nm SHG - Skin dermis