Univerza v LjubljaniFakulteta za matematiko in fiziko

Seminar 1

Two-photon excitation microscopy

Author: Petra CotarMentor: prof. dr. Janez Strancar

Ljubljana, may 2018

AbstractIn this seminar I will explain the principle of two-photon excitation end its application inmicroscopy. This is an alternative method to confocal microscopy. It is mostly used for

examination of biological samples and allows deep tissue imaging with less damage to thesample.

Contents

1 Introduction 2

2 Fluorescence 22.1 Two-photon excitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3 Two-photon excitation microscopy 43.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.2 Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.3 Photoacoustic imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4 Fluorophores 8

5 Conclusion 9

1 Introduction

The first observation fluorescence was reported by Sir John Frederick William Herschel in1845. But multiple photon fluorescence was first predicted by Mari Goppert-Mayer in herdoctoral dissertation in 1931. It took 32 years for her theory to be proven right. In 1963there was first report on two-photon excitation by Kaiser and Garret. After that there wasmany other examples. Two-photon spectroscopy has become an important tool for studyingmolecule structure. Two-photon excitation microscopy was invented by Denk et al. in 1990[1]. It allowed dipper and noninvasive observation of biological specimens. Other advantagesand limitations will also be discussed in these seminar.

2 Fluorescence

Fluorescence is a form of luminescence. It occurs when a material, called fluorophor, absorbslight and then emits it. Electron is excited to a higher energy level (S1) and when it returnsto a ground state (S0) a photon is emitted. The process of fluorescence is usually illustratedby the Jablonski diagram. As shown in the figure 1, photon usually excite electron in one ofthe higher vibration states of S1. Molecule than relax to the lowest vibrational level. Thisoccurs in ∼10−12 s and it is called internal conversion. After that electron returns to one ofthe higher vibrational states of S0. This occurs in around 10−8 s and it is called fluorescencelifetime (τ). Lastly, electron returns to the lowest vibrational state of S0. As we se onthe Jablonski diagram, there are also other possibilities for molecule relaxation. It can alsorelax through triplet states (T1), vibrational states or can be excited to higher energy levels.Relaxation from a state T1 is slow ∼ 10−3. If the molecule goes in these state we do notget any signal. This is also called dark period. Interactions with other molecules can alsoprevent fluorescence form happening (quenching). From Jablonski diagram we see, that theenergy of emitted photon is smaller than the energy of absorbed photon. That means thatthe emission light has a longer wavelength. This change in wavelength is called Stokes shift.Important characteristic of fluorophores is quantum yield. It is a ratio of emitted photonsto the number ob absorbed photons. Fluorophores with higher quantum yield have brighteremission. It is given by the equation

Q =Γ

Γ + knr.

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Figure 1: Jablonski diagram. It shows different ways of excitation end relaxation. ICSstands for intersystem crossing and IC for internal conversion [3].

The rate constants Γ and knr both depopulate the excited state. Where knr is the rate fornon-radiative decay and Γ radiative decay (emission rate of fluorophore). As we mentionpreviously, the lifetime of fluorophors is around 10 ns, and it is calculated as

τ =1

Γ + knr.

Another characteristic of fluorophors is photo bleaching. With time fluorescence is fading.Therefore each fluorophor has a limited number of excitation cycles [2].

2.1 Two-photon excitation

Electron can be also excited to a higher state with two photon (or even more). Two photonshave to be absorbed nearly simultaneously to excite electron to S1. In that case each photonhas 1/2 of the energy that is needed to excite the molecule. In comparison with one-photonexcitation (1PE), that means double the wavelength. First photon excites the electron to avirtual state. This is a very short-lived state (between ps and fs). If the second photon is’tabsorbed in this time, the electron returns to the ground state. If the photon density is nothigh enough we don’t get any signal. That gives us a cut-off in signal. Since we need twophotons for 2PE, the amount of light absorbed is proportional to the square of intensity (for1PE it is linear dependency). That is why 2PE only occurs where the density of photons isthe highest and we get only a small volume of fluorescence. With 1PE we get fluorescenceall along the laser beam. This is shown in the figure 2.

There ia also a different cross sections for 2PE. For 1PE we have σ1 in units cm−2 andit ranges from 10−15 to 10−17 cm−2. For 2PE the cross-section is in units cm4 s/photon orGM. Where 1 GM = 10−50 cm4 s/photon. The unit is named after Maria Gopperd-Mayerwho first described the concept of 2PE. The physical origin of 2PE cross-section can beunderstood if we look at the expression for the number of photons absorbed per second. For1PE that is (with units in parentheses)

NA1(photon/s) = σ1(cm2)I(photons/cm2s)

and for 2PENA2(photon/s) = σ2I

2(photons/cm2s)2.

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Figure 2: Comperison of a one-photon and two-photon excitation. On the left there is anexperimental illustration of fluorescence in both cases. And on the right there is a schematicillustration and a graph of intensity. We see fluorescence all along the laser beam in a 1PEand only a dot in a 2PE [2].

From here we see that the unit of σ2 must be cm4 s/photon. Excellent fluorophores have across-section larger than 100 GM (rhodamine B) [2].

3 Two-photon excitation microscopy

Two-photon microscopy started in 1990 and it is now widely used. It is mostly used forobserving living specimens. The first problem with realization of the theory was the lightsource used for excitation. As mentioned, there has to be a large photon density (1020 -1030 photons/cm2s), which is achieved with high power lasers. That is ∼ 106 times higherflux as needed for 1PE used in confocal microscopy. At that power we would also damagethe specimen. Therefore the two-photon microscopy become well-established with the use ofpowerful pulsed lasers. The most used are titanium-sapphire (Ti-Sa) lasers. They generateshort pulses with a typical duration of a few ten femtoseconds and frequency around 80MHz, with wavelength from 700 nm to 1000 nm. Power during the pulse is high enoughto cause fluorescence. The average power (around 2 W) is low enough to not damage thespecimen. Other component of two-photon microscope are similar to the setup where 1PEis used (confocal microscopy). A schematic of a typical microscope is shown in figure 3. Itshows the path of excitation light (in red) from laser on to the dichroic mirror and troughthe objective on to the specimen. There it creates fluorecence. Emitted light (in blue) passestrough objective and dichroic mirror on to the sensor. Since it can only observe one point atthe time, it uses Galvanometer-drive to move the laser beam and scan the whole specimen.Dichroic mirrors let the light of some wavelength pass trough and reflects all others. It isused to separate excitation and emission light. Photon sensors can be a photomultiplier tube,an avalanche photodiode or a CCD. There is also a mechanism that allows the specimen tobe moved up and down. That allows to observe the deeper layers [1].

3.1 Advantages

There are a few reasons to use 2PE instead of 1PE (confocal microscopy). First was alreadymentioned in previous chapter. With 2PE we get fluorescence in a small area, as small as 0.1

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Figure 3: Simple schematic of a two-photon excitation microscope. Red lines repriset exci-tation light and the blue emitted light [1].

µm. Which means, that we only get signal from one point and there is no out of focus signal.With 1PE we get a bigger excitation volume. With 1PE we get fluorescence in a larger area,that also mean photobleaching and photodamage in parts of the specimen that we are notobserving. So we can destroy parts of the specimen before even getting any information fromit. That is all improved with a small excitation volume of 2PE [5].

With 2PE we can observe thicker specimens. Limit for 1PE is around 100 µm, but with2PE we can image at depths up to 1 mm. This arises because of a few reasons. The firstone is the effect of scattering. It is less detrimental to two-photon microscopy as in thecase of confocal. In 1PE even the scattered light can cause the fluorescence and we can getunwanted signal. There is a pinhole, that should block the out of focus light. Problem is,that the emitted light also scatters. So the pinhole can let trough the light we do not wantand blocs the one we would like. 2PE only gives fluorescence in the focus. So even if thephotons scatter, we still know that they came from the point we are observing. This givestechniques using 2PE betters contrast and allows deeper imaging, since this effect increaseswith depth. If there is more tissue there is more scattering. This is demonstrated in figure 4.The second reason is that the light with longer wavelength scatter less in biological samples.Since excitation photons in 2PE have two times bigger wavelength than photons for 1PE,they will scatter less. Third reason is smaller absorption of excitation light in two-photonmicroscopy. In confocal microscopy fluorescence is generated trough the specimen and thatmeans that some photons get absorbed before the focal plane. In two-photon microscopymore photons reach the focal plane. Comparison of both methods is shown in figure 5.Some methods of application deep tissue imaging are immune cell dynamics, neuron imaging,studying kidneys... [6].

Excitation with UV light is hard with 1PE because photons have a high enough energyto damage living specimens and are more scattered. There are also special optics neededfor UV microscopy. We get ride of all this problems with 2PE since we use light with lowerenergy. That lowers scattering and photodamage [4].

Two-photon microscopy is also the best option for imaging structures in a living cells.This is because of multiple reasons we already mentioned. It makes less photodamage and we

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Figure 4: Schematic showing the comparison between confocal (A) and two-photon mi-croscopy (B). In A the excitation light (blue) is scattered and makes fluorescence in differentlocations. From each of that point there is emitted light (green). There is some light thatcomes from the focal plane and goes trough the detector (a). Some light form the focal planegets scattered and is blocked by the pinhole (c). There is also some out of focus light (b, d,e). Some of it is blocked (b and d) and some is let trough because of the scattering (e). In Bthere is excitation light that is also scattered (red) but the fluorescence only happen in thefocal plane (b and d do not caouse fluorescence). All the emitted light (green) is collected,even the scattered (a and c)[6].

can image deeper, which is spatially important since we can’t manipulate the specimen. Avery nice example of this is an experiment done on mammalian embryos. Researchers wantedto observe embryos through their development and maintain viability. They used hamsterembryos and observe them with 2PE microscopy and confocal microscopy (1PE). Confocalimaging inhibited development after only 8 hours. Some cells did not even divide once. Theyalso had embryos without added fluorophores. Their development was also inhibited underthe laser used for 1PE. That suggests that the light directly affects the embryos, withoutexcitation of fluorophores. With 2PE microscopy they were able to observe the embryodevelopment. They imaged the cells every 2.5 minute for 24 h. Later (after transferred into females), the embryo developed in to healthy animal [7]. The comparison of developmentunder both condition is shown in figure 6 on the left. The right side of figure 6 showsdevelopment of the embryo.

Another advantage of these method is in vivo imaging. Example of that is examinationof neuronal networks. It is done with Ca2+ imaginge. It allows a real-time analyses ofindividual cells trough the skull. Two-photon microscopy is also used to study in vivo bloodflow. All these experiments were done on rodents [9].

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Figure 5: Comparison of two different methods, 1PE (confocal) and 2PE. The third row isalso 2PE but it was made with a different detector, non-descanned detector (NDD), thatimproves image quality. We get better images with 2PE, especially dipper in the specimen[6].

3.2 Limitations

One limitation of this method is the laser. Because of their high price, there is usually onlyone excitation laser in the system. So we can only examine with one excitation specter.

There is also more photobleaching in 2PE microscopy. Because of that, we can only imagethe specimen a limited amount of times. Then we have to prepare a new one. With 1PEwe observe photobleaching that increase linearly with excitation power and with 2PE thereis higher-order dependency. This is especially detrimental for thinner samples. Because ofhigh energy lasers there are also more molecules that get excited to higher states. If we lookback at the Jablonski diagram (figure 1), there are also states higher than S1. So the photongets excited to higher state from S1 or even T1 and that doesn’t give us any signal. Thateffect is smaller with 1PE since the density of photons is smaller and the molecule have timeto relax to ground state before the second photon is absorbed. [4].

2PE microscopy is limited with diffraction. Even though the excitation volume is small,the point-spread function is wider because of smaller wavelengths. That gives us slightlyworse spatial resolution as the confocal microscopy. One way to improve that is with STED- stimulated emission depletion microscopy. If we combine these two methods we can getsub-diffraction resolution [8].

3.3 Photoacoustic imaging

Another way to use 2PE is photoacustic imaging (PAT). PAT is used for molecules are notfluorophores. This method takes advantage of the fact that the energy of an absorbed photoncauses rise in temperature. Because of the heating the medium expands. When the photonexit the medium starts to cool down, therefore starts to decrees its size. This expansion andcontraction propagates ultra sound waves and that is the signal we detect. The two-photonexcitation photoacoustic imaging detects signal caused by two-photon absorption. Becauseof 2PE we get a localized absorption. Ultrasonic waves are scattered much less then photons,

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Figure 6: On the left side there is a comparison of hamster embryo development usingconfocal microscopy (first row) and two-photon microscopy (second row). It shows embryosat the beginning (a and b) and at the end of imaging sequence (c after 8 h and d after 24h). We can see that the cells observed with confocal microscopy did not divide. On theright side there is a long-term imaging of the embryos with two-photon microscopy. Theyare taken at the beginning (A), after 8.5 h (B, arrow pointing to mitotic spindle), after 24h (C) and after 82 h (D, taken with a Nomarski microscope). The last is an image (E) of afetus developed from one of these embryos (black eyed), next to an uterine mate. First scalebar = 45 µm and the second = 4.75 mm [7].

which allows us a deeper imaging, to a few mm [10]. There was a experiment that observedfluorescence and photoacoustic signal of Rhodamine B at the same time (both were inducedsimultaneously) [11].

4 Fluorophores

With 2PE microscopy we usually observe fluorophores. Some specimens already have moleculesthat fluorescent; those are called natural or intrinsic fluorophores. Frequently the moleculesdon’t have them or they don’t emit enough signal. In that case we have to label moleculeswith extrinsic probes. Probes are made specially for the molecule or part of cell that wewant to observe. They are made so that they interact or binde to the part we want. Proteinare usually labeled with chromophores, membranes with DPH and so on. Their character-istic can change once binded with another molecule. Example of an intrinsic fluorophor isenzyme NADH that is found in living cells. Structure of NADH is shown in figure 7. Lifetime of NADH is near 0.4 second. Upon binding it increases for 1.2 ns and his quantumyield increases four times. His absorption and emission peaks are at 340 nm and 460 nm.As mention before, one od the intrinsic fluorophores is DPH. Membranes can be also labeledby covalent attachment of probes to the lipids. One of those probes is shown in figure 7(Texas Red-PE). Probes attach to the phospholipids in membranes. The fluorophore TexasRed is often used for long wavelength absorption and high photostability. In cells we often

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want to observe DNA. It has a very weak intrinsic emission, too weak for imaging. Thereare several probes that spontaneously binds to DNA. We can see one of them in figure 7,Ethidium Bromide (EB) with excitation end emission wavelength 514 nm and 533 nm. EBfluorescence is increased about 30 times once binded to DNA. His lifetime also increases from1.7 ns to 20 ns. EB’s aromatic rings interact with base pairs in DNA [2].

Figure 7: NADH is an intrinsic fluorophore found in living cells. Texas Red and EthidiumBromide are extrinsic probes used to label membranes and DNA [2].

One of the most important property of probes is photostability. You want slow photo-bleaching, so that the probe can be used longer. To get better photostability or label newmolecule, you have to synthesis new probes. Those are usually made based one the expe-rience and prior results. It is hard to predict fluorescence or even calculate it, because ofall the atoms that compose molecules. There is also interaction betwen molecules that canchange fluorescence. Fluorophores usually have aromatic rings and long carbon tales. Youalso can’t just take 1PE probes and use it for 2PE. Because of different selection rules not allfluorophores that are good for 1PE are also good for 2PE; but bright 1PE are usually alsogood for 2PE. Even if they are good for both methods, their absorption specter is usuallydifferent [5].

5 Conclusion

Two-photon microscopy has enabled advances in biological imaging, but there are also tech-niques that are used to improve the quality. We already talked about one thnique, STEDthat improve spatial resolution. We also mentioned that we can only image one point at thetime and have to scan the hole specimen. This can be improved with scanless two-photonmicroscopy (SLM). This system simultaneously targets several tens of points and shortensthe time needed for imaging the whole specimen [12]. There are already some microscopesthat uses three-photon excitation. It does need higher energies but it gives even smaller focusvolume and can image even deeper (3.5 mm) that two-photon. Even four-photon excitationcould be used. With higher numbers we need even powerful lasers that can damage thespecimen.But with that we would probably get cell damage because of the high energy ofthe laser [13].

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[9] C. Stosiek, O. Garaschuk, K. Holthoff and A. Konnerth, PNAS 100, 7319 (2003)

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[12] T. E. Holy, Frontiers in neuroscience 3, 333 (2009)

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