- Email: [email protected]
Nonlinear microscopy
C. R. Acad. Sci. Paris, t. 2, Série IV, p. 1153–1160, 2001 Physique appliquée/Applied physics (Biophysique/Biophysics)
IMAGERIE ACOUSTIQUE ET OPTIQUE DES MILIEUX BIOLOGIQUES OPTICAL AND ACOUSTICAL IMAGING OF BIOLOGICAL MEDIA
Nonlinear microscopy Jerome MERTZ
DOSSIER
Laboratoire de neurophysiologie et nouvelles microscopies, INSERM EPI 00-02, Paris, France E-mail: [email protected] (Reçu le 30 juin 2001, accepté le 11 juillet 2001)
Abstract.
We describe the basic principles of nonlinear optical microscopies based on two-photon excited fluorescence and on second-harmonic generation. Particular attention is given to the physical mechanisms underlying molecular second-harmonic generation, and the features unique to its signal contrast. We provide an overview of some applications of nonlinear microscopy including the visualization of molecular ‘flip–flop’ dynamics in membranes, high-resolution measurements of inter-membrane separations, and highsensitivity membrane potential imaging. Future developments of nonlinear microscopy are briefly considered. 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS non linear microscopy / two-photon fluorescence / second harmonic generation / optical biopsy / biological tissues
Microscopie non linéaire Résumé.
Nous décrivons les principes de base de la microscopie non linéaire : d’abord la fluorescence à deux photons qui est devenue une méthode très répandue dans les laboratoires, puis plus en détail, la microscopie du second harmonique en insistant sur les mécanismes physiques et le contraste spécifique de cette détection. Nous discutons ensuite de quelques applications de la microscopie non linéaire comme la mesure de la dynamique des molécules polaires, la mesure de distance intermembranaires et l’imagerie potentiel membranaire. Nous concluons en évoquant quelques développements futurs de la microscopie non linéaire. 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS micoscopie non linéaire / fluorescence biphotonique / génération du second harmonique / biopsie optique / tissus biologiques
Introduction Nonlinear microscopy, by its very nature, requires high optical intensities and has had to await the invention of the pulsed laser to come of age. By far the most well known form of nonlinear microscopy resulted from the pioneering work of Denk and Webb [1,2] in which they showed that a mode-locked laser beam, when focused into a sample, generated fluorescence only at the focal center. This ‘spot’ of fluorescence could be considered a 3-dimensional pixel, or voxel. By scanning the spot through the sample, a 3-dimensional image could then be constructed. Since its first demonstration in 1989, the two-photon Note présentée par Guy L AVAL. S1296-2147(01)01260-4/FLA 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS. Tous droits réservés
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laser scanning microscope based on this simple principle has become a laboratory standard [3,4] and has recently been commercialized. It should be mentioned, however, that several years prior to the invention of two-photon microscopy, another form of nonlinear microscopy had been utilized by various laboratories [5,6]. This microscopy was based on the generation of second harmonic light, either from surfaces or from mesoscopic tissue structures such as rat-tail tendons [7]. Because of difficulties in signal interpretation and perhaps because of its seemingly obscure utility, at least in biological imaging, second-harmonic microscopy has gone relatively unnoticed until only very recently. The purpose of this chapter is to briefly describe the basic principles behind nonlinear microscopy. Because several excellent reviews of two-photon excitation fluorescence (2PEF) microscopy are readily available, most of this article will be devoted to second-harmonic generation (2HG) microscopy, with particular emphasis on how it compares to and contrasts with 2PEF microscopy. New developments in the field will be briefly evoked at the end of the article. 1. 2HG versus 2PEF microscopy: principles The term second-harmonic generation is usually reserved for coherent phenomena arising in bulk volumes, such as crystals, or extended surfaces. In these cases, the radiating source is appropriately characterized by its nonlinear susceptibility. In contrast, the term hyper-Rayleigh scattering is usually reserved for (elastic) nonlinear scattering at the molecular level, and the sources are appropriately characterized by their nonlinear polarizabilities [8]. Inasmuch as 2HG from a macroscopic source represents the coherent summation of hyper-Rayleigh scattering from the microscopic source constituents, the two terms will be used interchangeably here at the expense of semantic rigor. Moreover, since it is common practice to quantify the efficiency of 2PEF by molecular cross sections [9], the same will be done for 2HG for purposes of comparison. It should be kept in mind, however, that the concept of a 2HG cross section is only applicable to a single isolated hyper-Rayleigh scatterer. When many scatterers are implicated, this concept becomes ambiguous since the resultant 2HG varies widely depending on how the scatterers are spatially distributed, as will be discussed below. When laser excitation is incident on a single molecule, it may be absorbed or scattered. Nonlinear versions of these phenomena are depicted in figure 1. In the case of 2PEF, two photons are simultaneously incident (within about a femtosecond) and are resonantly absorbed since their combined energies falls within the molecular absorption band. Some of this energy is dissipated to the solvent or to nuclear redistribution, and the rest may be available for fluorescence [10]. The phenomenon of absorption followed by fluorescence emission is generally incoherent in that the phases of the excitation and fluorescence photons are generally uncorrelated. In the case of 2HG, the excitation photons are not absorbed but instead are scattered into a single outgoing emission photon. Because the scattering is essentially instantaneous, the phases of the excitation and emission photons are tightly correlated in this case and the phenomenon is coherent. The difference between incoherence and coherence leads to fundamental differences in the natures of 2PEF versus 2HG. In particular, radiating molecules are effectively non-interacting when they emit incoherently, meaning that their total emission power simply scales with their number. This is not the case when they emit coherently. For example, if two molecules at the same position radiate in phase, then because of constructive interference their total radiated power is four times the power from an individual molecule,
Figure 1. Two photons incident on a molecule can be absorbed (left) or scattered (right), leading to the generation of fluorescence (2PEF) or second-harmonic light (2HG).
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whereas if they radiate out of phase, then because of destructive interference their total radiated power is zero. It is clear here that the molecules cannot be regarded as independent when they emit coherently. The situation is further complicated when the radiation phase depends on the molecule orientation or on its position in space, both of which arise in 2HG. A particularity of dipolar 2HG is that it requires a non-centrosymmetric source [8]. On a molecular level, this means that a hyperpolarizable molecule must exhibit ‘charge transfer’ [11], that is, there must be a difference between the dipole moments of the excited and ground states. The direction of this charge transfer imparts a well-defined orientation to the molecule. Depending on whether this direction is parallel or antiparallel to the incident field polarization, the resultant 2HG is driven either in-phase or out-of-phase. In the case of a population of 2HG radiating molecules, constructive interference then becomes only possible if the molecules exhibit a global alignment. A very effective technique for obtaining such an alignment consists in labeling lipid membranes with amphiphilic dye molecules consisting of polar and non-polar endgroups spanned by a non-centrosymmetric chromophore. Because of their geometry, these molecules readily insert themselves into lipid membranes with a preferred orientation, as shown in figure 2. It should be noted that the radiative properties of the chromophore are usually very sensitive to local environment. For example, there can easily be a factor of ten increase in the fluorescence quantum efficiency of the chromophore when it passes from an aqueous (polar) to a lipid (non-polar) environment [10]. When the driving field polarization is along the molecular axis, the 2HG cross-section of a single molecule, defined through P2HG = 12 σ2HG I 2 , where P2HG is the second-harmonic power (photons/s) and I is the incident laser intensity (photons/s/area) is related to the molecular hyperpolarizability β by [12]: σ2HG =
4ω 5 |β|2 3πnε30 c5
m4 ·(photon/s)−1
In general, σ2HG is small, much smaller than its fluorescence counterpart σ2PEF . For example, even when β is resonantly enhanced by its transition energy occurring within the molecule absorption band (as shown in figure 1), σ2HG is about four orders of magnitude smaller than σ2PEF for the molecule Di-6-ASPBS in a lipid membrane. For single-molecule detection, therefore, 2HG is at a considerable disadvantage. For the detection of many molecules, on the other hand, 2HG can be relatively efficient owing to its quadratic dependence on the number of radiating molecules, assuming these are aligned. Figure 3, for example, illustrates the emission spectrum of Di-6-ASPBS molecules in a membrane that undergo both 2PEF excitation (right lobe) and 2HG (left peak). The areas under the respective spectral features are about the same, indicating that the powers are comparable. The exact number of molecules that contribute to 2HG, or even to 2PEF, is a matter of definition. In the case of excitation with a tightly focused laser beam, the case usually encountered in scanning microscopy, this number can be unambiguously defined based on a Gaussian approximation of the laser beam profile about the focal center [13]. According to this definition, the spatial resolutions of both contrast modes are the same. It should be pointed out, however, that in addition to scaling quadratically with the number of radiating molecules, 2HG power critically depends on how these molecules are distributed relative to
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Figure 2. Molecular structure of Di-6-ASPBS and its asymmetric insertion geometry in a lipid bilayer (perfusion from top).
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OPTICAL AND ACOUSTICAL IMAGING OF BIOLOGICAL MEDIA
Figure 3. Simultaneously acquired 2HG (left peak) and 2PEF (right lobe) emission spectra from labeled membrane. From [35].
Figure 4. Radiation patterns of fluorescence (2PEF) and second-harmonic light (2HG) by focused excitation of amphiphilic dye molecules in a membrane.
one another. For example, when molecules are uniformly distributed on a membrane surface illuminated from the side, then the total 2HG power is significantly smaller than when the same number of radiating molecules are concentrated exactly at the focal center (neglecting aggregation effects such as dimerization, self-quenching, etc.). Moreover, the resultant 2HG also varies considerably in its angular distribution: for uniformly distributed molecules the 2HG is emitted along two forward directed off-axis lobes (see figure 4), whereas for molecules concentrated at a point, it adopts a simple dipole radiation profile. This sensitivity of the 2HG radiation pattern to molecular spatial distribution is a direct consequence of its coherent nature [14]. 2. 2HG versus 2PEF microscopy: applications The applications of 2PEF microscopy are well established. They range from deep high resolution imaging in scattering media, localized molecular uncaging [15,16] or photo bleaching [17], micro-lithography [18], etc., and will not be dwelt upon here since references on the matter are exhaustive [19–21]. In contrast, the applications of 2HG microscopy have been relatively unexplored. Three such applications will be considered here, which are meant to highlight the differences between 2HG and 2PEF. These applications are readily illustrated with model membranes such as Giant Unilamelar Vescicles (GUVs). 2.1. Measurement of flip–flop dynamics When amphiphilic molecules such as Di-6-ASPBS described above are perfused onto a GUV, they initially only label the outer membrane leaflet and the polar headgroups all face outward (upward in figure 4). This is a condition of maximum asymmetry, and therefore leads to maximum 2HG. With time, however, the molecules undergo thermally induced ‘flips’ to the inner membrane and eventually the populations in the outer and inner membranes become equal. This equilibrium condition has lost all its asymmetry and therefore cannot produce 2HG. The energy barrier for molecular flipping is high, typically tens of KT depending both on the molecule and lipid structures, meaning that the rate of redistribution
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Figure 5. Fluorescence (2PEF) and second-harmonic light (2HG) from Di-6-ASPBS molecules that are initially asymmetrically distributed in a DOPC membrane, but progressively become symmetrically distributed with time (room temperature). From [12].
is slow. Nonetheless, this rate is readily measurable by monitoring the decay of the 2HG signal with time [12,22]. It should be noted that the attendant 2PEF signal, which may be obtained simultaneously, is dependent only on the total number of radiating molecules regardless of whether they are in the outer or inner membrane leaflets. As such, 2HG provides a signature of the difference in populations between the inner and outer leaflets, whereas 2PEF provides the signature of their sum. This latter signature can vary as the result of perfusion dynamics or photo bleaching, and can provide an accurate normalization for the 2HG signal (see figure 5). 2.2. Measurement of intermembrane distance An alternative interpretation to the above 2HG response to ‘flip–flop’ is that molecules in the outer leaflet radiate with a given phase, whereas molecules in the inner leaflet, because they are oppositely oriented, radiate with the inverse phase. As a result, if the populations in the two leaflets are equal, then the net 2HG from both leaflets completely destructively interferes. This interpretation is correct because the inner and outer leaflets are essentially at zero distance from one another. On the other hand, if the two leaflets were separated by a microscopic distance of the order of the 2HG wavelength, then the above scenario changes radically. Because the 2HG emission from each leaflet is off axis, a microscopic distance between the leaflets occasions a difference in the optical path-lengths between the leaflets and the observer. As a result, what was destructive interference at zero inter-leaflet separation becomes constructive interference at a non-zero inter-leaflet separation. In this way, the 2HG signal provides an excellent measure of the inter-leaflet separation [12]. In practice, of course, instead of separating the inner and outer leaflets, it is much easier to bring two GUVs with labeled outer leaflets in close proximity, as shown in figure 6. The ‘hot spots’ apparent in this figure correspond to the inter-membrane distance where the 2HG interference is maximally constructive. This distance is given by approximately 0.6 times the excitation beam waist at the focus, corresponding typically to a few hundreds of nanometers. For smaller distances, the 2HG decreases roughly linearly. As such, 2HG provides an excellent measure of membrane separations over ranges that are inaccessible both to diffraction limited imaging and to short range interactions such as fluorescence resonance energy transfer (FRET).
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Figure 6. 2HG produced by two labeled membranes in close proximity. When membranes adhere, 2HG destructively interferes. When they are separated by 0.6 times the excitation beam waist (w0 ), 2HG constructively interferes resulting in ‘hot spots’ apparent in image of contacting GUVs.
2.3. Membrane potential imaging Perhaps one of the most promising applications of 2HG is its capacity to image electric fields across membranes. For example, the difference in electric potential between the inside and outside of living cells is typically several tens of millivolts. Supposing that the electric field is uniform inside the cell membrane, this would correspond to electric fields on the order of 105 V/cm. In actuality, the electric fields are far from being uniform. Cell membranes consist predominantly of DOPC lipids which possess zwitterionic headgroups. These create ‘dipole layers’ at the membrane surfaces where the electric fields vary rapidly and can be much larger than in the uniform field approximation. Suffice to say that the fields can be large enough to significantly perturb the hyperpolarizabilities of various types of 2HG chromophores [23,24]. Sensitivities of the order of 50% increases in 2HG for 100 mV changes in membrane potential can readily be demonstrated in model systems, as illustrated in figure 7. Such sensitivities are about an order of magnitude higher that those obtained with fluorescence based techniques relying on electrochromism. In cellular systems, however, such sensitivities are considerably harder to demonstrate largely because of problems with photo-toxicity and artifactual responses unrelated to membrane potential. Nonetheless, they have been reported, for example, by Loew and co-workers using Nernstian membrane potential variations induced by changes in extracellular potassium concentrations [25,26]. It should be emphasized that the
Figure 7. 2HG signal from a GUV labeled with ‘charge-transfer’ molecules. Membrane potential of GUV is controlled by an intra-vesicular electrode (not seen because it is at a different plane of focus).
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photo-physical mechanisms governing the membrane potential sensitivities of 2HG markers still remain poorly understood.
New developments in nonlinear microscopy are many-fold. Several groups, for example, have demonstrated the extension of nonlinear microscopy to higher orders. In particular, three-photon excited microscopy (3PEF) has been used to image autofluorescent endogenous molecules in cells, such as serotonin or indoleamines [27,28]. The molecules are normally one-photon excited in the UV range, which causes extensive photo damage. With the use of three-photon excitation, this range can be avoided. Similarly, thirdharmonic generation microscopy has become a promising tool for the imaging endogenous species [29,30]. 3HG has the advantage over 2HG that it does not require non-centrosymmetric sources, and hence is not limited to the imaging of interfaces (though interfaces may certainly enhance the 3HG signal). Finally, very recently, microscopy based on nonlinear coherent anti-Stokes–Raman scattering (CARS) has made its appearance [31]. This technique, also particularly applicable to the imaging of endogenous species, presents the added advantage of excellent molecular discrimination. With regard to exogenous markers, considerable headway has been made in the design of new molecules that are specifically optimized for nonlinear microscopy. This includes the development of quadrupolar dyes with enhanced two-photon absorption cross sections [32–34], for both imaging and optical limitation applications, and the development of molecules with enhanced first hyperpolarizabilities for 2HG applications. These new developments should bear their fruit in the very near future. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26]
Denk W., Strickler J.H., Webb W.W., Science 248 (1990) 73. Xu C., Zippel W., Shear J.B., Williams R.M., Webb W.W., Proc. Natl. Acad. Sci. USA 93 (1996). Svoboda K. et al., Nature 385 (1997) 161–165. Squirrell J.M., Wokosin D.L., White J.G., Bavister B.D., Nature Biotechnol. 17 (1999) 763–767. Hellwarth R., Christensen P., Opt. Commun. 12 (1974) 318–322. Gannaway J.N., Sheppard C.J.R., Opt. Quant. Electron. 10 (1978) 435–439. Freund I., Deutsch M., Sprecher A., Biophys. J. 50 (1986) 693–712. Boyd R.W., Nonlinear Optics, Academic Press, London, 1992. Xu C., Webb W.W., J. Opt. Soc. Am. B 13 (1996) 481–491. Lakowicz J.R., Principles of Fluorescence Spectroscopy, Kluwer Academic, New York, 1999. Loew L.M., Simpson L.L., Biophys. J. 34 (1981) 353. Moreaux L., Sandre O., Charpak S., Blanchard-Desce M., Mertz J., Biophys. J. 80 (2001) 1568–1574. Moreaux L., Sandre O., Mertz J., J. Opt. Soc. Am. B 17 (2000) 1685–1694. Mertz J., Moreaux L., Opt. Commun., in press. Denk W., Proc. Nat. Acad. Sci. USA 91 (1994) 6629–6633. Brown E.B., Shear J.B., Adams S.R., Tsien R.Y., Webb W.W., Biophys. J. 76 (1999) 489–499. Brown E.B., Wu E.S., Zipfel W., Webb W.W., Biophys. J. 77 (1999) 2837–2849. Cumpston B.H., Ananthavel S.P., Barlow S., Dyer D.L., Ehrlich J.E., Erksine L.L., Heikal A.A., Kuebler S.M., Lee I.-Y.S., McCordMaughon D., Qin J.H.R., Rumi M., Wu X.-L., Marder S.R., Perry J.W., Nature 398 (1999) 51–54. Denk W., Svoboda K., Neuron 18 (1997) 351–357. Xu C., Webb W.W., in: Lakowicz J.R. (Ed.), Topics in Fluorescence Spectroscopy, Vol. 5, Plenum Press, New York, 1997, p. 471. Mertz J., Eur. Phys. J. D (1998) 53–66. Srivastava A., Eisenthal K.B., Chem. Phys. Lett. 292 (1998) 345–351. Huang J.Y., Lewis A., Loew L.M., Biophys. J. 53 (1988) 665–670. Bouevitch O., Lewis A., Pinevsky I., Wuskell J.P., Loew L.M., Biophys. J. 65 (1993) 672–679. Peleg G., Lewis A., Linial M., Loew L.M., Proc. Natl. Acad. Sci. USA 96 (1999) 6700–6704. Campagnola P.J., Wei M., Lewis A., Loew L.M., Biophys. J. 77 (1999) 3341–3349.
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3. New directions in nonlinear microscopy
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[27] Lakowicz J.R., Gryczynski I., in: Lakowicz J.R. (Ed.), Topics in Fluorescence Spectroscopy, Vol. 5, Plenum Press, New York, 1997, p. 87. [28] Maiti S., Williams R.M., Shear J.B., Zipfel W.R., Watt W.W., Science 24 (1997) 530–532. [29] Yelin D., Silberberg Y., Opt. Express 5 (8) (1999) 169. [30] Muller M., Squier J., Wilson K.R., Brakenhoff G.J., J. Microscopy 191 (1998) 266. [31] Volkmer A., Cheng J.-X., Sunney Xie X., Phys. Rev. Lett. 87 (2001) 023901–023903. [32] Albota M.D., Beljonne D., Bredas J.L., Ehrlich J.E., Fu J.-Y., Heikal A.A., Hess S.A., Kogej T., Levin M.D., Marder S.R., McCordMaughon D., Perry J.W., Roeckel H., Rumi M., Subramaniarn G., Webb W.W., Wu X.-L., Xu C., Science 281 (1998) 1653–1656. [33] Ventelon L., Charier S., Moreaux L., Mertz J., Blanchard-Desce M., Angew. Chem. Int. Ed. 40 (2001) 2098–2101. [34] Marder S.R., Beratan D.N., Cheng L.-T., Science 252 (1991) 103. [35] Moreaux L., Sandre O., Blanchard-Desce M., Mertz J., Opt. Lett. 25 (2000) 3220–3222.
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IMAGERIE ACOUSTIQUE ET OPTIQUE DES MILIEUX BIOLOGIQUES OPTICAL AND ACOUSTICAL IMAGING OF BIOLOGICAL MEDIA
Nonlinear microscopy Jerome MERTZ
DOSSIER
Laboratoire de neurophysiologie et nouvelles microscopies, INSERM EPI 00-02, Paris, France E-mail: [email protected] (Reçu le 30 juin 2001, accepté le 11 juillet 2001)
Abstract.
We describe the basic principles of nonlinear optical microscopies based on two-photon excited fluorescence and on second-harmonic generation. Particular attention is given to the physical mechanisms underlying molecular second-harmonic generation, and the features unique to its signal contrast. We provide an overview of some applications of nonlinear microscopy including the visualization of molecular ‘flip–flop’ dynamics in membranes, high-resolution measurements of inter-membrane separations, and highsensitivity membrane potential imaging. Future developments of nonlinear microscopy are briefly considered. 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS non linear microscopy / two-photon fluorescence / second harmonic generation / optical biopsy / biological tissues
Microscopie non linéaire Résumé.
Nous décrivons les principes de base de la microscopie non linéaire : d’abord la fluorescence à deux photons qui est devenue une méthode très répandue dans les laboratoires, puis plus en détail, la microscopie du second harmonique en insistant sur les mécanismes physiques et le contraste spécifique de cette détection. Nous discutons ensuite de quelques applications de la microscopie non linéaire comme la mesure de la dynamique des molécules polaires, la mesure de distance intermembranaires et l’imagerie potentiel membranaire. Nous concluons en évoquant quelques développements futurs de la microscopie non linéaire. 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS micoscopie non linéaire / fluorescence biphotonique / génération du second harmonique / biopsie optique / tissus biologiques
Introduction Nonlinear microscopy, by its very nature, requires high optical intensities and has had to await the invention of the pulsed laser to come of age. By far the most well known form of nonlinear microscopy resulted from the pioneering work of Denk and Webb [1,2] in which they showed that a mode-locked laser beam, when focused into a sample, generated fluorescence only at the focal center. This ‘spot’ of fluorescence could be considered a 3-dimensional pixel, or voxel. By scanning the spot through the sample, a 3-dimensional image could then be constructed. Since its first demonstration in 1989, the two-photon Note présentée par Guy L AVAL. S1296-2147(01)01260-4/FLA 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS. Tous droits réservés
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laser scanning microscope based on this simple principle has become a laboratory standard [3,4] and has recently been commercialized. It should be mentioned, however, that several years prior to the invention of two-photon microscopy, another form of nonlinear microscopy had been utilized by various laboratories [5,6]. This microscopy was based on the generation of second harmonic light, either from surfaces or from mesoscopic tissue structures such as rat-tail tendons [7]. Because of difficulties in signal interpretation and perhaps because of its seemingly obscure utility, at least in biological imaging, second-harmonic microscopy has gone relatively unnoticed until only very recently. The purpose of this chapter is to briefly describe the basic principles behind nonlinear microscopy. Because several excellent reviews of two-photon excitation fluorescence (2PEF) microscopy are readily available, most of this article will be devoted to second-harmonic generation (2HG) microscopy, with particular emphasis on how it compares to and contrasts with 2PEF microscopy. New developments in the field will be briefly evoked at the end of the article. 1. 2HG versus 2PEF microscopy: principles The term second-harmonic generation is usually reserved for coherent phenomena arising in bulk volumes, such as crystals, or extended surfaces. In these cases, the radiating source is appropriately characterized by its nonlinear susceptibility. In contrast, the term hyper-Rayleigh scattering is usually reserved for (elastic) nonlinear scattering at the molecular level, and the sources are appropriately characterized by their nonlinear polarizabilities [8]. Inasmuch as 2HG from a macroscopic source represents the coherent summation of hyper-Rayleigh scattering from the microscopic source constituents, the two terms will be used interchangeably here at the expense of semantic rigor. Moreover, since it is common practice to quantify the efficiency of 2PEF by molecular cross sections [9], the same will be done for 2HG for purposes of comparison. It should be kept in mind, however, that the concept of a 2HG cross section is only applicable to a single isolated hyper-Rayleigh scatterer. When many scatterers are implicated, this concept becomes ambiguous since the resultant 2HG varies widely depending on how the scatterers are spatially distributed, as will be discussed below. When laser excitation is incident on a single molecule, it may be absorbed or scattered. Nonlinear versions of these phenomena are depicted in figure 1. In the case of 2PEF, two photons are simultaneously incident (within about a femtosecond) and are resonantly absorbed since their combined energies falls within the molecular absorption band. Some of this energy is dissipated to the solvent or to nuclear redistribution, and the rest may be available for fluorescence [10]. The phenomenon of absorption followed by fluorescence emission is generally incoherent in that the phases of the excitation and fluorescence photons are generally uncorrelated. In the case of 2HG, the excitation photons are not absorbed but instead are scattered into a single outgoing emission photon. Because the scattering is essentially instantaneous, the phases of the excitation and emission photons are tightly correlated in this case and the phenomenon is coherent. The difference between incoherence and coherence leads to fundamental differences in the natures of 2PEF versus 2HG. In particular, radiating molecules are effectively non-interacting when they emit incoherently, meaning that their total emission power simply scales with their number. This is not the case when they emit coherently. For example, if two molecules at the same position radiate in phase, then because of constructive interference their total radiated power is four times the power from an individual molecule,
Figure 1. Two photons incident on a molecule can be absorbed (left) or scattered (right), leading to the generation of fluorescence (2PEF) or second-harmonic light (2HG).
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whereas if they radiate out of phase, then because of destructive interference their total radiated power is zero. It is clear here that the molecules cannot be regarded as independent when they emit coherently. The situation is further complicated when the radiation phase depends on the molecule orientation or on its position in space, both of which arise in 2HG. A particularity of dipolar 2HG is that it requires a non-centrosymmetric source [8]. On a molecular level, this means that a hyperpolarizable molecule must exhibit ‘charge transfer’ [11], that is, there must be a difference between the dipole moments of the excited and ground states. The direction of this charge transfer imparts a well-defined orientation to the molecule. Depending on whether this direction is parallel or antiparallel to the incident field polarization, the resultant 2HG is driven either in-phase or out-of-phase. In the case of a population of 2HG radiating molecules, constructive interference then becomes only possible if the molecules exhibit a global alignment. A very effective technique for obtaining such an alignment consists in labeling lipid membranes with amphiphilic dye molecules consisting of polar and non-polar endgroups spanned by a non-centrosymmetric chromophore. Because of their geometry, these molecules readily insert themselves into lipid membranes with a preferred orientation, as shown in figure 2. It should be noted that the radiative properties of the chromophore are usually very sensitive to local environment. For example, there can easily be a factor of ten increase in the fluorescence quantum efficiency of the chromophore when it passes from an aqueous (polar) to a lipid (non-polar) environment [10]. When the driving field polarization is along the molecular axis, the 2HG cross-section of a single molecule, defined through P2HG = 12 σ2HG I 2 , where P2HG is the second-harmonic power (photons/s) and I is the incident laser intensity (photons/s/area) is related to the molecular hyperpolarizability β by [12]: σ2HG =
4ω 5 |β|2 3πnε30 c5
m4 ·(photon/s)−1
In general, σ2HG is small, much smaller than its fluorescence counterpart σ2PEF . For example, even when β is resonantly enhanced by its transition energy occurring within the molecule absorption band (as shown in figure 1), σ2HG is about four orders of magnitude smaller than σ2PEF for the molecule Di-6-ASPBS in a lipid membrane. For single-molecule detection, therefore, 2HG is at a considerable disadvantage. For the detection of many molecules, on the other hand, 2HG can be relatively efficient owing to its quadratic dependence on the number of radiating molecules, assuming these are aligned. Figure 3, for example, illustrates the emission spectrum of Di-6-ASPBS molecules in a membrane that undergo both 2PEF excitation (right lobe) and 2HG (left peak). The areas under the respective spectral features are about the same, indicating that the powers are comparable. The exact number of molecules that contribute to 2HG, or even to 2PEF, is a matter of definition. In the case of excitation with a tightly focused laser beam, the case usually encountered in scanning microscopy, this number can be unambiguously defined based on a Gaussian approximation of the laser beam profile about the focal center [13]. According to this definition, the spatial resolutions of both contrast modes are the same. It should be pointed out, however, that in addition to scaling quadratically with the number of radiating molecules, 2HG power critically depends on how these molecules are distributed relative to
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Figure 2. Molecular structure of Di-6-ASPBS and its asymmetric insertion geometry in a lipid bilayer (perfusion from top).
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OPTICAL AND ACOUSTICAL IMAGING OF BIOLOGICAL MEDIA
Figure 3. Simultaneously acquired 2HG (left peak) and 2PEF (right lobe) emission spectra from labeled membrane. From [35].
Figure 4. Radiation patterns of fluorescence (2PEF) and second-harmonic light (2HG) by focused excitation of amphiphilic dye molecules in a membrane.
one another. For example, when molecules are uniformly distributed on a membrane surface illuminated from the side, then the total 2HG power is significantly smaller than when the same number of radiating molecules are concentrated exactly at the focal center (neglecting aggregation effects such as dimerization, self-quenching, etc.). Moreover, the resultant 2HG also varies considerably in its angular distribution: for uniformly distributed molecules the 2HG is emitted along two forward directed off-axis lobes (see figure 4), whereas for molecules concentrated at a point, it adopts a simple dipole radiation profile. This sensitivity of the 2HG radiation pattern to molecular spatial distribution is a direct consequence of its coherent nature [14]. 2. 2HG versus 2PEF microscopy: applications The applications of 2PEF microscopy are well established. They range from deep high resolution imaging in scattering media, localized molecular uncaging [15,16] or photo bleaching [17], micro-lithography [18], etc., and will not be dwelt upon here since references on the matter are exhaustive [19–21]. In contrast, the applications of 2HG microscopy have been relatively unexplored. Three such applications will be considered here, which are meant to highlight the differences between 2HG and 2PEF. These applications are readily illustrated with model membranes such as Giant Unilamelar Vescicles (GUVs). 2.1. Measurement of flip–flop dynamics When amphiphilic molecules such as Di-6-ASPBS described above are perfused onto a GUV, they initially only label the outer membrane leaflet and the polar headgroups all face outward (upward in figure 4). This is a condition of maximum asymmetry, and therefore leads to maximum 2HG. With time, however, the molecules undergo thermally induced ‘flips’ to the inner membrane and eventually the populations in the outer and inner membranes become equal. This equilibrium condition has lost all its asymmetry and therefore cannot produce 2HG. The energy barrier for molecular flipping is high, typically tens of KT depending both on the molecule and lipid structures, meaning that the rate of redistribution
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Figure 5. Fluorescence (2PEF) and second-harmonic light (2HG) from Di-6-ASPBS molecules that are initially asymmetrically distributed in a DOPC membrane, but progressively become symmetrically distributed with time (room temperature). From [12].
is slow. Nonetheless, this rate is readily measurable by monitoring the decay of the 2HG signal with time [12,22]. It should be noted that the attendant 2PEF signal, which may be obtained simultaneously, is dependent only on the total number of radiating molecules regardless of whether they are in the outer or inner membrane leaflets. As such, 2HG provides a signature of the difference in populations between the inner and outer leaflets, whereas 2PEF provides the signature of their sum. This latter signature can vary as the result of perfusion dynamics or photo bleaching, and can provide an accurate normalization for the 2HG signal (see figure 5). 2.2. Measurement of intermembrane distance An alternative interpretation to the above 2HG response to ‘flip–flop’ is that molecules in the outer leaflet radiate with a given phase, whereas molecules in the inner leaflet, because they are oppositely oriented, radiate with the inverse phase. As a result, if the populations in the two leaflets are equal, then the net 2HG from both leaflets completely destructively interferes. This interpretation is correct because the inner and outer leaflets are essentially at zero distance from one another. On the other hand, if the two leaflets were separated by a microscopic distance of the order of the 2HG wavelength, then the above scenario changes radically. Because the 2HG emission from each leaflet is off axis, a microscopic distance between the leaflets occasions a difference in the optical path-lengths between the leaflets and the observer. As a result, what was destructive interference at zero inter-leaflet separation becomes constructive interference at a non-zero inter-leaflet separation. In this way, the 2HG signal provides an excellent measure of the inter-leaflet separation [12]. In practice, of course, instead of separating the inner and outer leaflets, it is much easier to bring two GUVs with labeled outer leaflets in close proximity, as shown in figure 6. The ‘hot spots’ apparent in this figure correspond to the inter-membrane distance where the 2HG interference is maximally constructive. This distance is given by approximately 0.6 times the excitation beam waist at the focus, corresponding typically to a few hundreds of nanometers. For smaller distances, the 2HG decreases roughly linearly. As such, 2HG provides an excellent measure of membrane separations over ranges that are inaccessible both to diffraction limited imaging and to short range interactions such as fluorescence resonance energy transfer (FRET).
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Figure 6. 2HG produced by two labeled membranes in close proximity. When membranes adhere, 2HG destructively interferes. When they are separated by 0.6 times the excitation beam waist (w0 ), 2HG constructively interferes resulting in ‘hot spots’ apparent in image of contacting GUVs.
2.3. Membrane potential imaging Perhaps one of the most promising applications of 2HG is its capacity to image electric fields across membranes. For example, the difference in electric potential between the inside and outside of living cells is typically several tens of millivolts. Supposing that the electric field is uniform inside the cell membrane, this would correspond to electric fields on the order of 105 V/cm. In actuality, the electric fields are far from being uniform. Cell membranes consist predominantly of DOPC lipids which possess zwitterionic headgroups. These create ‘dipole layers’ at the membrane surfaces where the electric fields vary rapidly and can be much larger than in the uniform field approximation. Suffice to say that the fields can be large enough to significantly perturb the hyperpolarizabilities of various types of 2HG chromophores [23,24]. Sensitivities of the order of 50% increases in 2HG for 100 mV changes in membrane potential can readily be demonstrated in model systems, as illustrated in figure 7. Such sensitivities are about an order of magnitude higher that those obtained with fluorescence based techniques relying on electrochromism. In cellular systems, however, such sensitivities are considerably harder to demonstrate largely because of problems with photo-toxicity and artifactual responses unrelated to membrane potential. Nonetheless, they have been reported, for example, by Loew and co-workers using Nernstian membrane potential variations induced by changes in extracellular potassium concentrations [25,26]. It should be emphasized that the
Figure 7. 2HG signal from a GUV labeled with ‘charge-transfer’ molecules. Membrane potential of GUV is controlled by an intra-vesicular electrode (not seen because it is at a different plane of focus).
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photo-physical mechanisms governing the membrane potential sensitivities of 2HG markers still remain poorly understood.
New developments in nonlinear microscopy are many-fold. Several groups, for example, have demonstrated the extension of nonlinear microscopy to higher orders. In particular, three-photon excited microscopy (3PEF) has been used to image autofluorescent endogenous molecules in cells, such as serotonin or indoleamines [27,28]. The molecules are normally one-photon excited in the UV range, which causes extensive photo damage. With the use of three-photon excitation, this range can be avoided. Similarly, thirdharmonic generation microscopy has become a promising tool for the imaging endogenous species [29,30]. 3HG has the advantage over 2HG that it does not require non-centrosymmetric sources, and hence is not limited to the imaging of interfaces (though interfaces may certainly enhance the 3HG signal). Finally, very recently, microscopy based on nonlinear coherent anti-Stokes–Raman scattering (CARS) has made its appearance [31]. This technique, also particularly applicable to the imaging of endogenous species, presents the added advantage of excellent molecular discrimination. With regard to exogenous markers, considerable headway has been made in the design of new molecules that are specifically optimized for nonlinear microscopy. This includes the development of quadrupolar dyes with enhanced two-photon absorption cross sections [32–34], for both imaging and optical limitation applications, and the development of molecules with enhanced first hyperpolarizabilities for 2HG applications. These new developments should bear their fruit in the very near future. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26]
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[27] Lakowicz J.R., Gryczynski I., in: Lakowicz J.R. (Ed.), Topics in Fluorescence Spectroscopy, Vol. 5, Plenum Press, New York, 1997, p. 87. [28] Maiti S., Williams R.M., Shear J.B., Zipfel W.R., Watt W.W., Science 24 (1997) 530–532. [29] Yelin D., Silberberg Y., Opt. Express 5 (8) (1999) 169. [30] Muller M., Squier J., Wilson K.R., Brakenhoff G.J., J. Microscopy 191 (1998) 266. [31] Volkmer A., Cheng J.-X., Sunney Xie X., Phys. Rev. Lett. 87 (2001) 023901–023903. [32] Albota M.D., Beljonne D., Bredas J.L., Ehrlich J.E., Fu J.-Y., Heikal A.A., Hess S.A., Kogej T., Levin M.D., Marder S.R., McCordMaughon D., Perry J.W., Roeckel H., Rumi M., Subramaniarn G., Webb W.W., Wu X.-L., Xu C., Science 281 (1998) 1653–1656. [33] Ventelon L., Charier S., Moreaux L., Mertz J., Blanchard-Desce M., Angew. Chem. Int. Ed. 40 (2001) 2098–2101. [34] Marder S.R., Beratan D.N., Cheng L.-T., Science 252 (1991) 103. [35] Moreaux L., Sandre O., Blanchard-Desce M., Mertz J., Opt. Lett. 25 (2000) 3220–3222.
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