Second-Harmonic Imaging Microscopy for Visualizing Biomolecular Arrays in Cells, Tissues and Organisms

PERSPECTIVE FOCUS ON OPTICAL IMAGING Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms Paul J Campagnola & Leslie M Loew Although the nonlinear optical effect known as second- preserves the coherence of the laser light. Frequency-doubling crystals harmonic generation (SHG) has been recognized since the are commonly used to produce visible laser light from near-infrared earliest days of laser physics and was demonstrated through a lasers or UV light from visible lasers. Box 1 provides a simplified sum- microscope over 25 years ago, only in the past few years has mary of key equations underlying the theory of SHG. it begun to emerge as a viable microscope imaging contrast Biological materials can be highly polarizable and often assemble mechanism for visualization of cell and tissue structure and into large, ordered noncentrosymmetric structures. Indeed, it has function. Only small modifications are required to equip a been known for 20 years that collagen can produce SHG signals1 and standard laser-scanning two-photon microscope for second- for 15 years that biological membranes might be good general scaf- http://www.nature.com/naturebiotechnology harmonic imaging microscopy (SHIM). Recent studies of the folds for noncentrosymmetric arrays of SHG-active molecules2,3. three-dimensional in vivo structures of well-ordered protein However, it is only in the past few years that it has been shown that assemblies, such as collagen, microtubules and muscle high-resolution SHG images can be obtained with instrumentation myosin, are beginning to establish SHIM as a nondestructive similar to that used for TPEF microscopy4–11. Like that of two-photon imaging modality that holds promise for both basic research absorption, the amplitude of SHG is proportional to the square of the and clinical pathology. Thus far the best signals have been incident light intensity. Therefore, SHIM has the same intrinsic opti- obtained in a transmitted light geometry that precludes in vivo cal sectioning characteristic as TPEF microscopy. Thus, a new three- measurements on large living animals. This drawback may be dimensional microscope contrast mechanism that does not require addressed through improvements in the collection of SHG excitation of fluorescent molecules has been made available to the signals via an epi-illumination microscope configuration. In biological community. addition, SHG signals from certain membrane-bound dyes The properties of SHG offer several advantages for live cell or tissue have been shown to be highly sensitive to membrane potential. imaging. Because SHG does not involve excitation of molecules, it Although this indicates that SHIM may become a valuable tool should not suffer, in principle, from phototoxicity effects or photo- © Group 2003 Nature Publishing for probing cell physiology, the small signal size would limit bleaching, both of which limit the usefulness of fluorescence the number of photons that could be collected during the microscopy, including two-photon fluorescence microscopy, for the course of a fast action potential. Better dyes and optimized imaging of living specimens. (There can be collateral damage, how- microscope optics could ultimately lead to the imaging of ever, if the incident laser light also produces two-photon excitation of neuronal electrical activity with SHIM. chromophores in the specimen.) Another advantage is that many intrinsic structures produce strong SHG, so labeling with exogenous SHIM is based on the familiar nonlinear optical effect called SHG, also molecular probes is not required. On the other hand, electrochromic commonly called frequency doubling. This phenomenon requires membrane dyes can be used to produce SHG that is highly sensitive to intense laser light passing through a highly polarizable material with a membrane potential. This may allow new optical approaches to be noncentrosymmetric molecular organization—most typically an developed for mapping electrical activity in complex neuronal sys- inorganic crystal. The second-harmonic light emerging from the tems. Excitation uses near-infrared wavelengths, allowing excellent material is at precisely half the wavelength of the light entering the depth penetration, and thus this method is well suited for studying material. Thus, the SHG process within the nonlinear optical material thick tissue samples. changes two near-infrared incident photons into one emerging visible Information about the molecular organization of chromophores, photon at exactly twice the energy (and half the wavelength). including dyes and structural proteins, can be extracted from SHG As opposed to two-photon-excited fluorescence (TPEF), in which imaging data in several ways. SHG signals have well-defined polariza- some of the incoming energy is lost during relaxation of the excited tions, and thus SHG polarization anisotropy can be used to determine state, SHG does not involve an excited state, is energy conserving and the absolute orientation and degree of organization of proteins in tissues. In addition, TPEF images can be collected in a separate data channel simultaneously with SHG. Correlation between the SHG and Center for Biomedical Imaging Technology, Department of Physiology, University TPEF images provides the basis not only for molecular identification of Connecticut Health Center, Farmington, Connecticut 06030, USA. of the SHG source but also for probing radial and lateral symmetry Correspondence should be addressed to L.M.L. ([email protected]). within structures of interest. These special characteristics of SHIM as Published online 31 October 2003; doi:10.1038/nbt894 well as their current limitations are reviewed in this article. 1356 VOLUME 21 NUMBER 11 NOVEMBER 2003 NATURE BIOTECHNOLOGY PERSPECTIVE Because SHG is a coherent process, most of Box 1 Theoretical background and fundamental equations the signal wave propagates with the laser. The exact ratio of the forward to backward signal The nonlinear polarization for a material is defined by: is dependent upon the sample characteristics. P = χ(1) ⊗ E + χ(2) ⊗ E ⊗ E + χ(3) ⊗ E ⊗ E ⊗ E + ... (1) For thin samples, such as tissue culture cells, essentially the entire signal is directed for- where P represents the induced polarization vector, E represents the vector electric field, ward. At higher sample turbidity, some of the χ(i) is the i th-order nonlinear susceptibility tensor and ⊗ represents a combined tensor SHG is scattered backward. The use of an product and integral over frequencies. The χ(i) corresponds to optical effects as follows: upright microscope has some advantages for • 1st-order process: absorption and reflection SHG imaging over that of an inverted geome- • 2nd-order process: SHG, sum and difference frequency generation, hyper-Rayleigh try. The optical path is simpler for trans col- • 3rd-order process: multiphoton absorption, third harmonic generation, coherent lection and this geometry also makes it more anti-Stokes Raman scattering straightforward to implement the additional The second nonlinear susceptibility is a bulk property and related to the molecular external optics required for polarization hyperpolarizability, β, by: anisotropy measurements (described below). Commercial titanium sapphire femtosecond χ(2) = N <β> (2) s lasers are ideal for SHG (and TPEF) because of the high repetition rate (80 MHz) and high where Ns is the density of molecules and the brackets denote an orientational average, which shows the need for an environment lacking a center of symmetry. peak powers (and low pulse energies) and the The second-harmonic intensities in such media will scale as follows: broad tunability throughout the near infrared (700–1,000 nm). Because SHG intensities are ∝ 2τ χ(2) 2 SHGsig p ( ) (3) typically smaller than those of TPEF, it is important to optimize the collection and τ where p and are the laser pulse energy and pulse width, respectively. detection efficiency for both signal isolation http://www.nature.com/naturebiotechnology The magnitude of the SHG intensity can be strongly enhanced when the energy of the and detection electronics. –ω –ω SHG signal (2h ) is in resonance with an electronic absorption band (h ge). Within the SHG imaging can also determine molecu- β two-level system model, the first hyperpolarizability, , and thus SHG efficiency is given by lar symmetries by use of polarization analysis. SHG polarization anisotropy measurements 3e2 ω ƒ ∆µ β ≈ —– —————————––ge ge ge (4) are made with a Glan Laser Polarizer through –3 2 2 2 2 2h [ωge – ω ][ωge – 4ω ] which the data is obtained by maintaining the same input laser polarization and obtaining ω ƒ ∆µ where e is electron charge and ge, ge and ge are the energy difference, oscillator images with the analyzing polarizer oriented strength (that is, integral of the absorption spectrum) and change in dipole moment both parallel and perpendicular to the laser between the ground and excited states, respectively. Because of the denominator in this polarization. In addition, radial and lateral equation, resonance-enhanced SHG has a dependence on the wavelength of the incident symmetries are probed by rotating the plane light similar to the two-photon excitation spectrum. of polarization of the laser with half (λ/2) and quarter (λ/4) wave plates. © Group 2003 Nature Publishing History and instrumentation Endogenous imaging of structural protein arrays The first reports of the integration of SHG and microscopy appear to Recently, it has been observed that

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