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Multiphoton spectroscopy


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  • 1. w w w. s p e c t r o s c o p y o n l i n e . c o m Januar y 2011 Spectroscopy 26(1) 1Lasers and Optics InterfaceMultiphoton Spectroscopy[Author: Please provide a brief two- or three sentence abstract.]Youngjae Kim, Joseph Salhany, and Alain VilleneuveT here are many types of nonlinear optical techniques, and several depend on the spatial and temporal over- lap of multiple optical pulses at different wavelengthson the target. For example, with techniques such as coher-ent anti-Stokes Raman scattering (CARS) spectroscopy and does not require various fluorophores for imaging contrast enhancement, which are usually used to label the molecules for fluorescent imaging. In fluorescent imaging, measure- ment sensitivity is usually limited by photobleaching and phototoxicity (5,6).stimulated Raman scattering (SRS) spectroscopy, which aregaining in popularity, the ability to alter at least one of the Two-Photon Excitation andwavelengths is essential. Many different systems exist today Second Harmonic Generation Microscopyaddressing the many microscopic and spectroscopic tech- In the two-photon excitation (TPE) and second harmonicniques being practiced; however, most are dedicated to one generation (SHG) processes, two photons interact togethersingle application. Fiber lasers are finding their way into ap- to generate blue-shifted signals (6–8). SHG is a coherentplications that in the past were only addressable by the larger technique in which energy is conservative, and with TPE,classic laser systems. Multiphoton spectroscopy is an area energy is lost through the relaxation stage as shown in thethat only recently began benefiting from the advances made energy level diagram in Figure fiber laser development and the potential value they offer. There are several advantages of TPE and SHG mi-Development activity in this area is growing at a rapid rate. croscopy when compared to conventional techniques such as f luorescent confocal microscopy, which is basedRaman Spectroscopy upon linear and one-photon absorption. In confocalScattered photons from molecules are grouped in two de- microscopy, f luorophores above and below the focalpending on the type of scattering: elastic scattering and plane are also excited; photobleaching takes place ininelastic scattering (1). In elastic scattering (also known as whole planes and, therefore, is not detected (8,9). Sig-Rayleigh scattering), the scattered photons all have the same nals from TPE are collected only at the vicinity of theenergy and frequency. In the latter case, however, we can focal points, which reduces the phototoxic effects andobserve small amounts of scattered photons at a lower or photobleaching. In the case of SHG, excitation of f luo-higher frequency than the injected photons after interac- rophores is not necessary, which makes it free from anytion with samples (molecular vibrations). Most of incident phototoxic effects and photobleaching (10,11). Anotherphotons on samples experience Rayleigh scattering in the advantage is related to increased depth of images. Near-spontaneous Raman process and only a small number IR lasers deployed in TPE and SHG microscopy applica-of photons contribute Raman signals (2). Because whole tions can propagate photons much deeper inside bio-samples — gases, liquids, gels, and solids — exhibit unique medical tissues, which enables high resolution imagesspectral responses, Raman spectroscopy has became a very in thicker samples (7,8,12,13).useful spectroscopic tool (2–4). The principles of TPE and SHG microscopy extend to Because Raman scattering is a phenomenon occurring cover multiphoton microscopy and higher harmonic genera-between photons and the chemical bonds between atoms, it tion microscopy (7,8).
  • 2. 2 Spectroscopy 26(1) Januar y 2011 w w w. s p e c t r o s c o p y o n l i n e . c o m copy is a useful tool in research areas such as biology, chemistry, and phys- ics (25). The temporal response of the sample is probed after the pump beam reaches the sample while the delay time of the probe beam is adjusted. The time resolution in pump-probe spectroscopy is dependent upon the pulse width. It is also possible to implement two-color pump-probe spectroscopy by deploying two lasers. Fluorescence lifetime imaging mi- croscopy (FLIM) is one of established technologies based on the two-colorFigure 1: Energy level diagram showing transition states for two-photon excitation (TPE), second pump-probe scheme (26,27). FLIMharmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS) and stimulated was developed as a technique thatRaman scattering (SRS). provides an enhanced image contrast over fluorescence microscopy. In con-CARS Spectroscopy neously offer a higher spectral resolu- ventional fluorescence microscopy,CARS is based on four-wave mixing tion as well as higher contrast between multiple fluorophores tagging differ-and is a third-order nonlinear phe- the CARS signals and the nonresonant ent molecules are excited by photonsnomenon where typically two lasers background (20–22). within their absorption bands, whichare deployed (14–16). If the pump and causes them to emit fluorescence inStokes fields (denoted as ωp and ωs, re- SRS Spectroscopy the emission bands. However, withspectively) are spatially and temporally SRS spectroscopy was realized by fluorescence microscopy, when theoverlapped in molecules with vibra- adopting another interesting four-wave emitted fluorescent spectra are placedtional frequency Ω matching ωp – ωs, mixing process, stimulated Raman close each other, each molecule can novibration of the molecules becomes scattering (23,24). To realize SRS in longer be visualized properly. FLIM,enhanced at ωp – ωs and the pump field vibrational molecules, two fields are on the other hand, can identify fluoro-becomes scattered to produce the anti- again used, the pump and the Stokes, phores through fluorophore-dependentStokes signal ωas at 2ωp – ωs. CARS which are synchronized and focused on lifetime measurement. With FLIMsignals are quadratically proportional the samples with vibrational frequency based on pump-probe techniques, theto the pump fields and are linearly de- Ω matching ωp – ωs. Instead of mea- pump beam excites fluorophores andpendent on the Stokes field. By virtue suring anti-Stokes signals as in CARS, the delayed probes experience stimu-of the coherent properties of CARS, stimulated Raman loss (SRL) of pump lated emissions. Instead of measuringthe resulting signal is orders of mag- field or stimulated Raman gain (SRG) the probe beam, the intensity variationnitude higher than the signal obtained of Stokes field is used. of the fluorescence is measured. Thisthrough spontaneous Raman tech- Unlike CARS spectroscopy, no back- technique does not require a high in-niques, which makes molecular real ground noise is present in SRS because tensity pump to saturate fluorophorestime imaging a possibility.[AUTHOR: the SRG and SRL happen only when and consequently reduces the risk ofSENSE OK?] Hyperspectral informa- the frequency difference between pump photobleaching.tion can also be obtained by deploy- and Stokes is equal to the vibrationaling two lasers; one should have a wide frequency. The SRG and SRL are pro- Terahertz Imaging and Sensingwavelength tuning capability while re- portional to the product of the pump Radiation in the terahertz range (wave-maining synchronized with the other. and Stokes intensities. Measurement of lengths from 30 µm to 3000 µm and The intrinsic properties of CARS the SRL is achieved by modulating the frequencies from 0.1 THz to 10 THz)differ from Raman spectroscopy be- Stokes field. Likewise, measurement of penetrate clothing and many other or-cause of the existence of a nonresonant the SRG is achieved by modulating the ganic materials and offers spectroscopicbackground that is unrelated to any pump field. SRS measurements require information, especially for materialsvibrational resonances. Because the the use of lock-in amplifiers for extract- that impact safety such as explosivesnonresonant background provides no ing the signal. and pharmacological substances (28). Ituseful information from the samples, it is also notable that terahertz waves havemust be suppressed as much as possible Pump-Probe Spectroscopy the characteristics of millimeter waves(17–19). Typically picosecond pulses Reflection, transmission, absorption, and IR waves. Various compoundswith narrow spectral width of a few and other characteristics of a sample respond differently when exposed towavenumbers are preferred for CARS can be examined by use of pump-probe radiation in the 0.1–3 THz range andspectroscopy because they can simulta- spectroscopy. Pump-probe spectros- each thus can be uniquely identified.
  • 3. w w w. s p e c t r o s c o p y o n l i n e . c o m Januar y 2011 Spectroscopy 26(1) 3Terahertz spectroscopy has been ac- Fourier domain. Fourier-domain OCT Synchronization between two lasers istively under research for applications is further categorized into two meth- achieved by placing optical delay linesin chemical sensing, noninvasive mo- ods: spectral domain and swept source in either of the two laser output paths.lecular imaging, and carrier dynamics OCT (SSOCT). The lasers and setups in use today as-measurement of semiconductors, di- Applications based on the acquisition sociated with this approach are largeelectrics, and nanomaterials. Terahertz of video-rate OCT images require both and complex and must be installedsignals also can be used for communi- a fast A-scan rate and high sensitivity. in a controlled and stable environ-cation, astronomy, and security. Fourier-domain OCT, which exhibits ment. Also, wavelength tuning of such Regarding terahertz sources, several 20–30 dB better sensitivity than time- systems requires either a temperaturedifferent techniques have been con- domain OCT, is commonly used for adjustment or crystal alignment thatsidered promising in the realization of video-rate imaging. requires longer than 1 s. Spectroscopyterahertz waves. When considering di- and hyperspectral imaging experi-rect methods, quantum cascade lasers, Laser System Requirements: What ments remain fundamentally limitedgas lasers, Schottky diodes, and Gunn Exists Today by the relatively slow wavelength tun-diodes fall into this range. Lasers that Each aforementioned application tech- ing speeds of lasers. Schemes based onemit terahertz waves directly typically nique requires a specific laser type and supercontinuum generation providerequire cryogenic cooling (28,29). configuration. For example, SSOCT a broadband spectrum that is then Terahertz signals can also be gener- requires lasers with wavelength tun- mixed with a narrowband pump, andated indirectly by difference frequency ing speeds at rates of tens of kilohertz a spectrometer is used to resolve themixing of two lasers at wavelengths that for real-time imaging applications. A vibrational spectra (32). Spectral imagesare very close together or by the use of longer coherence length (narrow in- have been achieved with simple systemsfemtosecond lasers. In both schemes, stantaneous linewidth) is also required but they lack the efficiency to imagephotoconductive antennas consisting to achieve deeper imaging for depths thick tissue. The development of laserof either a semiconductor substrate or of several millimeters or sometimes sources with characteristics tailoreda nonlinear optical crystal and onto up to tens of millimeters depending for the needs of nonlinear imaging andwhich a metallic antenna structure is on the sample. Wavelengths also need spectroscopy systems remains an activedeposited are gated by the applied opti- to be chosen carefully depending on area of beating signals or the femtosecond the properties of the samples to be For applications in life sciences andlaser pulses. Terahertz pulses usually analyzed. The optical bandwidth of the more specifically in clinical applications,are generated by the use of Ti:sapphire laser is important because the axial res- fiber-based lasers are very appealinglasers or femtosecond fiber lasers olution is linearly improved by optical because they are durable, robust, com-whereas continuous wave terahertz bandwidth; ideally, a bandwidth greater pact, and simpler to manage and theysources are generated by the use of two than 100 nm is demanded. There are a facilitate light delivery to the sample.continuous wave semiconductor lasers few companies today producing lasers However, techniques based upon coher-or two continuous wave fiber lasers that meet the specifications for SSOCT. ent Raman spectroscopy require a laser(29). These lasers usually are composed of a source capable of generating picosecond semiconductor gain medium and fast pulses that have a spectral width wellOptical Coherence Tomography wavelength filters. matched to the Raman linewidth of theOptical coherence tomography (OCT) However, lasers adapted for SSOCT vibrational molecules. So far, only a fewis a technique used to acquire the cross- applications cannot be deployed in technologies based on fiber lasers havesectional and noninvasive tomographic applications requiring nonlinear re- been proposed for nonlinear spectros-image of biological samples with high sponses from the samples because of copy techniques such as CARS and SRSaxial resolution and high speed (30). the quasi-continuous wave signals that imaging (33–35).The axial resolution is determined by are generated from SSOCT lasers. Con-the coherence length of the optical ventional lasers in use today for nonlin- Conclusionsource. A number of different types of ear spectroscopy applications consist of The field of multiphoton spectroscopyOCT techniques have been developed Ti:sapphire lasers because of the wide includes many techniques that apply toto acquire different pieces of infor- optical bandwidth they offer (799–1000 a broad range of applications. Laser sys-mation from the biological samples, nm), narrow pulse width (down to tems or laser setups today typically areincluding spectroscopic OCT, phase- a few femtoseconds), and high peak capable of addressing a single applicationsensitive OCT, optical Doppler tomog- power (higher than a few kilowatts). and a major effort is required when re-raphy (ODT), polarization-sensitive For nonlinear spectroscopy applica- configuring them for other applications.OCT, optical coherence microscopy tions requiring two lasers such as in Efforts toward development of sys-(OCM), optical coherence phase mi- CARS, SRS, and two-color pump probe tems that can be easily adapted for use incroscopy, and many more (31). Two applications, optical parametric oscil- multiple applications are highly desiredimplementation methods are available lators (OPO) pumped by a Ti:sapphire and one such area is in clinical applica-for OCT systems: time domain and laser are deployed as the sources. tions of spectroscopic imagery. Fiber
  • 4. 4 Spectroscopy 26(1) Januar y 2011 w w w. s p e c t r o s c o p y o n l i n e . c o mlasers are robust and provide a smaller (25) K.L. Hall et al., Opt. Lett. 17, 874–877footprint, transportability, and ease of (1989).use. We are seeing a growing trend in (26) C.Y. Dong et al., J. Biophys. 69, 2234–the use of fiber lasers for applications 2242 (1995).that formerly were the domain of larger (27) J.R. Lakowicz, Principles of Fluores-and more-complex systems. cence Spectroscopy, Third Edition (Springer, New York, 2006).References (28) P.H. Siegel, IEEE T. Microw. Theory(1) R.W. Boyd, Nonlinear Optics, third Tech. 50, 910–928 (2002). edition (Academic Press, New York, (29) M. Tonouchi, Nat. Photonics 1, 97–105 2008). (2007).(2) K. Kneipp et al., Chem. Rev. 99, 2957– (30) I. Hartl et al., Opt. Lett. 26, 608–610 (2001). 2975 (1999). (31) A.F. Fercher et al., Rep. Prog. Phys. 66,(3) J.T. Motz et al., J. Biomed. Opt. 10(3), 239–303 (2003). 031113 (2005). (32) T.W. Kee and M.T. Cicerone, Opt Lett.(4) A.S. Haka et al., J. Biomed. Opt. 14(5), 29(23), 2701–2701 ((2004). 054021 (2009). (33) K. Kieu, B.G. Saar, G.R. Holtom, X.S.(5) J.W. Lichtman et al., Nat. Methods 2, Xie, and F.W. Wise, Opt. Lett. 34, 2051– 910–919 (2005). 2053 (2009).(6) W. Denk et al., Science 248, 73–76 (34) M. Marangoni, A. Gambetta, C. Man- (1990). zoni, V. Kumar, R. Ramponi, and G. Ce-(7) L. Moreaux et al., Biophys. J. 80, 1568– rullo, Opt. Lett. 34, 3262–3264 (2009). 1574 (2001). (35) G. Krauss, T. Hanke, A. Sell, D. Traut-(8) W.R. Zipfel et al., Nat. Biotechnology lein, A. Leitenstorfer, R. Selm, M. Win- 21, 1369–1377 (2003). terhalder, and A. Zumbusch, Opt. Lett.(9) J.-A. Conchelle et al., Nat. Methods 2, 34, 2847–2849 (2009). 920–931 (2005).(10) P.J. Campagnola et al., Nat. Biotechnol. Youngjae Kim, Joseph Salhany, 21, 1356–1360 (2003). and Alain Villeneuve are with(11) D.A. Dombeck et al., Proc. Natl. Acad. Genia Photonics Inc., Lasalle, Québec, Sci. U.S.A. 100, 7081–7086 (2003). Canada. The authors can be contacted at(12) F. Helmchen et al., Nat. Methods 2, the following e-mail address: info@genia- 932–940 (2005). J. Mertz, Curr. Opin. Neurobiol. 14, 610–616 (2004).(14) J.X. Cheng et al., J. Phys. Chem B 108, 827–840 (2004).(15) F. Ganikhanov et al., Opt. Lett. 31, For more information on 1292–1294 (2006). this topic, please visit:(16) A.F. Pegoraro et al., Opt. Express 17, 2984–2996 (2009).(17) M. Jurna et al., Opt. Express 16, 15863–15869, 2009.(18) A. Volkmer et al., Phys. Rev. Lett. 87, 023901 (2001).(19) E.O. Potma et al., Opt. Lett. 31, 241– 243 (2006).(20) K. Kieu et al., Opt. Lett. 34, 2051–2053 (2009).(21) E.R. Andresen et al., Opt. Express 15, 4848–4856 (2007).(22) G. Krauss et al., Opt. Lett. 34, 2847– 2849 (2009).(23) C.W. Freudiger et al., Science 322, 1857–1861 (2008).(24) P. Nandakumar et al., N. J. Phys. 11, 033026 (2009).
  • 5. w w w. s p e c t r o s c o p y o n l i n e . c o m Januar y 2011 Spectroscopy 26(1) 5