NATURE PHOTONICS DOL:10.1038/NPHOTON.2013.361 REVIEW ARTICLES Remote focusing.Finally,most high-speed imaging has focused on 6. Chen,I.H.,Chu,S.W.Sun,C.K.,Cheng,P.C.Lin,B.L.Wavelength techniques for the rapid raster scanning of lateral images.Recent dependent damage in biological multi-photon confocal microscopy:A developments have led to the remote focusing'technique,which micro-spectroscopic comparison between femtosecond Ti:sapphire and allows rapid axial scanning of the beam0.This technique,when Cr:forsterite laser sources.Opt.Quant.Electron.34,1251-1266(2002). combined with a scan mirror system,permits novel access to bio- 1- Helmchen,F.Denk,W.Deep tissue two-photon microscopy.Nat.Methods 2,932-940(2005). logical systems,such as the ability to image an x-z plane or perform 8. Yaroslavsky,A.N.et al.Optical properties of selected native and coagulated high-speed 3D imagingRemote focusing operates by modify- human brain tissues in vitro in the visible and near infrared spectral range. ing the divergence of the beam at the back of the excitation objec- Phys.Med.Biol.47,2059-2073(2002). tive,usually by imaging the objective's stop to an upstream location, 9. Theer,P.Denk,W.On the fundamental imaging-depth limit in two where a second 'remote'objective is used in conjunction with a mir- photon microscopy.J.Opt.Soc.Am.A 23,3139-3149 (2006) 0 Kobat,D.et al.Deep tissue multiphoton microscopy using longer wavelength ror at its focus.It is this second,remotely located objective-mirror excitation.Opt.Express 17,13354-13364 (2009). combination that produces the necessary divergence at the excitation 11. Ntziachristos,V.Going deeper than microscopy:The optical imaging frontier objective when the distance between the remote objective and mirror in biology.Nat.Methods 7,603-614(2010). is adjusted.This configuration is similar to how a properly imaged 12. Barad,Y.,Eisenberg.H.,Horowitz,M.Silberberg.Y.Nonlinear scanning scan-relay system is designed,although such a system operates on the laser microscopy by third harmonic generation.Appl.Phys.Lett.70, 922-924(1997) angle at the back of the objective rather than the divergence.The abil- ity to control the axial focus of the microscope by employing remote Masihzadeh,O.,Schlup,P.&Bartels,R.A.Label-free second harmonic generation holographic microscopy of biological specimens.Opt.Express 18 focusing represents a dramatic step forwards for biological imaging, 9840-9851(2010). as this allows biologists to examine specimens in three dimensions 14. Zhuo,S.et al.Label-free monitoring of colonic cancer progression using without moving either the object under examination or the objec- multiphoton microscopy.Biomed.Opt.Express 2,615-619(2011). tive used for imaging(Fig.5)05.This change in imaging paradigm is 15. Segawa,H.et al.Label-free tetra-modal molecular imaging of living cells significant because moving the specimen requires expensive stages with CARS,SHG.THG and TSFG(coherent anti-Stokes Raman scattering, second harmonic generation,third harmonic generation and third-order and can introduce significant problems for 'registering real-world sum frequency generation).Opt.Express 20,9551-9557(2012) coordinates in individual frames with any previously captured data. Theer,P.,Hasan,M.T.Denk,W.Two-photon imaging to a depth of 1000 Although moving the objective might be considered as an alternative um in living brains by use of a Ti:Al,O,regenerative amplifier.Opt.Lett.28 to translating the specimen,such an endeavour degrades the image 1022-1024(2003). quality by moving away from the ideal objective position and can 17. Yue,S.,Slipchenko,M.Cheng.J.X.Multimodal nonlinear optical microscopy.Las.Photon.Rev.5,496-512 (2011). introduce vibrations that negatively impact the image quality. Chung.C.Y.,Boik,J.Potma,E.O.Biomolecular imaging with coherent nonlinear vibrational microscopy.Ann.Rev.Phys.Chem.64,77-99 (2013) Conclusion 19. Larson,A.Yeh,A.Ex vivo characterization of sub-10-fs pulses.Opt.Lett. The landscape of MPM has grown enormously since the initial appli- 31.1681-1683(2006). cation of TPEF microscopy by Denk et al,and as such,no single 20. Pestov,D.,Xu,B.,Li,H.Dantus,M.Delivery and characterization of sub- article can truly do justice to the broad range of new technologies and 8fs laser pulses at the imaging plane of a two-photon microscope.Proc.SPIE 7903,79033B(2011). novel explorations that have resulted.There are significant topics not addressed here,including pushing the resolution limitsspec- 21. Selm,R.,Krauss,G.,Leitenstorfer,A.Zumbusch,A.Simultaneous second- harmonic generation,third-harmonic generation,and four-wave mixing trally resolved imaging and multiphoton light sheet microscopy. microscopy with single sub-8 fs laser pulses.Appl.Phys.Lett.99, The future for MPM looks bright from many perspectives.New 181124(2011). femtosecond laser sources that operate reliably with exceptional ease Zipfel,W.,Williams,R.Webb,W.W.Nonlinear magic:Multiphoton are continuously being developed.In this regard,a significant new microscopy in the biosciences.Nature Biotechnol.21,1369-1377(2003). 23. benchmark has recently been achieved:a femtosecond laser suitable Mertz,J.Nonlinear microscopy:New techniques and applications.Curr. Opin.Neurobiol.14,610-616 (2004). for nonlinear microscopy can now be purchased at a price equiva- 24 Svoboda,K.Yasuda,R.Principles of two-photon excitation microscopy lent to that of a high-end microscope objective.Similarly,a broader and its applications to neuroscience.Neuron 50,823-839 (2006). class of optics are being optimized with specific application to fem- Sheetz,K.E.Squier,J.Ultrafast optics:Imaging and manipulating tosecond laser excitation and detection.These optical systems have biological systems.J.Appl.Phys.105,051101 (2009) higher throughput and are designed to deliver diffraction-limited 26. Carriles,R.et al.Invited review article:Imaging techniques for harmonic and focal spots and transform-limited pulse durations.As such,we can multiphoton absorption fluorescence microscopy.Rev.Sci.Instr.80, 081101(2009). envision future systems that will continue to push the boundaries of 27. Field,J.et al.Optimizing the fluorescent yield in two-photon laser scanning imaging,further compelling studies that will connect chemical and microscopy with dispersion compensation.Opt.Express 18, physiological processes to structure and function,which will pro- 13661-13672(2010). vide,for the first time,a comprehensive picture of organisms from Gannaway,J.N.Sheppard,C.J.R.Second-harmonic imaging in the the atomic to the macroscopic level. scanning optical microscope.Opt.Quant.Electron.10,435-439(1978) 29. Raghunathan,V.,Han,Y.,Korth,O.,Ge,N.H.Potma,E.Rapid vibrational imaging with sum frequency generation microscopy.Opt.Lett.30, 3891-3893(2011). References 30. Fu,D.et al.Quantitative chemical imaging with multiplex stimulated Ramar 1. Denk,W.,Strickler,J.H.Webb,W.W.Two-photon laser scanning scattering microscopy.J.Am.Chem.Soc.134,3623-3626(2012). fluorescence microscopy.Science 248,73-76(1990). 31. Zumbusch,A.,Holtom,G.R.Xie,X.S.Three-dimensional vibrational Peleg,G.,Lewis,A.,Linial,M.Loew,L.M.Nonlinear optical measurement imaging by coherent anti-Stokes Raman scattering.Phys.Rev.Lett.82, of membrane potential around single molecules at selected cellular sites. 4142-4145(1999). Proc.Natl Acad.Sci.USA 96,6700-6704(1999). Chen,H.et al.A multimodal platform for nonlinear optical microscopy and Chu,S.W.et al.In vivo developmental biology study using noninvasive microspectroscopy.Opt.Express 17,1282-1290 (2009). multi-harmonic generation microscopy.Opt.Express 11, 33. Becker,W.Bergmann,A.Lifetime-resolved imaging in nonlinear 3093-3099(2003). microscopy in Handbook of Biomedical Nonlinear Optical Microscopy (eds Cheng.A.,Goncalves,J.T.,Golshani,P.,Arisaka,K.Portera-Cailliau Masters,B.So,P.)499-556(Oxford University,2008). C.Simultaneous two-photon calcium imaging at different depths with 34 Becker,W.et al.Fluorescence lifetime imaging by time-correlated single spatiotemporal multiplexing.Nat.Methods 8,139-142(2011). photon counting.Microsc.Res.Tech.63,58-66(2004). Stelzer,E.H.et al.Nonlinear absorption extends confocal fluorescence 35. Driscoll,J.D.et al.Photon counting,censor corrections,and lifetime microscopy into the ultra-violet regime and confines the illumination imaging for improved detection in two-photon microscopy./Neurophysiol. volume.OpL.Co1mum.104,223-228(1994). 105,3106-3113(2011). NATURE PHOTONICS VOL7|FEBRUARY 2013 www.nature.com/naturephotonics 99 2013 Macmillan Publishers Limited.All rights reserved© 2013 Macmillan Publishers Limited. All rights reserved. NATURE PHOTONICS | VOL 7 | FEBRUARY 2013 | www.nature.com/naturephotonics 99 Remote focusing. Finally, most high-speed imaging has focused on techniques for the rapid raster scanning of lateral images. Recent developments have led to the ‘remote focusing’ technique, which allows rapid axial scanning of the beam101,102. This technique, when combined with a scan mirror system, permits novel access to bio￾logical systems, such as the ability to image an x–z plane or perform high-speed 3D imaging91,103,104. Remote focusing operates by modify￾ing the divergence of the beam at the back of the excitation objec￾tive, usually by imaging the objective’s stop to an upstream location, where a second ‘remote’ objective is used in conjunction with a mir￾ror at its focus. It is this second, remotely located objective–mirror combination that produces the necessary divergence at the excitation objective when the distance between the remote objective and mirror is adjusted. This configuration is similar to how a properly imaged scan-relay system is designed, although such a system operates on the angle at the back of the objective rather than the divergence. The abil￾ity to control the axial focus of the microscope by employing remote focusing represents a dramatic step forwards for biological imaging, as this allows biologists to examine specimens in three dimensions without moving either the object under examination or the objec￾tive used for imaging (Fig. 5)105. This change in imaging paradigm is significant because moving the specimen requires expensive stages and can introduce significant problems for ‘registering’ real-world coordinates in individual frames with any previously captured data. Although moving the objective might be considered as an alternative to translating the specimen, such an endeavour degrades the image quality by moving away from the ideal objective position and can introduce vibrations that negatively impact the image quality. Conclusion The landscape of MPM has grown enormously since the initial appli￾cation of TPEF microscopy by Denk et al.1 , and as such, no single article can truly do justice to the broad range of new technologies and novel explorations that have resulted. There are significant topics not addressed here, including pushing the resolution limits106–109, spec￾trally resolved imaging110 and multiphoton light sheet microscopy111. The future for MPM looks bright from many perspectives. New femtosecond laser sources that operate reliably with exceptional ease are continuously being developed. In this regard, a significant new benchmark has recently been achieved: a femtosecond laser suitable for nonlinear microscopy can now be purchased at a price equiva￾lent to that of a high-end microscope objective. Similarly, a broader class of optics are being optimized with specific application to fem￾tosecond laser excitation and detection. These optical systems have higher throughput and are designed to deliver diffraction-limited focal spots and transform-limited pulse durations. As such, we can envision future systems that will continue to push the boundaries of imaging, further compelling studies that will connect chemical and physiological processes to structure and function, which will pro￾vide, for the first time, a comprehensive picture of organisms from the atomic to the macroscopic level. References 1. Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990). 2. Peleg, G., Lewis, A., Linial, M. & Loew, L. M. Nonlinear optical measurement of membrane potential around single molecules at selected cellular sites. Proc. Natl Acad. Sci. USA 96, 6700–6704 (1999). 3. Chu, S. W. et al. In vivo developmental biology study using noninvasive multi-harmonic generation microscopy. Opt. Express 11, 3093–3099 (2003). 4. Cheng, A., Gonçalves, J. T., Golshani, P., Arisaka, K. & Portera-Cailliau, C. Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing. Nat. Methods 8, 139–142 (2011). 5. Stelzer, E. H. et al. Nonlinear absorption extends confocal fluorescence microscopy into the ultra-violet regime and confines the illumination volume. Opt. Commun. 104, 223–228 (1994). 6. Chen, I. H., Chu, S. W., Sun, C. K., Cheng, P. C. & Lin, B. L. Wavelength dependent damage in biological multi-photon confocal microscopy: A micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources. Opt. Quant. Electron. 34, 1251–1266 (2002). 7. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005). 8. Yaroslavsky, A. N. et al. Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range. Phys. Med. Biol. 47, 2059–2073 (2002). 9. Theer, P. & Denk, W. On the fundamental imaging-depth limit in two￾photon microscopy. J. Opt. Soc. Am. A 23, 3139–3149 (2006). 10. Kobat, D. et al. Deep tissue multiphoton microscopy using longer wavelength excitation. Opt. Express 17, 13354–13364 (2009). 11. Ntziachristos, V. Going deeper than microscopy: The optical imaging frontier in biology. Nat. Methods 7, 603–614 (2010). 12. Barad, Y., Eisenberg, H., Horowitz, M. & Silberberg, Y. Nonlinear scanning laser microscopy by third harmonic generation. Appl. Phys. Lett. 70, 922–924 (1997). 13. Masihzadeh, O., Schlup, P. & Bartels, R. A. Label-free second harmonic generation holographic microscopy of biological specimens. Opt. Express 18, 9840–9851 (2010). 14. Zhuo, S. et al. Label-free monitoring of colonic cancer progression using multiphoton microscopy. Biomed. Opt. Express 2, 615–619 (2011). 15. Segawa, H. et al. Label-free tetra-modal molecular imaging of living cells with CARS, SHG, THG and TSFG (coherent anti-Stokes Raman scattering, second harmonic generation, third harmonic generation and third-order sum frequency generation). Opt. Express 20, 9551–9557 (2012). 16. Theer, P., Hasan, M. T. & Denk, W. Two-photon imaging to a depth of 1000 μm in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024 (2003). 17. Yue, S., Slipchenko, M. & Cheng, J. X. Multimodal nonlinear optical microscopy. Las. Photon. Rev. 5, 496–512 (2011). 18. Chung, C. Y., Boik, J. & Potma, E. O. Biomolecular imaging with coherent nonlinear vibrational microscopy. Ann. Rev. Phys. Chem. 64, 77–99 (2013). 19. Larson, A. & Yeh, A. Ex vivo characterization of sub-10-fs pulses. Opt. Lett. 31, 1681–1683 (2006). 20. Pestov, D., Xu, B., Li, H. & Dantus, M. Delivery and characterization of sub- 8fs laser pulses at the imaging plane of a two-photon microscope. Proc. SPIE 7903, 79033B (2011). 21. 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Optimizing the fluorescent yield in two-photon laser scanning microscopy with dispersion compensation. Opt. Express 18, 13661–13672 (2010). 28. Gannaway, J. N. & Sheppard, C. J. R. Second-harmonic imaging in the scanning optical microscope. Opt. Quant. Electron. 10, 435–439 (1978). 29. Raghunathan, V., Han, Y., Korth, O., Ge, N. H. & Potma, E. Rapid vibrational imaging with sum frequency generation microscopy. Opt. Lett. 30, 3891–3893 (2011). 30. Fu, D. et al. Quantitative chemical imaging with multiplex stimulated Raman scattering microscopy. J. Am. Chem. Soc. 134, 3623–3626 (2012). 31. Zumbusch, A., Holtom, G. R. & Xie, X. S. Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering. Phys. Rev. Lett. 82, 4142–4145 (1999). 32. Chen, H. et al. A multimodal platform for nonlinear optical microscopy and microspectroscopy. Opt. Express 17, 1282–1290 (2009). 33. Becker, W. & Bergmann, A. Lifetime-resolved imaging in nonlinear microscopy in Handbook of Biomedical Nonlinear Optical Microscopy (eds Masters, B. & So, P.) 499–556 (Oxford University, 2008). 34. Becker, W. et al. Fluorescence lifetime imaging by time-correlated single photon counting. Microsc. Res. Tech. 63, 58–66 (2004). 35. Driscoll, J. D. et al. Photon counting, censor corrections, and lifetime imaging for improved detection in two-photon microscopy. J. Neurophysiol. 105, 3106–3113 (2011). NATURE PHOTONICS DOI: 10.1038/NPHOTON.2013.361 REVIEW ARTICLES