Photodynamic therapy by in situ nonlinear photon conversion


In photodynamic therapy, light is absorbed by a therapy agent (photosensitizer) to generate reactive oxygen, which then locally kills diseased cells. Here, we report a new form of photodynamic therapy in which nonlinear optical interactions of near-infrared laser radiation with a biological medium in situ produce light that falls within the absorption band of the photosensitizer. The use of near-infrared radiation, followed by upconversion to visible or ultraviolet light, provides deep tissue penetration, thus overcoming a major hurdle in treatment. By modelling and experiment, we demonstrate activation of a known photosensitizer, chlorin e6, by in situ nonlinear optical upconversion of near-infrared laser radiation using second-harmonic generation in collagen and four-wave mixing, including coherent anti-Stokes Raman scattering, produced by cellular biomolecules. The introduction of coherent anti-Stokes Raman scattering/four-wave mixing to photodynamic therapy in vitro increases the efficiency by a factor of two compared to two-photon photodynamic therapy alone, while second-harmonic generation provides a fivefold increase.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Nonlinear optical mechanisms for PDT.
Figure 2: Experimental design for nonlinear excitation of PDT.
Figure 3: Contributions of CARS/FWM and SHG nonlinear optical conversions to chlorin e6 excitation.
Figure 4: Cellular phototoxicity in response to nonlinear optical PDT excitation by CARS/FWM/TPA and TPA modes for different numbers of laser scans.
Figure 5: Cellular phototoxicity in response to nonlinear optical PDT excitation by SHG/TPA and TPA modes for different numbers of laser scans.


  1. 1

    Henderson, B. W. & Dougherty, T. J. Photodynamic Therapy: Basic Principles and Clinical Applications (Marcel Dekker, 1992).

    Google Scholar 

  2. 2

    Prasad, P. N. Introduction to Biophotonics (Wiley-Interscience, 2003).

    Google Scholar 

  3. 3

    Prasad, P. N. Introduction to Nanomedicine and Nanobioengineering (Wiley, 2012).

    Google Scholar 

  4. 4

    Pervaiz, S. & Olivo, M. Art and science of photodynamic therapy. Clin. Exp. Pharmacol. Physiol. 33, 551–556 (2006).

    Article  Google Scholar 

  5. 5

    Gupta, A. et al. Multifunctional nanoplatforms for fluorescence imaging and photodynamic therapy developed by post-loading photosensitizer and fluorophore to polyacrylamide nanoparticles. Nanomed. Nanotechnol. Biol. Med. 8, 941–950 (2012).

    Article  Google Scholar 

  6. 6

    Ohulchanskyy, T. Y. et al. Organically modified silica nanoparticles with covalently incorporated photosensitizer for photodynamic therapy of cancer. Nano Lett. 7, 2835–2842 (2007).

    ADS  Article  Google Scholar 

  7. 7

    Huang, Y.-Y. et al. In vitro photodynamic therapy and quantitative structure–activity relationship studies with stable synthetic near-infrared-absorbing bacteriochlorin photosensitizers. J. Med. Chem. 53, 4018–4027 (2010).

    Article  Google Scholar 

  8. 8

    DeRosa, M. C. & Crutchley, R. J. Photosensitized singlet oxygen and its applications. Coord. Chem. Rev. 233, 351–371 (2002)10.1016/S0010-8545(02)00034-6.

    Google Scholar 

  9. 9

    Brown, S. Photodynamic therapy—two photons are better than one. Nature Photon. 2, 394–395 (2008).

    ADS  Article  Google Scholar 

  10. 10

    Bhawalkar, J. D., He, G. S. & Prasad, P. N. Nonlinear multiphoton processes in organic and polymeric materials. Rep. Prog. Phys. 59, 1041–1070 (1996).

    ADS  Article  Google Scholar 

  11. 11

    Bhawalkar, J. D., Kumar, N. D., Zhao, C.-F. & Prasad, P. N. Two-photon photodynamic therapy. J. Clin. Laser Med. Surg. 15, 201–204 (1997).

    Article  Google Scholar 

  12. 12

    Fisher, W. G., Partridge, W. P. Jr, Dees, C. & Wachter, E. A. Simultaneous two-photon activation of type-I photodynamic therapy agents. Photochem. Photobiol. 66, 141–155 (1997).

    Article  Google Scholar 

  13. 13

    König, K., Riemann, I., Fischer, P. & Halbhuber, K.-J. Intracellular nanosurgery with near infrared femtosecond laser pulses. Cell. Mol. Biol. 45, 195–201 (1999).

    Google Scholar 

  14. 14

    Collins, H. A. et al. Blood-vessel closure using photosensitizers engineered for two-photon excitation. Nature Photon. 2, 420–424 (2008).

    Article  Google Scholar 

  15. 15

    Dichtel, W. R. et al. Singlet oxygen generation via two-photon excited FRET. J. Am. Chem. Soc. 126, 5380–5381 (2004).

    Article  Google Scholar 

  16. 16

    Oar, M. A. et al. Photosensitization of singlet oxygen via two-photon-excited fluorescence resonance energy transfer in a water-soluble dendrimer. Chem. Mater. 17, 2267–2275 (2005).

    Article  Google Scholar 

  17. 17

    Oar, M. A. et al. Light-harvesting chromophores with metalated porphyrin cores for tuned photosensitization of singlet oxygen via two-photon excited FRET. Chem. Mater. 18, 3682–3692 (2006).

    Article  Google Scholar 

  18. 18

    Kim, S., Ohulchanskyy, T. Y., Pudavar, H. E., Pandey, R. K. & Prasad, P. N. Organically modified silica nanoparticles co-encapsulating photosensitizing drug and aggregation-enhanced two-photon absorbing fluorescent dye aggregates for two-photon photodynamic therapy. J. Am. Chem. Soc. 129, 2669–2675 (2007).

    Article  Google Scholar 

  19. 19

    Idris, N. M. et al. In vivo photodynamic therapy using upconversion nanoparticles as remote-controlled nanotransducers. Nature Med. 18, 1580–1585 (2012).

    Article  Google Scholar 

  20. 20

    Jodele, S., Blavier, L., Yoon, J. M. & DeClerck, Y. A. Modifying the soil to affect the seed: role of stromal-derived matrix metalloproteinases in cancer progression. Cancer Metastas. Rev. 25, 35–43 (2006).

    Article  Google Scholar 

  21. 21

    Shoulders, M. D. & Raines, R. T. Collagen structure and stability. Annu. Rev. Biochem. 78, 929–958 (2009).

    Article  Google Scholar 

  22. 22

    Volkmer, A. Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering microscopy. J. Phys. D 38, R59–R81 (2005).

    ADS  Article  Google Scholar 

  23. 23

    Le, T. T., Huff, T. B. & Cheng, J.-X. Coherent anti-Stokes Raman scattering imaging of lipids in cancer metastasis. BMC Cancer 9, 42 (2009).

    Article  Google Scholar 

  24. 24

    Bozza, P. T. & Viola, J. P. B. Lipid droplets in inflammation and cancer. Prostag. Leukotr. Ess. Fatty Acids 82, 243–250 (2010).

    Article  Google Scholar 

  25. 25

    Pliss, A., Kuzmin, A. N., Kachynski, A. V. & Prasad, P. N. Biophotonic probing of macromolecular transformations during apoptosis. Proc. Natl Acad. Sci. USA 107, 12771–12776 (2010).

    ADS  Article  Google Scholar 

  26. 26

    Pliss, A., Kuzmin, A. N., Kachynski, A. V. & Prasad, P. N. Nonlinear optical imaging and Raman microspectrometry of the cell nucleus throughout the cell cycle. Biophys. J. 99, 3483–3491 (2010).

    ADS  Article  Google Scholar 

  27. 27

    Allison, R. R. et al. Photosensitizers in clinical PDT. Photodiag. Photodyn. Ther. 1, 27–42 (2004).

    Article  Google Scholar 

  28. 28

    Zenkevich, E. et al. Photophysical and photochemical properties of potential porphyrin and chlorin photosensitizers for PDT. J. Photochem. Photobiol. B 33, 171–180 (1996).

    Article  Google Scholar 

  29. 29

    Tolles, W. M., Nibler, J. W., McDonald, J. R. & Harvey, A. B. Review of the theory and application of coherent anti-Stokes Raman-spectroscopy (CARS). Appl. Spectrosc. 31, 253–271 (1977).

    ADS  Article  Google Scholar 

  30. 30

    Gubskaya, A. V. & Kusalik, P. G. The multipole polarizabilities and hyperpolarizabilities of the water molecule in liquid state: an ab initio study. Mol. Phys. 99, 1107–1120 (2001).

    ADS  Article  Google Scholar 

  31. 31

    Débarre, D. & Beaurepaire, E. Quantitative characterization of biological liquids for third-harmonic generation microscopy. Biophys. J. 92, 603–612 (2007).

    ADS  Article  Google Scholar 

  32. 32

    Deniset-Besseau, A. et al. Measurement of the second-order hyperpolarizability of the collagen triple helix and determination of its physical origin. J. Phys. Chem. B 113, 13437–13445 (2009).

    Article  Google Scholar 

  33. 33

    He, G. S., Zheng, Q., Baev, A. & Prasad, P. N. Saturation of multiphoton absorption upon strong and ultrafast infrared laser excitation. J. Appl. Phys. 101, 083108 (2007).

    ADS  Article  Google Scholar 

  34. 34

    Baev, A., Gel'mukhanov, F., Macák, P., Luo, Y. & Ågren, H. General theory for pulse propagation in two-photon active media. J. Chem. Phys. 117, 6214–6220 (2002).

    ADS  Article  Google Scholar 

  35. 35

    Dalton (2011). Release Dalton 2011. Available from

  36. 36

    Chen, P. et al. Two-photon excitation of chlorin-e6-C15 monomethyl ester for photodynamic therapy. Proc. SPIE 5630, 209–217 (2005).

    ADS  Article  Google Scholar 

  37. 37

    Fu, Y., Wang, H., Shi, R. Y. & Cheng, J.-X. Characterization of photodamage in coherent anti-Stokes Raman scattering microscopy. Opt. Express 14, 3942–3951 (2006).

    ADS  Article  Google Scholar 

  38. 38

    Juzeniene, A., Juzenas, P., Bronshtein, I., Vorobey, A. & Moan, J. The influence of temperature on photodynamic cell killing in vitro with 5-aminolevulinic acid. J. Photochem. Photobiol. B 84, 161–166 (2006).

    Article  Google Scholar 

  39. 39

    Kalluri, R. & Zeisberg, M. Fibroblasts in cancer. Nature Rev. Cancer 6, 392–401 (2006).

    Article  Google Scholar 

  40. 40

    Brown, E. et al. Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation. Nature Med. 9, 796–800 (2003).

    Article  Google Scholar 

  41. 41

    Zoumi, A., Yeh, A. & Tromberg, B. J. Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc. Natl Acad. Sci. USA 99, 11014–11019 (2002).

    ADS  Article  Google Scholar 

Download references


This work was supported in part by a grant from the Air Force Office of Scientific Research (grants no. 1096313-1-58130 and no. FA95500610398). J.Q. acknowledges support from the National Natural Science Foundation of China (61378091) and the National Basic Research Program of China (grant no. 2012CB825802).

Author information




A.V.K. provided technical and conceptual support and edited the manuscript. A.P. performed experiments, analysed data and wrote the manuscript. A.N.K. designed and performed experiments, analysed data and edited the manuscript. A.B. performed theoretical modelling and wrote the theoretical analysis. T.Y.O. designed experiments, analysed the data and wrote the manuscript. J.Q. performed experiments and edited the manuscript. P.N.P. supervised the analysis and edited the manuscript.

Corresponding authors

Correspondence to J. Qu or P. N. Prasad.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 903 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kachynski, A., Pliss, A., Kuzmin, A. et al. Photodynamic therapy by in situ nonlinear photon conversion. Nature Photon 8, 455–461 (2014).

Download citation

Further reading


Quick links

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing