Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Imaging chromophores with undetectable fluorescence by stimulated emission microscopy


Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging1,2. However, many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay3. Yet the detection of their absorption is difficult under a microscope. Here we use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, and report a new contrast mechanism for optical microscopy. In a pump–probe experiment, on photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, the intensity of which is concurrently increased. We extract the miniscule intensity increase with shot-noise-limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz frequency. The signal is generated only at the laser foci owing to the nonlinear dependence on the input intensities, providing intrinsic three-dimensional optical sectioning capability. In contrast, conventional one-beam absorption measurement exhibits low sensitivity, lack of three-dimensional sectioning capability, and complication by linear scattering of heterogeneous samples. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Principles of stimulated emission microscopy.
Figure 2: Characterizations of stimulated emission microscopy.
Figure 3: Imaging non-fluorescent chromoproteins and chromogenic reporter for gene expression.
Figure 4: Transdermal drug distribution in three-dimensional and microvascular imaging.

Similar content being viewed by others


  1. Pawley, J. B. Handbook of Biological Confocal Microscopy 3rd edn (Springer, 2006)

    Book  Google Scholar 

  2. Lakowicz, J. R. Principles of Fluorescence Spectroscopy (Plenum Press, 1983)

    Book  Google Scholar 

  3. Turro, N. J. Modern Molecular Photochemistry (University Science Books, 1991)

    Google Scholar 

  4. Einstein, A. On the quantum theory of radiation. Phys. Z. 18, 121 (1917)

    CAS  Google Scholar 

  5. Seigman, A. E. Laser 264–307 (University Science Books, 1986)

    Google Scholar 

  6. Hamilton, C. E., Kinsey, J. L. & Field, R. W. Stimulated emission pumping: new methods in spectroscopy and molecular dynamics. Annu. Rev. Phys. Chem. 37, 493–524 (1986)

    Article  ADS  CAS  Google Scholar 

  7. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994)

    Article  ADS  CAS  Google Scholar 

  8. Dong, C. Y., So, P. T., French, T. & Gratton, E. Fluorescence lifetime imaging by asynchronous pump-probe microscopy. Biophys. J. 69, 2234–2242 (1995)

    Article  ADS  CAS  Google Scholar 

  9. Cantor, C. R. & Schimmel, P. R. Biophysical Chemistry 361–374 (W. H. Freeman, 1980)

    Google Scholar 

  10. Moerner, W. E. & Kador, L. Optical detection and spectroscopy of single molecules in a solid. Phys. Rev. Lett. 62, 2535–2538 (1989)

    Article  ADS  CAS  Google Scholar 

  11. Ye, J., Ma, L. S. & Hall, J. L. Ultrasensitive detections in atomic and molecular physics: demonstration in molecular overtone spectroscopy. J. Opt. Soc. Am. B 15, 6–15 (1998)

    Article  ADS  CAS  Google Scholar 

  12. Freudiger, C. W. et al. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science 322, 1857–1861 (2008)

    Article  ADS  CAS  Google Scholar 

  13. Fu, D. et al. High-resolution in vivo imaging of blood vessels without labeling. Opt. Lett. 32, 2641–2643 (2007)

    Article  ADS  Google Scholar 

  14. Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990)

    Article  ADS  CAS  Google Scholar 

  15. Evans, C. L. & Xie, X. S. Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine. Annu. Rev. Anal. Chem. 1, 883–909 (2008)

    Article  CAS  Google Scholar 

  16. Rittweger, E., Rankin, B. R., Westphal, V. & Hell, S. W. Fluorescence depletion mechanisms in super-resolving STED microscopy. Chem. Phys. Lett. 442, 483–487 (2007)

    Article  ADS  CAS  Google Scholar 

  17. Du, H. et al. PhotochemCAD: A computer-aided design and research tool in photochemistry. Photochem. Photobiol. 68, 141–142 (1998)

    CAS  Google Scholar 

  18. Gurskaya, N. G. et al. GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS Lett. 507, 16–20 (2001)

    Article  CAS  Google Scholar 

  19. Chan, M. C. Y. et al. Structural characterization of a blue chromoprotein and its yellow mutant from the sea anemone Cnidopus japonicus . J. Biol. Chem. 281, 37813–37819 (2006)

    Article  CAS  Google Scholar 

  20. Zhang, J., Campbell, R. E., Ting, A. Y. & Tsien, R. Y. Creating new fluorescent probes for cell biology. Nature Rev. Mol. Biol. 3, 906–918 (2002)

    Article  CAS  Google Scholar 

  21. Miller, J. H. Experiments in Molecular Genetics 171–224 (Cold Spring Harbor Laboratory, 1972)

    Google Scholar 

  22. Cai, L., Friedman, N. & Xie, X. S. Stochastic protein expression in individual cells at the single molecule level. Nature 440, 358–362 (2006)

    Article  ADS  CAS  Google Scholar 

  23. Tremblay, J. F. et al. Photodynamic therapy with toluidine blue in Jurkat cells: cytotoxicity, subcellular localization and apoptosis induction. Photochem. Photobiol. Sci. 1, 852–856 (2002)

    Article  CAS  Google Scholar 

  24. Chelvanayagam, D. K. & Beazley, L. D. Toluidine blue-O is a Nissl bright-field counterstain for lipophilic fluorescent tracers Di-ASP, DiI and DiO. J. Neurosci. Methods 72, 49–55 (1997)

    Article  CAS  Google Scholar 

  25. McDonald, D. M. & Choyke, P. L. Imaging of angiogenesis: from microscope to clinic. Nature Med. 9, 713–725 (2003)

    Article  CAS  Google Scholar 

  26. Grinvald, A., Lieke, E., Frostig, R. D., Gilbert, C. D. & Wiesel, T. N. Functional architecture of cortex revealed by optical imaging of intrinsic signals. Nature 324, 361–364 (1986)

    Article  ADS  CAS  Google Scholar 

  27. Kleinfeld, D., Mitra, P. P., Helmchen, F. & Denk, W. Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex. Proc. Natl Acad. Sci. USA 95, 15741–15746 (1998)

    Article  ADS  CAS  Google Scholar 

  28. Clay, G. O., Schaffer, C. B. & Kleinfeld, D. Large two-photon absorptivity of hemoglobin in the infrared range of 780–880 nm. J. Chem. Phys. 126, 025102 (2007)

    Article  ADS  Google Scholar 

  29. Wang, W. et al. Femtosecond multicolor pump-probe spectroscopy of ferrous cytochrome c. J. Phys. Chem. B 104, 10789–10801 (2000)

    Article  CAS  Google Scholar 

Download references


We thank K. Lukyanov and A. Miyawaki for the gifts of chromoprotein gtCP and cjBlue plasmid DNA, respectively; Coherent Inc. for lending us a femtosecond optical parametric oscillator; and P. Choi for preparing X-gal E. coli cells. We also thank B. G. Saar, C. W. Freudiger, S. Basu, J. W. Lichtman and C. B. Schaffer for discussions, and R. Tsien for suggesting the use of chromoproteins. This work was supported by a National Science Foundation (grant CHE-0634788) and the US Department of Energy’s Basic Energy Sciences Program (DE-FG02-07ER15875).

Author Contributions W.M., S.L. and S.C. performed experiments and analysed data. R.R. constructed E. coli cells expressing chromoproteins. G.R.H. and S.C. helped to construct the laser systems. W.M., S.L. and X.S.X. conceived the concept, designed the experiments and wrote the paper.

Author information

Authors and Affiliations


Corresponding author

Correspondence to X. Sunney Xie.

Ethics declarations

Competing interests

[Competing interests: Harvard University has recently filed a US patent application (“Systems and Methods for Stimulated Emission Imaging.”) on behalf of X.S.X., W.M. and S.L. based on this work.]

Supplementary information

Supplementary Information

This file contains Supplementary Methods and Supplementary Figures 1-3 with Legends. (PDF 513 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Min, W., Lu, S., Chong, S. et al. Imaging chromophores with undetectable fluorescence by stimulated emission microscopy. Nature 461, 1105–1109 (2009).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing