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Imaging chromophores with undetectable fluorescence by stimulated emission microscopy

Abstract

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.

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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.

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Acknowledgements

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.

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Correspondence to X. Sunney Xie.

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[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.]

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Min, W., Lu, S., Chong, S. et al. Imaging chromophores with undetectable fluorescence by stimulated emission microscopy. Nature 461, 1105–1109 (2009). https://doi.org/10.1038/nature08438

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