Letter

Nature 461, 1105-1109 (22 October 2009) | doi:10.1038/nature08438; Received 28 March 2009; Accepted 13 August 2009

Imaging chromophores with undetectable fluorescence by stimulated emission microscopy

Wei Min1,2, Sijia Lu1,2, Shasha Chong1, Rahul Roy1, Gary R. Holtom1 & X. Sunney Xie1

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA
  2. These authors contributed equally to this work.

Correspondence to: X. Sunney Xie1 Correspondence and requests for materials should be addressed to X.S.X. (Email: xie@chemistry.harvard.edu).

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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