Fluorescence, that is,
spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in
optical imaging. However, many chromophores, such as
haemoglobin and
cytochromes, absorb but have undetectable
fluorescence because the
spontaneous emission is dominated by their fast non-radiative decay. 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.
