Imaging Chromophores With Undetectable Fluorescence by Stimulated Emission Microscopy

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Imaging Chromophores With Undetectable Fluorescence by Stimulated Emission Microscopy

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dc.contributor.author Min, Wei
dc.contributor.author Lu, Sijia
dc.contributor.author Chong, Shasha
dc.contributor.author Roy, Rahul
dc.contributor.author Holtom, Gary
dc.contributor.author Xie, Xiaoliang Sunney
dc.date.accessioned 2010-02-23T18:40:56Z
dash.embargo.terms 2010-04-23
dc.date.issued 2009
dc.identifier.citation Min, Wei, Sijia Lu, Shasha Chong, Rahul Roy, Gary R. Holtom, and Xiaoliang Sunney Xie. 2009. Imaging chromophores with undetectable fluorescence by stimulated emission microscopy. Nature: 461(7267): 1105-1109. en_US
dc.identifier.issn 0028-0836 en_US
dc.identifier.uri http://nrs.harvard.edu/urn-3:HUL.InstRepos:3693471
dc.description.abstract 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. en_US
dc.description.sponsorship Chemistry and Chemical Biology en_US
dc.language.iso en_US en_US
dc.publisher Nature Publishing Group en_US
dc.relation.isversionof doi:10.1038/nature08438 en_US
dc.relation.hasversion http://bernstein.harvard.edu/papers/Nature%2010.22.09.pdf en_US
dash.license LAA
dc.title Imaging Chromophores With Undetectable Fluorescence by Stimulated Emission Microscopy en_US
dc.type Journal Article en_US
dc.description.version Accepted Manuscript en_US
dc.relation.journal Nature en_US
dash.depositing.author Xie, Xiaoliang Sunney
dc.date.available 2010-04-23T07:31:11Z

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