Imaging chromophores with undetectable fluorescence by stimulated emission microscopy

• Published in: Nature (London) Vol. 461; no. 7267; pp. 1105 - 1109
• Main Authors: Min, Wei, Lu, Sijia, Chong, Shasha, Roy, Rahul, Holtom, Gary R., Xie, X. Sunney
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 22.10.2009; Nature Publishing Group
• Subjects: Absorption; Analysis; Animals; Biological and medical sciences; Blood vessels; Chromophores; Decay; Diverse techniques; Ear; Emission measurements; Emission spectra; Escherichia coli - metabolism; Fluorescence; Fundamental and applied biological sciences. Psychology; Gene Expression Profiling; Genes, Reporter - genetics; Haemoglobin; Hemoglobins - analysis; Humanities and Social Sciences; Imaging; Indigo Carmine; Indoles - metabolism; Lac Operon - genetics; Lasers; letter; Light microscopy; Medical imaging; Methods; Mice; Microscope and microscopy; Microscopy; Microscopy - methods; Molecular and cellular biology; Molecular Imaging - methods; Molecules; multidisciplinary; Noise; Photosensitizing Agents - analysis; Properties; Science; Science (multidisciplinary); Sensitivity and Specificity; Spectra; Spontaneous emission; Stimulated emission; Three dimensional; Tomography; United States; Cytochrome; Stimulated emission microscopy; Fluorescence; Hemoglobin; Hemoprotein; Imaging
• ISSN: 0028-0836; 1476-4687; 1476-4687
• DOI: 10.1038/nature08438

Super-resolution microscopy For imaging beyond the diffraction limit, to resolve tiny features in cells for example, researchers have so far had to rely on tagging the imaged object with fluorescent chromophores or on other microscopy techniques that do not exploit fluorescence but are much less sensitive. A team from the Department of Chemistry and Chemical Biology at Harvard has now developed an alternative approach known as stimulated emission microscopy, incorporating experimental techniques previously used in other multiphoton microscopies. The sensitivity of the method was demonstrated in applications including label-free microvascular imaging and monitoring lacZ gene expression with a chromogenic reporter. The technique is orders of magnitude more sensitive than absorption, is not subject to interference from other chromophores in the sample, and is amenable to three-dimensional sectioning. Importantly, all molecules are potential targets for stimulated emission microscopy, so it can be used to image non-fluorescing substances such as haemoglobin previously inaccessible to super-resolution microscopy. Imaging beyond the diffraction limit — to resolve tiny features in cells, for example — has had to rely on tagging the imaged substance with fluorescent chromophores or other techniques that are much less sensitive, like absorption. The use of stimulated emission (a property, unlike fluorescence, which all molecules can have) is now reported; sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, and fluorescence is not used. Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging 1 , 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 decay 3 . 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.