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Published August 2014 | Published
Journal Article Open

Label-free photoacoustic nanoscopy


Super-resolution microscopy techniques—capable of overcoming the diffraction limit of light—have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules.

Additional Information

© 2014 The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. Paper 140197R received Apr. 28, 2014; revised manuscript received Jul. 3, 2014; accepted for publication Jul. 11, 2014; published online Aug. 7, 2014. We thank the Tissue Culture Support Center and Yu Shrike Zhang for culturing melanoma cells and fibroblasts. We thank Kate Nelson and Howard Wynder from the Nano Research Facility for technical help with atomic force microscopy and transmission electron microscopy imaging, and sample preparation. This work was sponsored in part by National Institutes of Health grants DP1 EB016986, R01 CA186567, R01 EB016963, R01 CA157277, and R01 CA159959. L.V.W. has a financial interest in Microphotoacoustics Inc. and Endra Inc., which, however, did not support this work. K.M. has a financial interest in Microphotoacoustics Inc.

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