Wavelength-Dependent Differential Interference Contrast Microscopy: Multiplexing Detection Using Nonfluorescent Nanoparticles Yong Luo, Wei Sun, Yan Gu, Gufeng Wang, and Ning Fang* Ames LaboratorysUnited States Department of Energy and Department of Chemistry, Iowa State University, Ames, Iowa 50011 The wavelength dependence of plasmonic nanoparticles’ contrasts in differential interference contrast (DIC) mi- croscopy has been exploited previously for unambiguous identification and dynamic tracking of these nanoprobes in complex environments (Anal. Chem. 2009, 81, 9203- 9208). In the present study, the suitability of multiplexing detection in DIC microscopy was investigated systemati- cally with 19 kinds of nanoparticles of different materials and/or sizes. A unique DIC contrast spectrum was found for each kind of nanoparticle. Multiplexing detection was accomplished by measuring DIC contrasts at a minimum of two specific illumination wavelengths. The main ad- vantages of DIC microscopy for multiplexing detection over other nonfluorescence techniques, such as dark field microscopy and surface-enhanced Raman scattering, were demonstrated by differentiating four kinds of nanopar- ticles on the cell membrane while providing high-contrast images of both the nanoprobes and cell features. Multianalyte detection is desirable when developing biomedical and environmental assays. In particular, a number of multiplexing detection schemes based on spectroscopic analysis have been reported. 1-4 The most commonly used scheme employs organic dyes for fluorescence-based detection, which has proved its power in gene sequencing. 5,6 Luminescent quantum dots have been shown to be an excellent alternative to organic dyes for long-term multicolor imaging of live cells. 7 Raman spectroscopy is another suitable tool thanks to the narrow bandwidths of Raman fingerprints. 8-13 Nonfluorescent nanoparticles have emerged with increasing importance as nonbleaching probes for a variety of optical microscopies. 14 In dark-field microscopy, the Rayleigh scattering of light by nanoparticles results in bright spots over a dark background. Dark-field microscopy-based multiplex identification of biomarkers was demonstrated by Yu et al. using gold nanorods of three different aspect ratios 15 and by Hu et al. using silver nanospheres and gold nanorods. 16 Differential interference contrast (DIC) microscopy, which generates image contrasts for optical path length gradients in the sample, has been shown to have several advantages over other optical techniques in nanoparticle imaging. DIC microscopy allows the use of full objective and condenser apertures, thus providing the user with the highest lateral resolution and the shallowest depth of field. It also enables simultaneous imaging of nanopar- ticles and the cellular environment, eliminating the need of switching microscopy modes to correlate the probes and cell features. 17 Recently, we showed that single gold and silver nanoparticles can be selectively imaged in DIC microscopy by varying the illumination wavelength. 18 The dependence of the nanoparticles’ DIC contrasts on the illumination wavelength, material, size, and shape implies a new multiplexing detection scheme featuring DIC microscopy. In this study, we systematically investigated 19 types of nanoparticles: gold nanoparticles of 6 sizes, silver nanoparticles * To whom correspondence should be addressed. E-mail: nfang@iastate.edu. Phone: (515) 294-1127. Fax: (515) 294-0105. (1) Bake, K. D.; Walt, D. R. Annu. Rev. Anal. Chem. 2008, 1, 515–547. (2) Kunimura, S.; Kawai, J. Anal. Chem. 2007, 79, 2593–2595. (3) Phillips, T. E., Bargeron, C. B., Benson, R. C., Carlson, M. A., Fraser, A. B., Groopman, J. D., Ko, H. 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