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Creating attoliter detection volume by microsphere photonic nanojet andf l uorescence depletionCuifang Kuanga, ? , Yong Liu a,b , Xiang Hao a , Ding Luo a , Xu Liu aaState Key Lab of Modern Optical Instrumentation, Zhejiang University, Hangzhou 310027, ChinabDepartment of Physics, Zhaoqing University, Zhaoqing 526061, Chinaa b s t r a c t a r t i c l e i n f oArticle history:Received 22 August 2011Accepted 7 October 2011Available online 19 October 2011Keywords:Sub-diffractionPhotonic nanojetCylindrical polarizationFluorescence depletionIn this paper, a novel method is presented for creating a 3-dimensinal sub-diffraction effective observationvolume based on microsphere photonic nanojet and f l uorescence depletion effect. Using microsphere toachieve photonic nanojet focusing effect, the proposed method applies the radially polarized plane wave toexcite f l uorescence and the azimuthally polarized beam to obtain f l uorescence depletion f i eld. The effectivedetection volume of photonic nanojet can reach 0.002 μm 3 (2aL). Compared with conventional confocal mi-croscopy, this effective detection volume represents a reduction of almost 2 orders of magnitude. With sim-ple conf i guration based on cost-effective microspheres, the proposed method is theoretically proved to be apotential tool for the f l uorescence correlation spectroscopy (FCS) to have large analysis range and to investi-gate single molecule at high concentrations.Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.1. IntroductionThe detection and analysis of single molecule is very signif i cant inchemical, biochemical and biophysical applications, since it can reveallarge amount of information about dynamics and characteristics ofthe sample, such as reaction kinetic, molecules diffuse, and transientstates. Recently, many methods have been presented to study singlemolecule. Among them, Fluorescence Correlation Spectroscopy(FCS) is a powerful one, because it can in principle reveal informationabout all molecular processes that induce the change of the f l uores-cence intensity [1]. However, in practice, the diffraction limit of gen-eral optical instruments will restrict the analytical volume and thechoice of f l uorescence marks in FCS. The minimum detection volumethat can be produced by general confocal optical system is approxi-mately 200 aL and therefore require f l uorophore concentrations ofor even below nanomolar range to isolate individual molecules. Inorder to overcome such limits, two strategies have been proposed[2–11]. One is to shape the excitation beam by total internal ref l ectionf l uorescence microscopy, 4Pi microscopy, stimulated emission deple-tion microscopy (STED), or the interference effect on mirror. Theother one is to introduce photonic structures, such as paraboloid col-lector, solid immersion lens, microsphere, nanof l uidic channel, scan-ning near-f i eld optical microscopy (SNOM), and nanometricapertures.Due to the simple structure and low cost of microsphere, the FCSbased on latex microsphere has attracted many attentions. When amicrosphere is illuminated by plane wave, a special beam called“photonic nanojet” with high intensity, subwavelength transverse di-mensions and low divergence can be generated [12]. However, its di-mension along the optical axis is typically 2–3 μm. This limits theability of photonic nanojet to provide three-dimensional sub-wavelength light conf i nement. Although some studies have provedthat the property of photonic nanojet depends on parameters includ-ing the refractive index ratio of microsphere and background, thewavelength, intensity distribution, polarization state of illuminationlight, the diameter of microsphere and the refraction index distribu-tion of microsphere, the photonic nanojet makes it feasible for the ex-citation volume in FCS to break out diffraction limit. When themicrosphere is illuminated by a tightly focused Gaussian beam, theeffective detection volume is reduced by one order of magnitudebelow the diffraction limit and a 5-fold gain in the f l uorescence rateper molecule is obtained [9]. Combined with two-photon excitation,the signal from single molecule can be enhanced up to 10 times bya 3 μm diameter latex sphere while adding almost no photo-luminescence background [13]. However, compared with the FCSbased on SNOM, STED, nanof l uidics and nanoapertures, the effectivevolume of photonic nanojet must be squeezed to make the FCS appli-cable under high concentration conditions with large analysis range.In this paper, the FCS based on photonic nanojet and STED effect isproposed. The cylindrical polarization state is used to control the pho-tonic nanojet [14]. If we only consider the shape of focal spot, the mi-crosphere is basically similar to a high numerical aperture (NA)objective lens. If the microsphere is illuminated by linearly polarizedlight, the photonic nanojet generated will not be central symmetric inthe focal plane, while the radially polarized illumination light resultsin a squeezed nanojet, which has been proved in Ref.[15]. For theOptics Communications 285 (2012) 402–406? Corresponding author.E-mail address: cfkuang@zju.edu.cn (C. Kuang).0030-4018/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.doi:10.1016/j.optcom.2011.10.021Contents lists available at SciVerse ScienceDirectOptics Communicationsjournal homepage: www.elsevier.com/locate/optcom利用微球、光子、纳米射流和荧光损耗技术，建立起一套完整的检测系统