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1Institute of Photonics, Department of Optics and Optical Engineering, University ofScience and Technology of China, Hefei, Anhui 230026, China. 2School of Science,Southwest University of Science and Technology, Mianyang, Sichuan 621010, China.3State Key Laboratory of Modern Optical Instrumentation, College of Optical Scienceand Engineering, Zhejiang University, Hangzhou 310027, China. 4Department ofPolymer Science and Engineering, iChEM, University of Science and Technologyof China, Hefei, Anhui 230026, China. 5Key Laboratory of Environmental Opticsand Technology, Anhui Institute of Optics and Fine Mechanics, Chinese Academyof Sciences, Hefei 230031, China. 6Center for Fluorescence Spectroscopy, Depart-ment of Biochemistry and Molecular Biology University of Maryland School of Med-icine, 725 West Lombard St., Baltimore, MD 21201, USA.*These authors contributed equally to this work.†Corresponding author. Email: dgzhang@ustc.edu.cn

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Label-free surface-sensitive photonic microscopywith high spatial resolution using azimuthalrotation illumination

Yan Kuai1*, Junxue Chen2*, Xi Tang1, Yifeng Xiang1, Fengya Lu1, Cuifang Kuang3, Liang Xu3,Weidong Shen3, Junjie Cheng4, Huaqiao Gui5, Gang Zou4, Pei Wang1, Hai Ming1, Jianguo Liu5,Xu Liu3, Joseph R. Lakowicz6, Douguo Zhang1†

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Surface plasmon resonance microscopy (SPRM) with single-direction illumination is a powerful platform for bio-medical imaging because of its wide-field, label-free, and high-surface-sensitivity imaging capabilities. However,two disadvantages prevent wider use of SPRM. The first is its poor spatial resolution that can be as large asseveral micrometers. The second is that SPRM requires use of metal films as sample substrates; this introducesworking wavelength limitations. In addition, cell culture growth on metal films is not as universally available asgrowth on dielectric substrates. Here we show that use of azimuthal rotation illumination allows SPRM spatialresolution to be enhanced by up to an order of magnitude. The metal film can also be replaced by a dielectricmultilayer and then a different label-free surface-sensitive photonic microscopy is developed, which has morechoices in terms of the working wavelength, polarization, and imaging section, and will bring opportunities forapplications in biology.

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INTRODUCTIONAnalysis of the localization and dynamics of molecules and eventsoccurring near the surfaces of cells is very important in cell biology.For example, the plasma membrane represents the barrier that allmolecules must cross to enter or exit a cell, and large numbers ofbiological processes occur at or near this plasma membrane. Theseprocesses are difficult to image using traditional epifluorescence orconfocal microscopy techniques because image details near the cellsurface can easily be obscured by the fluorescence that originatesfrom the internal contents of the cell. In contrast, total internalreflection fluorescence microscopy (TIRFM) is particularly suitablefor imaging of plasma membranes because of its use of evanescentwaves for selective illumination and excitation of fluorophores with-in a restricted specimen region (1–4). The imaged section is thinner(at 100 to 200 nm) than the sections that are obtained with mostoptical sectioning techniques, such as confocal (5) or multiphoton mi-croscopy (6), where temporal resolutions are also limited by the needfor confocal scanning. Following the rapid development of plasmonicand nanophotonic technologies, a label-free surface-sensitive photonicmicroscopy technique, surface plasmon resonance microscopy (SPRM),has been developed to image the surfaces or interfaces of cells (7–9). Sur-face plasmons (SPs) can be seen as one kind of evanescent waves thatexist at the metal-dielectric interface, such as a metal sheet in air. Theyare also localized at the near-field region (such as 100 to 200 nm or

less) on the substrate. They are more sensitive to environmental changeson surfaces than the evanescent waves that are used in TIRFM (10, 11).The localized electric field of SPs is also stronger than that of the ev-anescent waves used in TIRFM. So, SPs can be used to develop highlysensitive label-free surface imaging methods such as SPRM.

When compared with the surface-sensitive microscopy techniquesof single-molecule TIRFM, SPRM offers the advantageous capabilitytomonitor individual binding events continuouslywithout the need toaccount for photo bleaching or blinking of the fluorophores. There-fore, in recent years, SPRM has found numerous applications in thestudy of biological targets, including cells (12), bacteria (13), viruses(7), DNA molecules (14), and proteins (15); the local electrochemicalreactions of heterogeneous surfaces (16, 17); and the catalytic reactionsof nanomaterials (18, 19). However, SPRM has obvious disadvantagesthat prevent the general use of this interesting technique. The first dis-advantage is its poor spatial resolution, which is caused by the SPspropagation length (which is much larger than the diffraction limit)along its propagating direction (12–19). The second disadvantage isthat only noble metal films can be used as substrates for the cells inSPRM. For these metal films, which are most commonly gold or silverfilms, the SPs act as the imaging source, but the surface plasmonresonance (SPR) angle will increase as the wavelength of the incidentbeam decreases (20). However, the numerical aperture (NA) of theobjective lens that is used in SPRM is finite, so SPRM can only uselong-wavelength excitation beams at wavelengths such as 633 or671 nm, which will certainly limit the potential applications of thishigh-surface-sensitivity imaging technique if shorter light wave-lengths [e.g., ultraviolet (UV) light] were needed in some of these ap-plications. In addition, cell cultures are more commonly grown on aglass substrate than on a metal film, which is another reason whyTIRFM ismore popular in cell biology applications than SPRM. In thiswork, to remove these two disadvantages, a galvanometric scanning sys-tem is adopted for use in the SPRM to realize azimuthal rotation illu-mination (ARI) for incidence angle scanning and azimuthal averaging,which combine to greatly enhance the spatial resolution of SPRM. Fur-thermore, we demonstrate a label-free surface-sensitive photonic

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microscopy method called Bloch surface wave microscopy (BSWM)with ARI, which uses the Bloch surface waves (BSWs) as the illumina-tion sources to image the objects on the surface. BSW can be seen as thedielectric analog of SP and has the same merits, such as surface sensi-tivity and electric field enhancement. BSW also has some uniquefeatures that make it different from the SP, such as polarization. BSWMuses an all-dielectricmultilayer (similar to the glass) for its substrate andhas no limitations in terms of working wavelength.

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RESULTSConfiguration for label-free surface-sensitivephotonic microscopyIn this study, we demonstrate and compare two surface-sensitive pho-tonic microscopy methods. The first is SPRM based on SPs on a thinsilver film, and the second is BSWM based on BSWs on a dielectricmultilayer. Like TIRFM, SPRM and BSWMare only sensitive to objectswithin the near-field region of the substrate, but not to all the objectsplaced on the substrate; in other words, these methods focus on theobjects (e.g., themembrane proteins) within the evanescent field (BSWsor SPs) rather than those within the entire cell, so they can be calledsurface-sensitive photonic microscopy methods. Figure 1A shows aschematic diagram of the surface-sensitive photonic microscopy setup,in which a polarizer and a half-wave plate are used to control the po-larization orientation of an incident laser beam at 635 nm. Using a pairof scanning galvanometers and a focusing lens, the expanded and col-limated laser beam can be focused on any point in the back focal plane(BFP) of the objective (100×,NAof 1.49). The expanded and collimatedbeamwill then exit the objective and strike the substrate at a given angle

Kuai et al., Sci. Adv. 2019;5 : eaav5335 29 March 2019

of incidence (q). The beam that is reflected from the substrate will thenbe collected by the same objective and imaged on a scientific com-plementary metal-oxide semiconductor (sCMOS) array detector usinga tube lens. Another polarizer was inserted before the detector to blockthe light that has not excited the SPs but has just been reflected by themetal film. Figure 1B shows the illumination configuration. When thefocused spot was fixed at a specific position on the BFP, a collimatedbeam propagated outward from the objective at a particular angle(q no rotating beam). If this angle (q) is equal to the SPR angle, thenthe setup operates as the conventional SPRM (12–19) in which theexcited SPs will propagate on the metal film in an azimuthal direc-tion (at the angle φ). For comparison with the proposed technique,the conventional microscopy is named SPRM with single-directionillumination (SPRM-SDI) here. In the proposed technique, the mo-tion of the scanning galvanometers from which the incident beamwill be reflected is controlled, thus allowing the focused spot to orbit(rotate) on the BFP of the objective to form a circular orbit. The exitangle (q) of this rotating beam can be controlled based on the radius(r) of the circular orbit. If the exit angle (q) is equal to the SPR angle,then SPs can be excited and can propagate on the metal plane at allazimuthal angles (φ) during the orbit of the focused spot [note herethat, at the azimuthal direction (φ = 0) that is parallel to the incidentpolarization, SPs cannot be excited because the incident beam hasS polarization relative to the plane of incidence; in S polarization, thepolarization orientation is perpendicular to the plane of incidence](20). Here, we call this proposed optical configuration SPRM withARI (SPRM-ARI). The use of ARI allows incidence angle scanningand azimuthal averaging to be conducted automatically by the detec-tor (camera) to enable capture of the final image. In the following

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Fig. 1. Schematic of the experimental setup and the substrates. (A) Experimental setup. (B) Illustration of SDI and ARI. BFP, back focal plane. When the laser beam isfocused on the BFP of the objective and this focal spot is fixed, it is then the SDI. When this focal spot is rotating and forms a ring on the BFP, it is then the ARI. (C) Thinsilver film–coated glass operating as the substrate for SPRM. (D) Dielectric multilayer PBG structure operating as the substrate for BSWM.

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work, the differences in the surface imaging performances of SPRM-SDI (conventional SPRM) and SPRM-ARI will be investigated anddiscussed.

For SPRM, a 45-nm-thick silver thin film coated on a glass sur-face was used as shown in Fig. 1C, where SPs can be excited at themetal-air interface. A dielectric multilayer that supports BSWs willalso be proposed to replace the silver thin film and enable the dem-onstration of another type of label-free surface-sensitive photonicmicroscopy, BSWM, which uses the same setup that is shown inFig. 1 (A and B). The dielectric multilayer photonic band gap (PBG)structure, where BSWs can be excited on the top surface, is shown inFig. 1D (21). The thicknesses of each dielectric layer and total numberof layers (14 layers) are exactly shown in Fig. 1D. Details of the fabri-cation process of the dielectric multilayer are described in the Materialsand Method section. BFP images of the silver thin film, the dielectricmultilayer, and the bare glass (substrate for TIRFM) are shown infig. S1, which verify the excitation of the SPs and BSWs on the firsttwo substrates, respectively (22). The angle-dependent excitationspectrum and the field characteristics of the SP and the BSW weresimulated as shown in fig. S2. The electromagnetic fields of both theSPs and the BSWs decay exponentially from the substrates and thusonly penetrate to depths of approximately 100 to 200 nm into thesample medium; this determines the section that can be imaged usingeach microscopy method and plays an important role in studiestoward the visualization of the spatiotemporal dynamics of moleculeslocated at or near the cell surface. Similar to the TIRFM, the imagedsection is also thinner (at 100 to 200 nm) when compared with thatobtained by most optical sectioning techniques, such as confocal ormultiphoton microscopy (5, 6).

For simplicity, in this investigation, polymer nanowires, microwires,and nanospheres were used as objects to test the surface-sensitive im-aging performances of the proposed photonic microscopes, althoughthey can certainly be replaced with real cells in practical applications.The nanowire is approximately 150 nm in diameter, while the di-ameter of the microwire is approximately 3 to 4 mm. The commercialnanospheres are approximately 50 nm in diameter. Scanning electronmicroscopy (SEM) images of a nanowire, a microwire, and a nano-sphere are shown in fig. S3.

Enhancing the spatial resolution of SPRM by using ARIFigure 2A shows a bright-field image of a nanosphere with a diam-eter of approximately 50 nm that is placed on the Ag film. This singlenanosphere is consistent with all previously reported works in that itproduces a wave-like pattern with parabolic tails on the image (Fig. 2B)acquired using conventional SPRM (SPRM-SDI) (7, 8, 13, 15, 18). Boththeoretical (23, 24) and experimental (18, 25, 26) investigations haveindicated that this wave-like pattern is due to the scattered SPs gen-erated by the nanosphere. This pattern can be regarded as the pointspread function (PSF) of the SPRM-SDI (27). The tails of thesecurved fringes are as long as several micrometers (Fig. 2D) in the direc-tion of plasmon propagation (along dashed line 1), which is consistentwith the results reported in (7), (18), and (25); the spatial resolution ofthe SPRM-SDI along the plasmon propagation direction is thus quitepoor and is of the same order as the SP propagation length on the silver-air interface at an incident wavelength of 635 nm (of micrometer scale)(7, 25, 27).

There are three irregular bright spots at the nanosphere’s position(Fig. 2B), and the distance between any two adjacent spots is approx-imately 460 nm (Fig. 2E). This distance can thus be regarded as the

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spatial resolution of the SPRM-SDI in the direction along dashedline 2. Therefore, from the pattern shown in Fig. 2B, we can concludethat the spatial resolution of the SPRM-SDI is very poor and that itdiffers along the different spatial directions; this will certainly induceartefacts in the images, particularly when the object to be imaged hasa complex shape. For example, as shown in Fig. 2G, when a windingnanowire is placed on the Ag film to be imaged, multiple fringes andscattering noise appear around the winding nanowire in the SPRM-SDIimage (Fig. 2H); this makes it difficult to recognize the nanowire if itsshape was not known before the measurement. This winding nanowireis similar to a DNA molecule, which is anisotropic. Consequently, thescattering of propagating SPs by a DNA molecule or a winding nano-wire is dependent on the orientation of the DNAmolecule or nanowirerelative to the plasmonic wave propagation direction, and this thendetermines the contrast and scattering patterns of the resulting SPRM-SDI images (14). This orientation is random and is in several directions,similar to the winding nanowire shown in Fig. 2G, so many irregularfringes and noise are contained in the SPRM-SDI image (Fig. 2H).

However, in the image of the single nanosphere acquired using theproposed SPRM-ARI, this wave-like pattern disappears and only twovery small sized, closed bright spots (Fig. 2C) appear. The reason forthis phenomenon can be explained as follows and will be analyzedwith a two-dimensional model. With ARI, SPs can be excited alongmultiple azimuthal directions on the silver surface, meaning that thefringes or tails induced by SPs scattering can then be azimuthally aver-aged or homogenized. As a result, the nanosphere’s location appearsto be much brighter than the other areas, which leads to the improvedspatial resolution. The reason for the appearance of two closed brightspots rather than a solid bright spot can be explained on the basis ofthe PSF for surface plasmon–coupled emission microscopy (SPCEM)(28, 29). As reported in (28, 29), a fluorescent nanoparticle on a thinAg film will form as a doughnut-shaped spot on the imaging plane ofthe SPCEMbecause the coupled emission has the same polarization(P polarization, where the orientation of the polarization is withinthe plane of incidence) in all the azimuthal directions and occurs ata single axial angle. This doughnut-shaped spot can be regarded asthe PSF of the SPCEM. The principle is the same in the case of thenanosphere on the Ag film. The azimuthally rotating illuminationwill cause SPs to be excited on the Ag films in most azimuthal di-rections when scattered by the nanosphere. Similar to the coupledemission from the fluorescent spots, the light scattered by this nano-sphere will also form a doughnut-shaped spot on the SPRM-ARI im-aging plane. In this work, a polarizer was inserted in front of thecamera, so that the doughnut-shaped spot was split into two brightspots, as shown in Fig. 2C. This splitting is the same behavior thatwould be observed when a polarizer is used after the radially or azi-muthally polarized beam, where the cross section of this doughnut-shaped beam would also split into two spots (30).

The two closed bright spots can be regarded as the PSF of the SPRM-ARI here (29). When the patterns shown in Fig. 2 (B and C) are com-pared, it is clear that the spatial resolution of the SPRM-ARI is muchbetter than that of the SPRM-SDI. The two closed bright spots are ap-proximately 370 nm in size (peak-to-peak distance; Fig. 2F, line 3), sowe can estimate that the spatial resolution of the SPRM-ARI is higherby up to approximately an order of magnitude along the plasmonpropagation direction when compared with that of the SPRM-SDI.The high spatial resolution of the SPRM-ARI is advantageous in re-cognizing complex samples, e.g., the winding nanowire that has beenresolved as shown in the SPRM-ARI image (Fig. 2I).

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It should be noted here that the rotation speed of the focused spoton the BFP can be as fast as approximately 1 ms per circle, meaningthat an SPRM-ARI image can be acquired in only 1 ms (this imageacquisition speed is also dependent on the sCMOS response speed,and the full frame rate of the sCMOS used here is 100 frames persecond). In our experiment, the rotation speed is set at 10 ms, whichis faster than the speed of the differential plasmonic imaging tech-nique. In that technique, the substrate is moved laterally by 500 nm;one image is captured before the lateral movement, and another isthen captured after the movement. The subtraction of one imagefrom the other removes the background interference patterns, and

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the final differential SPRM image is thus obtained (18). Thesemechanical movements and image processing steps will reduce thespeed with which the final SPRM images are obtained.

Mechanism of spatial resolution enhancement using ARIThe SPRM-SDI image of a nanosphere can be modeled and simulatedvia a three-dimensional numerical method such as the finite elementmethod or the finite-difference time-domain method. However, as re-ported in (18), the wave-like patternwith the parabolic tails shown in theSPRM-SDI image can be described using a simplified two-dimensionalmodel. In our work, using this simplified model, the mechanism for

Fig. 2. Images acquired using SPRM-SDI and SPRM-ARI. The Ag film operating as the substrate. A nanosphere with diameter of about 50 nm (A to C) and a windingpolymer nanowire (G to I) were placed on the Ag film. (A and G) Bright-field images. (B and H) Corresponding images acquired using the SPRM-SDI. (C and I)Corresponding images acquired using the SPRM-ARI. (D to F) Intensity profiles along the dashed lines 1, 2, and 3, respectively. On (E) and (F), the peak-to-peak distancesare about 460 and 370 nm, respectively. The scale bars on (A) and (G) are applicable to (B) and (C) and (H) and (I), respectively. The incident angle is fixed at q = 43° forexcitation of the SPs.

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spatial resolution enhancement by ARI-induced incidence anglescanning and azimuthal averaging can be demonstrated. In thismodel, the excited SPs can be described as a planar wave propagatingin one direction, which can then be written as

U1ðx; y;φÞ ¼ Ae�iðkcosðφÞxþksinðφÞyÞ ð1Þ

where φ is the propagation direction of the planar wave, k is the wave-number, and A is the amplitude of the planar wave.

Because the nanosphere is very small when compared with thewavelength of the SP, it can be approximated as a point scatter thatcreates a circular wave with an origin at the nanosphere and is giv-en by (18)

U2ðx; yÞ ¼ Be�are�ikrr ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffix2 þ y2

pð2Þ

Here, B is the amplitude of the scattered wave, depending on thescattering strength and the amplitude of the plan wave, and a is thedamping constant of the scattered wave. The term e− ar representthe decay of the scatter waves, and in the following calculation, thedamping constant is selected as 0.5. The SPRM-SDI image can thenbe described as the superposition of the planar and circular waves.

Uðx; y;φÞ ¼ U1ðx; y;φÞ þ U2ðx; yÞ¼ Ae�iðkcosðφÞxþksinðφÞyÞ þ Be�are�ikr ð3Þ

The intensity of the image is then given by

jUðx; y;φÞj2 ¼ jAj2 þ jBj2e�2ar þ 2ABe�ar½cosðkcosðφÞxþ ksinðφÞy � krÞ� ð4Þ

The final terms in the brackets produce an interference patternand a long tail, as shown in Fig. 3 (A to C), where the planar wavepropagation direction (φ) values are defined as 0°, 45°, and 90°, re-spectively. The patterns show that the orientation of the parabolictail rotates with the change in the angle (φ).

In contrast, in SPRM-ARI, the propagation directions of the planarwave (φ) range from 0° to 360° at high speed to obtain the final imagebecause of the use of ARI, so the intensity of the images capturedusing SPRM-ARI can be described using Eq. 5

Iðx; yÞ ¼ ∫φ¼2p

φ¼0jUðx; y;φÞj2dφ ð5Þ

The calculated image is shown in Fig. 3D, in which the parabolictails have been averaged or removed, and only a single bright spot,similar to the Airy spot for conventional microscopy, is obtained.These simulated images verify that the spatial resolution of SPRMcan be greatly enhanced by the introduction of ARI. A more rigorouscalculation of the SPRM-SDI and SPRM-ARI images based on thetheory reported in (23, 24) was also performed as shown in the Sup-plementary Materials, which gives out the same conclusion. ARI willinduce the incidence angle scanning and azimuthal averaging andthen enhance the spatial resolution of the SPRM.

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Replacing the silver film with a dielectric multilayer todevelop BSWMAs described earlier, a thin metal film must be used as the substratefor the objects under study (such as cells) in SPRM-SDI and SPRM-ARI. It is well known in cell biology that the techniques used to culturecells on glass substrates are more mature and generally applicable thanthose for growth on metal films. On the basis of our experiences insurface wave–related experiments, a thinmetal film is not as stable orrobust as a dielectric film, it will be damaged a little after each use andcannot be used for many times. Therefore, in the following experi-ment, we will demonstrate that the metal film can be replaced with adielectric multilayer (in which the top layer is SiO2, i.e., the samematerial as glass) to develop a new type of label-free surface-sensitivephotonic microscopy: BSWM. BSWs can be excited using this di-electric multilayer structure. This dielectric multilayer is much stableand can be used formany times in experiments. Similar to SPs, BSWsare also electromagnetic surface waves and are excited at the interfacebetween the dielectric multilayer and the surrounding medium (31).These BSWs can be regarded as the dielectric analog of SPs, whichmeans that they are also highly sensitive to their environment andcan be used to develop a label-free imaging method (32, 33).

In this work, we present a BSWMtechniquewith both SDI (BSWM-SDI) and ARI (BSWM-ARI) for the first time. Different from theBSWM reported in (34), which was developed to image the propa-gation characters (amplitude and phase ) of the excited BSWs, ourBSWM technique uses the BSWs as the optical source to illuminatethe objects (such as the nanosphere or nanowires) placed on the di-electric multilayer. By collecting the scattering signals from theseobjects, we will realize the label-free surface-sensitive imaging ofthese objects, but not imaging of the BSWs themselves. In our exper-imental setup, only one objective was used for both the excitation ofthe BSWs and the following imaging of the objects. For comparisonwith the SPRM, we used the same incident beam for the BSWMexperiments. The setup is the same as that used for SPRM (Fig. 1A).The only difference here is that the silver film has been replaced bythe all-dielectric multilayer, as shown in Fig. 1D. This dielectric multi-layer sustains S-polarized BSWs at a wavelength of 635 nm (fig. S1C).The objects to be imaged are a polymeric nanowire on a polymeric mi-crowire (Fig. 4, A to C) and a winding nanowire (Fig. 4, D to F). Thereare many fringes on the images (Fig. 4, B and D) that were acquiredusing BSWM-SDI, and these fringes obscure the images of the wires.In contrast, the wires are seen very distinctly on the images (Fig. 4, Cand F) that were acquired using BSWM-ARI. Different from the bright-field image shown in Fig. 4A, in Fig. 4C, the nanowire is invisible in theareas where it crosses the microwire because when the nanowire is onthemicrowire and is far away from the dielectricmultilayer surface; thissection of the nanowire cannot be imaged using BSWs. The phenomenaverify that, in a manner same as the SPRM images, BSWM images alsofocus on the targets within the evanescent field (100 to 200 nm) only,rather than those contained in the entire sample, which made it ideallysuited to analyze the localization and dynamics of molecules and eventsoccurring near the plasma membrane. Our results also verify that theARI can enhance the spatial resolution of the BSWM.

Differences of the BSWM-ARI from the SPRM-ARIIn this section, we provide detailed descriptions on the differencesof the BSWM-ARI from the SPRM-ARI, which are both wide-fieldlabel-free surface-sensitive photonic microscopy methods of high spa-tial resolution. First, as shown in Fig. 1, the setups used for the SPRM

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Fig. 3. Simulated images captured using SPRM-SDI and SPRM-ARI. The superposition of a planar wave (the illumination source) and a circular wave caused byscattering from a nanosphere led to the image shape that was observed in SPRM. In (A) to (C), the planar wave is propagating along a single direction defined as φ = 0°,45°, and 90°, respectively, which corresponds to the illumination source used in SPRM-SDI. In (D), the propagation direction of the planar wave rotates from 0° to 360°,similar to the illumination source used in SPRM-ARI. The simulated area had dimensions of 6 mm by 6 mm.

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Fig. 4. Images acquired using BSWM-SDI and BSWM-ARI working with S-polarized BSWs. The dielectric multilayer operating as the substrate (Fig. 1D) sustainsS-polarized BSWs at a wavelength of 635 nm. (A and D) Bright-field images. (B and E) Corresponding images acquired using BSWM-SDI. (C and F) Correspondingimages acquired using BSWM-ARI. In (A) to (C), a polymeric nanowire was located on a polymeric microwire. These two straight wires were on the dielectric multilayer.In (D) to (F), a winding polymeric nanowire was placed on the dielectric multilayer. The scale bars shown on (A) and (D) are applicable to (B) and (C) and (E) (F),respectively. The angle of incidence was fixed at q = 47°, at which the BSWs can be excited. Different from the bright-field image (A), in the BSWM-ARI image (C),the nanowire is invisible in the areas where it crosses the microwire (labeled with the oval), showing the surface-sensitive imaging capability of the BSWM-ARI.

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and BSWM are the same, and the only real difference is the materialused for the substrate. In SPRM, a thin metal film such as a gold orsilver film was used. In BSWM, an all-dielectric multilayer structurewas used, where the top layer is SiO2, that is, the same material as thecommonly used glass. Glass substrates are commonly known to beused almost universally for applications in cell biology or biomedicaloptics, rather than as metal substrates. The technique used for surfacefunctionalization of the glass (SiO2) is both generally applicable andmature. Metal films may sometimes alter the conformation of theobjects to be imaged or affect the measurements because of the exis-tence of metal atoms on the substrate. Metal films may also presentcorrosion and oxidation rapidly in certain liquid or gaseous environ-ments, which will certainly affect the surface roughness of the thinmetal film and thus affect the image quality of the SPRM. However,metal films are conductive, so SPRM can be used to image local electro-chemical currents (16). This type of imaging can also be realized if thedielectric multilayer is coated using a conductive dielectric layer suchas indium tin oxide.

The second difference is that SPs can only be sustained in P polar-ization. BSWs can be sustained in both P polarization (Fig. 5) and Spolarization (Fig. 4), depending on the thickness and the refractiveindex of each of the dielectric layers (35). In our experiments, anotherdielectric multilayer (with structural parameters that are shown inFig. 5A) was used as the substrate for BSWM-ARI, where P-polarizedBSWs can be excited at the interface between the air and the top SiO2

layer (35). The resonant dip in the angle-dependent reflection curve(Fig. 5B) and the dark arcs in the reflected BFP image of this dielectric

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multilayer (Fig. 5C) verified the existence of the P-polarized BSWs (22).When a single polymeric nanosphere with a diameter of 50 nm wasplaced on this dielectric multilayer (Fig. 5D), it could be imaged anddetected, as shown in the BSWM-ARI image in Fig. 5E, meaning thatBSWM can also work in the P-polarized mode.

Third, the incident wavelength and angle requirements of SPRMand BSWM are different. In SPRM, SPs can be sustained at the metal-air (or metal-water) interface at wavelengths ranging from UV to thenear-infrared light band. However, because of the limited width of thePBG for any given dielectric multilayer, BSWs can only be sustainedwithin a limited range of wavelengths (21). As described earlier, bothSPRM and BSWM are based on the use of an oil-immersed objective,which is used for the excitation of the SPs and BSWs, in a mannersimilar to the well-known Kretschmann configuration (20). The NAof the objective is limited, and the most commonly used value is NA =1.49, which corresponds to a collection angle of up to 80° [arcsine(1.49/1.515) = 80°]. For SP excitation, a shorter incident wavelengthwill result in a larger SPR angle. For example, at a gold-water interface,the SPR angle at a wavelength of 633 nm is approximately 72°, but thisSPR angle will increase to 81° when the incident wavelength is reducedto 561 nm. At shorter wavelengths, SPs cannot be excited using thisobjective, and thus, the SPRM will not work. Most reported SPRMswork at longer wavelengths, e.g., 633 or 671 nm (7, 8, 14, 16, 18). Incontrast, the appropriate design of the PBG of the dielectric multilayerwill allow BSWs to be excited on the dielectric multilayer at both shortand long wavelengths, and the BSW resonance angle can be shifted toa small angle because there are many potential material choices for the

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Fig. 5. BSWM-ARI operating with P-polarized BSWs. (A) The dielectric multilayer operating as the substrate for BSWM. The thickness of each layer is as labeled in theillustrations. The refractive index of the Si3N4 is 2.65 + i0.005, which is higher than that of the Si3N4 used in Fig. 1D. (B) Angle-dependent excitation spectrum. ne is therefractive index of the environment. When ne changes from 1.0 to 1.1, the resonant dip in the P-polarized BSWs will shift, demonstrating the sensing ability of the BSWs.(C) Reflected BFP image of the dielectric multilayer, in which the pair of dark arcs verifies the excitation of the P-polarized BSWs. The double-headed arrow line on (C)indicates the orientation of the incident polarization. A nanosphere was placed on this dielectric multilayer, and (D) shows the bright-field images. (E) Correspondingimage acquired using BSWM-ARI. The scale bar shown on (D) is also applicable to (E). The angle of incidence is fixed at approximately q = 43° for excitation of theP-polarized BSWs, and the incident wavelength is 635 nm.

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all-dielectric multilayer and the thickness of each of these layers can becontrolled precisely. For example, BSWs can even be excited in theUV band with small angles of incidence (36). BSWM therefore pro-vides greater choice of working wavelengths, which can be used toenhance the sensitivity of the photonic microscopy process. For ex-ample, if the biological sample to be imaged has an absorption peakat a short wavelength, e.g., at 532 nm, then the surface-sensitive pho-tonic microscopy operating at this wavelength will be more sensitiveto changes in the sample than the microscopies working at otherwavelengths. Furthermore, light beams at shorter wavelengths are fa-vorable for fluorescence excitation applications because many of thefluorophores used in cell biology can only be excited at short wave-lengths. The experimental setup used for BSWM is the same as thatused for TIRFM, except for the substrate, so BSWM can easily beswitched to TIRFM after inclusion of a fluorescence filter when anillumination beam at a shorter wavelength is used. This means thatboth label-free and label-based surface-sensitive measurementscan be performed using the same setup, which is useful for on-siteobservations.

In our experiments, a third dielectricmultilayer, as shown in Fig. 6A,was used as the substrate for BSWM and could sustain S-polarizedBSWs in water when the incident wavelength was only 532 nm. Tothe best of our knowledge, this wavelength is shorter than all previous-ly reported wavelengths used in SPRM and also verifies that BSWMcan work at short wavelengths and in liquid environments. The black

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arcs shown on the reflected BFP image of this multilayer (Fig. 6B)demonstrate that BSWs can be excited at the interface between the wa-ter and the top SiO2 layer (22). When a nanosphere was placed on thisdielectric multilayer, it could also be imaged or detected on both theBSWM-SDI and BSWM-ARI images (Fig. 6, D and E). The sizes ofthe bright spots on the images captured via BSWM-ARI were also in-vestigated, as displayed in fig. S4. Figure S4 shows that the spot sizesin Fig. 5E and Fig. 6E are approximately 350 and 380 nm, respective-ly. These spot sizes are similar to that shown in Fig. 2C, thus demon-strating that the spatial resolution of BSWM-ARI is similar to that ofSPRM-ARI. While the spatial resolution for both BSWM-ARI andSPRM-ARI is limited by diffraction, these methods offer the meritsof wide-field and label-free imaging capabilities.

As reported in (21), the excitation angle (q) for the BSWs can betuned with the structure dimensions, especially the top dielectric layer.The penetration depth of the BSWs into the top space can be de-

scribed using a function of angle (q), l0=4p*ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðn0*sinqÞ2 � ðneÞ2

qÞ,

where l0 is the wavelength of the incident light in vacuum, n0 is re-fractive index of the substrate, and ne is the refractive index of the me-dium on the upper space, e.g., air or water. Then, the imaged sectionof the surface-sensitive photonic microscopy is determined by thepenetration depth of the BSWs. So the imaged section of BSWM-ARI can also be tuned, as demonstrated earlier. For the three dielectricmultilayers used here, the excitation angles (q) of BSWs are 47°, 43°,

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Fig. 6. BSWM operating in short wavelength and liquid environments. (A) Dielectric multilayer PBG structure operating as the substrate for BSWM. The upperspace is filled with water, and the incident wavelength is 532 nm. The thickness of each layer is as labeled in the illustrations. (B) Reflected BFP image of the dielectricmultilayer, in which the pair of dark arcs verifies the excitation of BSWs at the interface between the water and the top SiO2 layer. The double-headed arrow line on(B) represents the orientation of the incident polarization. A nanosphere was placed on this dielectric multilayer. (C) Bright-field image. (D and E) Correspondingimages acquired using BSWM-SDI and BSWM-ARI, respectively. The scale bar on (C) is applicable to (D) and (E). The angle of incidence was fixed at approximately q = 66° forexcitation of the BSWs in the water.

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and 66°, corresponding to the penetration depths, 105, 194, and 110 nm,respectively, of the evanescent fields, which will be useful for observ-ing the cells in different depths.

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DISCUSSIONBoth SPs and BSWs are well-known phenomena that have beenexploited for sensitive detection of biomolecular kinetics on surfaces.Extending these schemes to microscopy has presented some resolutionissues due to optical wave coupling to either the SPs or the BSWs, bothof which, with point coupling, lead to sets of trailing interference pat-terns. Although it is plausible that some of those imaging artefacts canbe removed by proper software computations, we have performed thistask experimentally by using the ARI (37–39), thus greatly improvingthe spatial resolution of the images. These high-spatial-resolutionimages are obtained without image processing and without mechanicalmovement of the samples for differential processing, as would be usedin SPRM-SDI; thismeans that imaging speed can be increased, whichis helpful during real-time observation. Themechanism of the spatialresolution enhancement by using ARI is analyzed with a simple two-dimensional model.

We have also used an all-dielectric multilayer to replace the com-monly used thin metal film substrates and have developed a new typeof surface-sensitive photonic microscopy called BSWM. BSWM isboth versatile and generally applicable. As demonstrated in this study,in addition to long wavelength operation (e.g., at 635 nm, which iscommonly used in SPRM), BSWM can also operate at shorter wave-lengths (e.g., 532 nm here), which cannot be realized in SPRM. Theoptical properties of the BSWs, such as their polarization states andpenetration depths (corresponding to the thickness of the imaged sec-tion of the microscopy), can be tuned precisely via the thicknesses ofthe materials used for the dielectric layers. For example, BSWMoperation in both P- and S-polarized modes is demonstrated in ourwork, and the thickness of the imaged section for BSWMhas also beentuned from 100 to 200 nm using the structural dimensions. In con-trast, the polarization of SPs is fixed at the P-polarized mode, andthe thickness of the imaged section of the SPRM cannot be tunedvia the thickness of the metal film.

Because they are sensitive to environmental changes on surfaces,both SPs and BSWs have been used to develop the high-performancesensors by monitoring the angular shift of the reflected light from themetal film or dielectric multilayer (10, 11, 32, 33). As far as the sensingfunction is concerned, if an additional CMOS or a quadrant diode wasincluded in our experimental setup to record the optical signals on theBFP of the objective, the angular shift of the reflected light from themetal film or dielectric multilayer can be also monitored (40), thenour setup can also work as a sensing instrument. It is a sensor basedon an objective but not a bulk prism. Thus, we anticipate that thesurface-sensitive photonic microscopy method with ARI will have abroad range of applications in both surface imaging and sensing, par-ticularly for the analysis of the localization and dynamics ofmoleculesand events located at or near cells because of its wide-field, label-free,and high-sensitivity surface imaging capabilities.

MATERIALS AND METHODSExperimental setupA schematic of the setup, which is based on a commercial invertedmicroscopy setup, is shown in Fig. 1A. The laser beam at the wave-

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length of 635 nm comes from a commercial laser diode. The scanninggalvanometer was from Thorlabs Inc., and its scanning speed can becontrolled via software. The objective (100×, NA of 1.49) was fromNikon, Japan. The Neo sCMOS detector was from Andor OxfordInstruments (UK).

Sample preparationThe first dielectric multilayers were fabricated by plasma-enhancedchemical vapor deposition (PlasmaPro System 100, Oxford) of SiO2

and Si3N4 on a standard microscope cover glass (0.17 mm thick) ata vacuumpressure of <0.1mtorr and at a temperature of 300°C. In thiscase, SiO2 was the low (L) refractive index dielectric, and Si3N4 was thehigh (H) refractive index dielectric. These dielectric layers had thick-nesses of 105 and 88 nm, respectively. A total of 14 dielectric layerswere deposited on top of each other. The thickness of the top SiO2

layer was approximately 240 nm. Another two dielectric multilayerstructures were fabricated in the same way.

The microwires and nanowires were fabricated using the electro-spinning technique. The nanowires were electrospun from a 2.0-mlsolution of formic acid that contained 0.6 g of Nylon 6. The micro-wires were electrospun from a 1.7-ml tetrahydrofuran solutionwith 0.5 g of hydrogel D4 (HydroMed D4, a commercially availablepolyurethane-based hydrogel). Both solutions were stocked in a 2-mldisposable syringe. A voltage of 10 kV was applied to a stainless steelneedle with a flat tip from a high-voltage power supply. The feed ratewasmaintained using a syringe pump. The feed rate was 0.1mmmin−1,and the internal diameter of the needle used for fabrication of the nano-wireswas 0.19mm. For themicrowires, the feed rate was 0.2mmmin−1,and the internal diameter of the needle was 0.50 mm. These wires werecollected at a distance of 10 cm from the tip of the needle and thentransferred to the substrates. The microwire was transferred to the sub-strate first and was followed by the nanowire, so that the nanowire wason top of the microwire.

The nonfluorescent polystyrene nanosphere (3050A), which hasa diameter of approximately 46 ± 2 nm, was bought from ThermoFisher Scientific (www.thermofisher.com/order/catalog/product/3050A), and the attachment of these spheres to the dielectric multi-layer and the Ag film was realized using the MET One Aerosol Par-ticle Generator 255 Gen.

SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/3/eaav5335/DC1Fig. S1. Surface waves sustained by Ag film or dielectric multilayer.Fig. S2. Angle-dependent excitation spectra and field characteristics for the SPs and BSWs.Fig. S3. SEM images of the polymeric nanowire and microwire and the nanospheres.Fig. S4. Sizes of the bright spots on images captured using BSWM-ARI.Fig. S5. The model used for a more rigorous calculation.Fig. S6. Simulated SPRM-SDI and SPRM-ARI images based on a rigorous calculation.

REFERENCES AND NOTES1. D. Axelrod, Cell-substrate contacts illuminated by total internal reflection fluorescence.

J. Cell Biol. 89, 141–145 (1981).2. A. L. Mattheyses, S. M. Simon, J. Z. Rappoport, Imaging with total internal reflection

fluorescence microscopy for the cell biologist. J. Cell Sci. 123, 3621–3628 (2010).3. M. W. Allersma, L. Wang, D. Axelrod, R. W. Holz, Visualization of regulated exocytosis with

a granule-membrane probe using total internal reflection microscopy. Mol. Biol. Cell 15,4658–4668 (2004).

4. J. Boulanger, C. Gueudry, D. Münch, B. Cinquin, P. Paul-Gilloteaux, S. Bardin, C. Guérin,F. Senger, L. Blanchoin, J. Salamero, Fast high-resolution 3D total internal reflection

9 of 10

SC I ENCE ADVANCES | R E S EARCH ART I C L E

on March 29, 2020

http://advances.sciencemag.org/

Dow

nloaded from

fluorescence microscopy by incidence angle scanning and azimuthal averaging.Proc. Natl. Acad. Sci. U.S.A. 111, 17164–17169 (2014).

5. J.B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 1995).6. W. Denk, J. H. Strickler, W. W. Webb, Two-photon laser scanning fluorescence microscopy.

Science 248, 73–76 (1990).7. S. Wang, X. Shan, U. Patel, X. Huang, J. Lu, J. Li, N. Tao, Label-free imaging, detection, and

mass measurement of single viruses by surface plasmon resonance. Proc. Natl. Acad.Sci. U.S.A. 107, 16028–16032 (2010).

8. B. Huang, F. Yu, R. N. Zare, Surface plasmon resonance imaging using a high numericalaperture microscope objective. Anal. Chem. 79, 2979–2983 (2007).

9. A. R. Halpern, J. B. Wood, Y. Wang, R. M. Corn, Single-nanoparticle near-infrared surfaceplasmon resonance microscopy for real-time measurements of DNA hybridizationadsorption. ACS Nano 8, 1022–1030 (2014).

10. J. Homola, S. S. Yee, G. Gauglitz, Surface plasmon resonance sensors. Sens. Actuators B 54,3–15 (1999).

11. B. Liedberg, C. Nylander, I. Lunström, Surface plasmon resonance for gas detection andbiosensing. Sens. Actuators 4, 299–304 (1983).

12. W. Wang, Y. Yang, S. Wang, V. J. Nagaraj, Q. Liu, J. Wu, N. Tao, Label-free measuring andmapping of binding kinetics of membrane proteins in single living cells. Nat. Chem. 4,846–853 (2012).

13. K. Syal, R. Iriya, Y. Yang, H. Yu, S. Wang, S. E. Haydel, H.-Y. Chen, N. Tao, Antimicrobialsusceptibility test with plasmonic imaging and tracking of single bacterial motions onnanometer scale. ACS Nano 10, 845–852 (2016).

14. H. Yu, X. Shan, S. Wang, H. Chen, N. Tao, Plasmonic imaging and detection of single DNAmolecules. ACS Nano 8, 3427–3433 (2014).

15. A. M. Maley, Y. Terada, S. Onogi, K. J. Shea, Y. Miura, R. M. Corn, Measuring proteinbinding to individual hydrogel nanoparticles with single-nanoparticle surface plasmonresonance imaging microscopy. J. Phys. Chem. C 120, 16843–16849 (2016).

16. X. Shan, U. Patel, S. Wang, R. Iglesias, N. Tao, Imaging local electrochemical current viasurface plasmon resonance. Science 327, 1363–1366 (2010).

17. X. Shan, X. Huang, K. J. Foley, P. Zhang, K. Chen, S. Wang, N. Tao, Measuring surfacecharge density and particle height using surface plasmon resonance technique.Anal. Chem. 82, 234–240 (2010).

18. X. Shan, I. Díez-Pérez, L. Wang, P. Wiktor, Y. Gu, L. Zhang, W. Wang, J. Lu, S. Wang,Q. Gong, J. Li, N. Tao, Imaging the electro catalytic activity of single nanoparticles.Nat. Nanotechnol. 7, 668–672 (2012).

19. Y. Fang, H. Wang, H. Yu, X. Liu, W. Wang, H.-Y. Chen, N. Tao, Plasmonic imaging ofelectrochemical reactions of single nanoparticles. Acc. Chem. Res. 49, 2614–2624 (2016).

20. H. Raether, Surface Plasmons on Smooth and Rough Surfaces and on Gratings(Springer-Verlag, 1988), pp. 91–116.

21. P. Yeh, A. Yariv, C.-S. Hong, Electromagnetic propagation in periodic stratified media.I. General theory. J. Opt. Soc. Am. 67, 423–438 (1977).

22. D. Zhang, R. Badugu, Y. Chen, S. Yu, P. Yao, P. Wang, H. Ming, J. R. Lakowicz, Back focalplane imaging of directional emission from dye molecules coupled to one-dimensionalphotonic crystals. Nanotechnology 25, 145202 (2014).

23. A. Demetriadou, A. A. Kornyshev, Principles of nanoparticle imaging using surfacePlasmons. New J. Phys. 17, 013041 (2015).

24. A. Demetriadou, The impact of natural modes in plasmonic imaging. Sci. Rep. 5,18247 (2015).

25. H. Yu, X. Shan, S. Wang, N. Tao, Achieving high spatial resolution surface plasmonresonance microscopy with image reconstruction. Anal. Chem. 89, 2704–2707 (2017).

26. H. Yu, X. Shan, S. Wang, H. Chen, N. Tao, Molecular scale origin of surface plasmonresonance biosensors. Anal. Chem. 86, 8992–8997 (2014).

27. W. Wang, Imaging the chemical activity of single nanoparticles with optical microscopy.Chem. Soc. Rev. 47, 2485–2508 (2018).

28. W. T. Tang, E. Chung, Y.-H. Kim, P. T. C. So, C. J. R. Sheppard, Surface-plasmon-coupledemission microscopy with a spiral phase plate. Opt. Lett. 35, 517–519 (2010).

29. Y. Chen, D. Zhang, L. Han, G. Rui, X. Wang, P. Wang, H. Ming, Surface plasmon-coupledemission microscopy with a polarization converter. Opt. Lett. 38, 736–738 (2013).

Kuai et al., Sci. Adv. 2019;5 : eaav5335 29 March 2019

30. Q. Zhan, Cylindrical vector beams: From mathematical concepts to applications.Adv. Opt. Photonics 1, 1–57 (2009).

31. L. Yu, E. Barakat, T. Sfez, L. Hvozdara, J. Di Francesco, H. P. Herzig, Manipulatingbloch surface waves In 2D: A platform concept-based fat lens. Light Sci. Appl. 3, e124(2014).

32. F. Giorgis, E. Descrovi, C. Summonte, L. Dominici, F. Michelotti, Experimentaldetermination of the sensitivity of Bloch surface waves based sensors. Opt. Express 18,8087–8093 (2010).

33. V. Paeder, V. Musi, L. Hvozdara, S. Herminjard, H. P. Herzig, Detection of proteinaggregation with a Bloch surface wave based sensor. Sens. Actuators B 157, 260–264(2011).

34. E. Descrovi, E. Barakat, A. Angelini, P. Munzert, N. De Leo, L. Boarino, F. Giorgis,H. P. Herzig, Leakage Radiation Interference Microscopy. Opt. Lett. 38, 3374–3376 (2013).

35. J. Chen, D. Zhang, P. Wang, H. Ming, J. R. Lakowicz, Strong polarization transformation ofbloch surface waves. Phys. Rev. Appl. 9, 024008 (2018).

36. R. Badugu, J. Mao, S. Blair, D. Zhang, E. Descrovi, A. Angelini, Y. Huo, J. R. Lakowicz, Blochsurface wave-coupled emission at ultra-violet wavelengths. J. Phys. Chem. C 120,28727–28734 (2016).

37. M. D. Koch, A. Rohrbach, Label-free imaging and bending analysis of microtubules byROCS microscopy and optical trapping. Biophys. J. 114, 168–177 (2018).

38. D. Ruh, J. Mutschler, M. Michelbach, A. Rohrbach, Superior contrast and resolution byimage formation in rotating coherent scattering (ROCS) microscopy. Optica 5, 1371–1381(2018).

39. F. Jünger, P. v. Olshausen, A. Rohrbach, Fast, label-free super-resolution live-cellimaging using rotating coherent scattering (ROCS) microscopy. Sci. Rep. 6, 30393(2016).

40. F. Gittes, C. F. Schmidt, Interference model for back-focal-plane displacement detectionin optical tweezers. Opt. Lett. 23, 7–9 (1998).

AcknowledgmentsFunding: This work was supported by NSFC (91544218, 61427818, 11774330, 21574120, and21774115), MOST (2016YFA0200601 and 2013CBA01703), the Science and TechnologicalFund of Anhui Province for Outstanding Youth (1608085J02 and 1608085J01), the LongshanAcademic Talent Research Supporting program of SWUST (17LZX626), and the FundamentalResearch Funds for the Central Universities (WK2340000084 and WK2030380008), AnhuiProvincial Science and Technology Major Projects (18030901005), Anhui Initiative in QuantumInformation Technologies, the foundation of Key Laboratory of Environmental Optics andTechnology of Chinese Academy of Sciences (grant no. 2005DP173065-2019-XX). This workwas also supported by grants from the National Institute of Health (GM125976, GM129561,EB018959, and S10OD019975). The work was partially carried out at the University of Scienceand Technology of China’s Centre for Micro and Nanoscale Research and Fabrication. D.Z.acknowledges Q. Zhan of the University of Dayton, X. Liu and C. Qian of USTC for the helpfuldiscussions. Author contributions: D.Z. conceived the ideas, supervised the work, and draftedthe manuscript. Y.K. carried out the optical experiments. J.C. and X.T. performed the numericalcalculations. All authors discussed the results and contributed to the writing and editing ofthe manuscript. Competing interests: The authors declare that they have no competinginterests. Data and materials availability: All data needed to evaluate the conclusions inthe paper are present in the paper and/or the Supplementary Materials. Additional datarelated to this paper may be requested from the authors.

Submitted 26 September 2018Accepted 7 February 2019Published 29 March 201910.1126/sciadv.aav5335

Citation: Y. Kuai, J. Chen, X. Tang, Y. Xiang, F. Lu, C. Kuang, L. Xu, W. Shen, J. Cheng, H. Gui,G. Zou, P. Wang, H. Ming, J. Liu, X. Liu, J. R. Lakowicz, D. Zhang, Label-free surface-sensitivephotonic microscopy with high spatial resolution using azimuthal rotation illumination. Sci.Adv. 5, eaav5335 (2019).

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rotation illuminationLabel-free surface-sensitive photonic microscopy with high spatial resolution using azimuthal

Gui, Gang Zou, Pei Wang, Hai Ming, Jianguo Liu, Xu Liu, Joseph R. Lakowicz and Douguo ZhangYan Kuai, Junxue Chen, Xi Tang, Yifeng Xiang, Fengya Lu, Cuifang Kuang, Liang Xu, Weidong Shen, Junjie Cheng, Huaqiao

DOI: 10.1126/sciadv.aav5335 (3), eaav5335.5Sci Adv 

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