Focal adhesions are critical cell membrane components that regulate adhesion and migration and have cluster dimensions that correlate closely with adhesion engagement and migration speed. We utilized a label-free approach for dynamic, long-term, quantitative imaging of cell–surface interactions called photonic resonator outcoupler microscopy (PROM) in which membrane-associated protein aggregates outcoupled photons from the resonant evanescent ﬁeld of a photonic crystal biosensor, resulting in a highly localized reduction of the reﬂected light intensity. By mapping the changes in the resonant reﬂected peak intensity from the biosensor surface, we demonstrate the ability of PROM to detect focal adhesion dimensions. Similar spatial distributions can be observed between PROM images and ﬂuorescence-labeled images of focal adhesion areas in dental epithelial stem cells. In particular, we demonstrate that cell–surface contacts and focal adhesion formation can be imaged by two orthogonal label-free modalities in PROM simultaneously, providing a general-purpose tool for kinetic, high axial-resolution monitoring of cell interactions with basement membranes. and disassembly of a FA, the size of the FA cluster varies Introduction Focal adhesions (FAs), or cell–matrix adhesions, are and is highly correlated with the level of adhesion 13,29 large specialized proteins that are typically located at the engagement and migration speed . For example, non- interface between the cell membrane and extracellular mature focal complexes (FXs) are initially formed at the 1–24 matrix (ECM) (Fig. 1a, b) . FAs are critical for sup- leading edge of the cell (e.g., in the lamellipodia area) and porting the cell membrane structure and regulating signal are usually <0.2 µm . As the lamellipodia withdraws from transmission between the cytoskeleton (e.g., actin) and the leading edge, many FXs disassemble and release transmembrane receptors (e.g., integrins) during adhesion adhesion proteins back to the inner cell body, whereas 16–24 2 and migration . Monitoring the response of FA clus- some of the FXs grow larger (typically 1–10 µm ) and ters to drugs is one important mechanism by which the assemble into mature FA clusters by recruiting adapter 19,29 action of pharmaceutical compounds may be evaluated, proteins . Once the remaining FAs are in place, they particularly where approaches that enable characteriza- may form stationary attachment points by binding to the tion to be performed with a small number of cells are ECM, and a cell may utilize these anchors to migrate over 22,25–28 especially valuable . During the dynamic assembly the ECM by pushing and pulling the entire cellular 18,21,23 body . This insight into the dynamics of FA cluster formation and dissociation has been made possible by technical advances in the ﬁeld of ﬂuorescence and super Correspondence: Brian T. Cunningham (email@example.com) 1 30–36 Department of Bioengineering, University of Illinois at Urbana-Champaign, resolution microscopy . Optical modalities, including Urbana, IL 61801, USA total internal reﬂection ﬂuorescence microscopy, Micro and Nanotechnology Laboratory, University of Illinois at Urbana- photoactivation localization microscopy (PALM), Champaign, Urbana, IL 61801, USA Full list of author information is available at the end of the article © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Shift Zhuo et al. Light: Science & Applications (2018) 7:9 Page 2 of 15 ab Focal adhesion Evanescent field Extra cellular matrix Photonic crystal surface Photonic crystal surface Cell non-attached Cell attached Δλ (PWS) Cell BG ΔI (PIS) Photonic crystal Cell 0.9 biosensor 0.8 0.8 0.6 624 626 628 Objective lens 0.4 Wavelength (nm) Cylindrical 0.2 lens Dichroic 0 mirror 610 620 630 640 650 Wavelength (nm) Spectrometer Polarized LED beam splitter Mirror CCD camera Tube lens Fig. 1 Principle of the molecular-dynamics for cell attachment on a photonic crystal (PC) biosensor in photonic resonator outcoupler microscopy (PROM). Schematic representation of the molecular mechanism a before and b after a live cell attaches to the PC biosensor surface. c The principle of PROM imaging system. Inset: spectra shift before and after the cell attaches to the PC surface 17,37–39 stochastic optical reconstruction microscopy, and inter- micropillar substrates) , mechanical probing of cells 40,41 ferometric PALM, coupled with ﬂuorescence tagging of (e.g., atomic force microscopy) , and single molecular the element(s) of FA clusters via administration of ﬂuor- techniques (e.g., tension sensors) have allowed the escently labeled antibodies or incorporation of ﬂuorescent quantiﬁcation of molecular tension forces within FA reporter genes by transfection of cells, along with progress clusters as well as FA-mediated traction and adhesion made in single particle tracking algorithms, have allowed forces. researchers to quantify FA-associated parameters, such as Understanding the dynamics of FA formation and FA areas and sizes (x–y dimensions), FA architectures changes in FA-associated parameters is beneﬁcial not only (x–y–z dimensions), FA turnover rates, and spatio- for understanding the fundamentals of biology but also temporal distributions of FA complexes. Additionally, for the ﬁeld of biosensor diagnostics and screening for 36,43,44 developments in traction force measurements (e.g., based clinical applications . Changes in FA-associated on two-dimensional (2D) hydrogel substrates or parameters, such as FA sizes and traction forces, have Reflectance (AU) Reflectance (AU) Zhuo et al. Light: Science & Applications (2018) 7:9 Page 3 of 15 been linked to critical cellular processes, including value (PIV) from the PC before and after live cell metastasis, apoptosis, and chemotaxis, as well as pathol- attachment to acquire the peak intensity shift (PIS) at 9,29,36,45–47 ogies of cancers and other diseases . As such, each local voxel volume (Fig. 1a, b). Images of the PIS monitoring the response of FA clusters to drugs, for reveal highly localized and easily observable loci of protein example, is an important mechanism by which the action clusters that correlate with the spatial distribution and 22,25–28,36 of pharmaceutical compounds may be evaluated , size of FAs observed by ﬂuorescence microscopy. We hypothesize that the observed reduction in reﬂected and high-throughput approaches that enable the char- acterization of small cell populations in real time are intensity from the PC is mainly caused by the outcoupling especially valuable for these applications. Currently of resonant standing wave photons via scattering. available techniques largely make use of ﬂuorescence Because this imaging modality operates using an inde- tagging to mark individual FA proteins, which entails pendent sensing mechanism that obtains contrast through temporal limitations imposed by photobleaching and the formation of protein clusters at the cell–ECM inter- challenges associated with accurate quantitation and face that are capable of outcoupling light from the PC 9,11,48 long-term analysis . New tools are therefore required biosensor surface, we name this technique photonic to study the dynamic behavior of FA clusters and their resonator outcoupler microscopy (PROM) (Fig. 1c). In interaction with the ECM to characterize changes in FA our previous study, we report the ﬁrst observation of a dynamics in live cells in situ. However, determining the reduced and highly localized reﬂected intensity in the dynamic activity of a FA cluster is challenging, especially context of nanoparticles with optical absorption at the with all of the FA proteins that are simultaneously active resonant wavelength of the PC . However, these obser- during the in situ assembly and disassembly processes in vations were made with very high contrast and highly live cells. Although a variety of approaches have been localized metal absorbers (for plasmonic nanoparticles) or utilized to investigate these processes, the detailed titanium dioxide (TiO ) nanoparticle dielectric scatterers. mechanism of FA assembly and disassembly in live cells, Although the reduced reﬂected resonant intensity from including the variability of the FA dimension, is poorly the high-contrast surface-attached TiO scatterers (the 9,11,48 understood . For instance, ﬂuorescent tags are often refractive index of TiO nanoparticles (n = ~2.4) is 2 TiO2 used to mark individual FA proteins, but due to the much larger than that of the surrounding water medium temporal limitations imposed by photobleaching, accurate (n = ~1.333)) was the ﬁrst observation with PROM, this quantitation and long-term analysis are exceedingly dif- study is the ﬁrst to use PROM to observe scattered out- ﬁcult to perform, whereas the cytotoxicity of ﬂuorescent coupling from very low contrast FAs in live cell mem- tags compromises the viability of the cells under study. branes (averaged cell n = 1.35–1.38) to the surrounding cell Here we describe a label-free optical sensing approach medium (n = ~1.333). Using dental epithelial stem cells that combines two optical modalities to quantify the FA- attached to a ﬁbronectin-coated ECM surface as a associated parameters that are critical for characterizing representative example, we demonstrate that PWS and spatiotemporal distribution patterns and the strength of PIS images of the same cells display distinct and com- FA clusters in real time. In our previous studies on cell plementary information. Although the regions with the imaging by photonic crystal enhanced microscopy greatest PWS are at the cell–surface interface in which (PCEM), we utilized an imaging modality in which the uniformly distributed regions with the greatest surface reﬂected resonant peak wavelength value (PWV) was engagement occur, the regions with the greatest PIS measured over the imaging ﬁeld-of-view to derive images represent the formation of highly concentrated protein of the peak wavelength shift (PWS) that occur when cells clusters at the cell–surface interface that are capable of 49–52 attach to the photonic crystal (PC) surface . In these scattering photons. Therefore, we introduce PROM as a studies, we describe how the engagement of the cell quantitative, dynamic, and label-free approach to observe membrane components with the surface of the PC results the formation and evolution of FA cluster areas that are in highly localized shifts in the resonant reﬂected wave- otherwise challenging to observe with other available length from the biosensor , as well as the design of a imaging modalities, particularly for repeated observations modiﬁed brightﬁeld (BF) microscope that enables visua- of the same cell population for extended time periods. lization of cell–surface attachments with a ~0.6 × 0.6 µm pixel size. The PWS image sequences clearly show the Materials and methods evolution of cell attachment through the engagement of PC biosensor the lipid bilayer cell membrane and internal cell- The PCs used in this study are subwavelength nanos- associated proteins within the ~200 nm deep evanescent tructured surfaces with a periodic modulation in the ﬁeld region of the PC. In this study, we demonstrate a refractive index that acts as a narrow bandwidth resonant novel and orthogonal imaging modality within PCEM in optical reﬂector at one speciﬁc resonance wavelength 49,50,52–55 which we measure the resonant reﬂected peak intensity (Fig. 1) . The high reﬂection efﬁciency of the PC Zhuo et al. Light: Science & Applications (2018) 7:9 Page 4 of 15 ab c d s n d =d +d n g s (Medium) d g s n d =d 1 s d =d g d =0 (Slab) d =0 (UVCP) 0 0 k z z TE TM n’ y n’ (Substrate) x e fg PIV PWV 1.02 Linear Linear 0.9 1 626 0.98 0.8 625 1.33 1.34 1.35 1.36 1.37 0.8 1.34 1.36 624 626 628 630 Refractive index (AU) Refractive index (AU) Wavelength (nm) 0.1 0.6 n =1.333 n =1.343 0 0.4 n =1.353 PIS PWS n =1.363 2 –0.1 0.2 Linear Linear n =1.373 580 600 620 640 660 1.33 1.34 1.35 1.36 1.37 1.33 1.34 1.35 1.36 1.37 Wavelength (nm) Refractive index (AU) Refractive index (AU) hi j PWV PIV Linear 1 Linear 628 0.9 1 626 0.8 0.3 0.8 0.8 0.7 624 626 628 630 0 200 400 0 200 400 0.2 Wavelength (nm) NP radius (nm) NP radius (nm) 0.6 r =50 nm 0.1 0.4 1 r =100 nm PWS r =250 nm PIS 0.2 r =500 nm Linear Linear –0.1 –1 580 600 620 640 660 0 200 400 0 200 400 Wavelength (nm) NP radius (nm) NP radius (nm) Fig. 2 Principle of peak intensity shift (PIS) and peak wavelength shift (PWS) on a PC surface. SEM images of a fabricated PC biosensor with a side views of the cross-section (inset: zoomed-in side view) and b top views (inset: zoomed-in top view). c FDTD simulation model of the PC surface (side view of the cross-section). d Simpliﬁed model as a waveguide on the PC surface (side view of the cross-section). e Normalized spectra with different background refractive indices (n = 1.333, 1.343, 1.353, 1.363, 1.373) on a PC surface without dielectric nanoparticles (inset: zoomed-in peak of the reﬂection spectra). Corresponding f peak intensity shift (PIS) (inset: peak intensity value (PIV)) and g peak wavelength shift (PWS) (inset: peak wavelength value (PWV)). h Normalized spectra with different sizes (radius of 50, 100, 250, 500 nm) of dielectric nanoparticles on the PC surface (inset: zoomed-in peak of the reﬂection spectra). Corresponding i PIS (inset: PIV) and j PWS (inset: PWV). Scale bar: 200 nm Reflectance (AU) Reflectance (AU) Reflectance (AU) Reflectance (AU) Normalized PIS (AU) Normalized PIS (AU) Normalized PIV (AU) Normalized PIV (AU) PWS (nm) PWS (nm) PWV (nm) PWV (nm) V Zhuo et al. Light: Science & Applications (2018) 7:9 Page 5 of 15 at the resonant wavelength (Fig. 1c) is the result of the Fabrication and preparation of the PC surface formation of surface-conﬁned electromagnetic standing A room temperature replica molding approach is used waves that extend into the surrounding medium in the to fabricate the PC on a glass substrate using a quartz 53–80 form of an evanescent electromagnetic ﬁeld . The mold template with a negative volume image of the photonic band gap of the PC strictly limits the lateral desired grating structure (fabricated with e-beam litho- propagation of light. Thus the PC exhibits a strong optical graphy and reactive ion etching). First, the quartz mold conﬁnement of incident light into an inﬁnitesimal volume template is thoroughly cleaned with a piranha solution (a that selectively interacts with surface-adsorbed cell com- mixture of sulfuric acid (H SO ) and hydrogen peroxide 2 4 ponents while being insensitive to the components of the (H O ), H SO :H O = 3:1) for approximately 3 hours to 2 2 2 4 2 2 cell body that are not engaged with the surface. Simula- remove organic residues from the surface of the master tions (Fig. 2) performed using the ﬁnite-difference time- template. The glass substrate is cleaned in an ultrasonic domain (FDTD) method show the spatial distribution of bath three times with isopropyl alcohol (IPA), acetone and the resonant electromagnetic ﬁeld, which extends ~200 deionized (DI) water for 1 min in each solvent and then nm into the aqueous medium at the top of the PC. Pre- dried with nitrogen gas and treated with oxygen plasma. vious research has demonstrated that a speciﬁc location Second, the liquid UVCP is deposited between the quartz on the PC surface has a resonant reﬂected wavelength that mold template and glass substrate, and a high intensity can be independently measured from neighboring regions UV lamp is used to cure the liquid polymer to a solid and that the local PWV is determined by the dielectric state. After peeling the grating replica away from the permittivity of the biomaterial that is adsorbed at that quartz mold template, the nano-patterned surface is speciﬁc location . The PC surface can therefore act as a attached to a glass cover slip with an adhesive. Then PC proxy for a biological surface with a built-in capacity to fabrication is completed by reactive sputter deposition detect changes in the cell membrane components of cells (PVD 75, Kurt J. Lesker, Jefferson Hills, PA, USA) of a that attach to the PC within the evanescent ﬁeld, pro- high refractive index thin ﬁlm (TiO ) atop the grating viding a compelling platform for adhesion phenotyping of structure. Scanning electron microscopic (SEM) images of single cells (see Supplementary Materials Section S-1 for a cross-sectional view and a top view of the structure details). PC biosensor surfaces are inexpensively fabri- are shown in Fig. 2a, b, respectively. Next, before cell cated uniformly over large surface areas by a room tem- attachment experiments, the PC is cleaned in an ultra- perature nanoreplica molding process, as described sonic bath with IPA and DI water for 1 min each, followed 54,55 previously in refs. by drying with nitrogen gas. The PC is then treated , and are incorporated onto glass 49,50,52,81 microscope slides, described in refs. . with oxygen plasma to facilitate attachment of a liquid containment gasket formed from polydimethylsiloxane. Finally, the PC surface is hydrated with a phosphate- Modeling the PC surface for sensor design and simulation buffered saline solution and coated with a layer of A numerical electromagnetics simulation package ECM molecules (e.g., ﬁbronectin) to promote cellular (FDTD, Lumerical Solutions, Inc., Vancouver, BC, attachment. Canada) is used to calculate the distribution of a resonant evanescent ﬁeld on the PC biosensor surface. In our Photonic resonator outcoupler microscopy previous studies, the PC surface was modeled as an ideal The PROM instrument is a modiﬁed BF microscope 50,52 case with a rectangular nanostructure for simplicity . that uses a line-scanning approach to measure the spatial To more accurately represent the fabricated structure distribution of optical spectra across a PC surface with a (Fig. 2a, b), the model used in this study incorporates a submicron spatial resolution in the axial direction for 49,51 sidewall slope in a trapezoidal shape. As shown in Fig. 2c, label-free imaging (Fig. 1c) . An optical ﬁber-coupled d, the PC consists of a one-dimensional ultraviolet-cur- light-emitting diode is used as the light source, and a line- able polymer (UVCP) grating surface structure (refractive proﬁled (polarized perpendicular to the grating structure) index n = 1.46, grating depth d = 120 nm, period Λ = light beam illuminates the PC biosensor from below 0 g 400 nm, duty cycle f = 41.6%, sidewall angle θ = 85°) through a microscope objective lens (e.g., 10×). Illumi- g g coated with a thin ﬁlm of TiO (refractive index n = 2.4, nation from below eliminates the possibility of the scat- 2 1 slab thickness d = 61 nm, duty cycle f = 50%, sidewall tering, absorption, and meniscus reﬂection and refraction s s angle θ = 82°) to generate a resonant reﬂected narrow- of materials in the cell media or cell body from effecting a band mode at a wavelength near λ = ~626 nm. The resonant reﬂected signal to the PC surface. The reﬂected adhesion of FAs on the PC surface is also modeled in light, containing the resonant reﬂected spectrum, passes FDTD, where the FA is represented as a homogeneous through the objective lens in the opposite direction and and lossless sphere (n = ~1.46, radius range 50–500 through the entrance slit of an imaging spectrometer and FA nm) composed of many protein molecules (Fig. 2e–j). is ﬁnally collected by a charge-coupled device camera, Zhuo et al. Light: Science & Applications (2018) 7:9 Page 6 of 15 which records the resonant reﬂected spectrum from each locally quench the PC resonance; (2) concentrated local pixel across the illuminated line on the PC surface. A high regions of high dielectric permittivity that can outcouple spatial resolution in the axial direction is obtained due to resonantly conﬁned light by scattering . Interestingly, by the shallow evanescent ﬁeld of the PC (~200 nm). The analyzing PROM data during cell attachment, we resolution in the lateral direction is determined by the observed the characteristics of PIV images that differ lateral propagation distance of resonant-coupled photons, substantially from PWS images. Although optical resulting in the detection of distinct surface-attached absorption at the PC resonant wavelength will efﬁciently objects for widely dispersed features at the micron size reduce the PIV in a highly localized manner, the protein scale . The same ﬁeld of view can be re-scanned and lipid components of cells and cell membranes do not repeatedly to generate a sequence of images for the display strong absorption in the visible wavelength range. same cells. The current shortest available scan interval for Human tissues and live cells exhibit strong absorption in our instrument is ~10 s for ~100 × 100 µm , which is the infrared wavelength range; however, these wave- limited by the exposure time and speed of the motorized lengths are not utilized in our detection approach and scan stage. While characterizing the resolution perfor- thus are unlikely to be the dominant factors that con- mance through the intentional introduction of dielectric tribute to PIV reduction. Additionally, though metallic and metallic nanoparticles of a variety of sizes (30–500 elements comprise a small fraction of a cell’s atomic nm), we observe that dielectric objects not only induce a constituency, metal atoms are present as ions rather than shift in the PWV but also a reduction in the resonant peak as clusters that are capable of optical absorption in the intensity . When a cell attaches to the PC surface, the visible part of the spectrum. Scattering occurs when light peak resonant wavelength red-shifts from a lower wave- is forced to deviate from its original trajectory due to length (before cell attachment, e.g., λ = ~626 nm) to a localized non-uniformities in its propagation medium, BG higher wavelength (after cell attachment, e.g., λ = ~628 which occur, for example, when light propagating through cell nm). At the same time, the resonant reﬂection efﬁciency, water is reﬂected or refracted by a particle with a greater as measured by the peak intensity, changes from a higher refractive index. A highly concentrated region (e.g., FA PIV (before cell attachment, e.g., ~90% normalized to a cluster) with a greater refractive index than its sur- peak reﬂectance of 100% for the PC immersed in water) to roundings (e.g., cell media) generates more localized and a lower value (after cell attachment, e.g., 80% as a efﬁcient scattering than a diffuse region with a gradual normalized PIV). A negative PIV shift indicates that gradient in the refractive index. Scattering effects also proteins in some areas of the cell bind to transmembrane become stronger when the size of a region with a proteins (e.g., integrins) to form more substantial FA refractive index contrast increases. Because the cross- clusters. The PROM instrument and sensor structure sectional area of a FA cluster is typically 0.2–1.0 µm ,we measure the resonant reﬂection characteristics of the can expect to observe measurable differences in the PC via the spectrum obtained from each ~0.6 × 0.6 µm scattering efﬁciency of membrane-associated protein pixel area, representing a total ﬁeld of view region clusters as they form, change size, and subsequently dis- (e.g., ~300 × 300 µm ) of the PC surface. sipate. Light scattering from internal cell components, such as organelles and mitochondria, has recently been Stem cell culture utilized to achieve imaging contrast in the context of Murine dental epithelial stem cells (mHAT9a) changes that occur in precancerous cells that express were maintained in Dulbecco’s Modiﬁed Eagle phenotypic changes due to the expression of mutant 82–84 Medium supplemented with 10% fetal bovine serum genes . In PROM, we detect the modulation of and 1% Penicillin–Streptomycin. Stem cells were membrane-associated scattering that occurs due to FA cultured at a temperature of 37 °C and supplied formation by utilizing the ability of localized high with an environment of 5% CO humidiﬁed air during refractive index protein clusters to produce image imaging. contrast by reducing the reﬂection efﬁciency of a PC biosensor. Results and Discussion After analyzing the mechanism of PIV reduction, we Electromagnetic computer modeling of resonant study the useful cellular information that can be uniquely outcoupling from a PC by FA extracted from the measurement of this physical quantity. It is important to understand the physical mechanism Our hypothesis is that the dominant cause for the mea- that is responsible for the PIS in the context of cell sured PIV reduction is light scattering rather than attachment. Theoretical and experimental analyses sug- absorption. Thus a scattering model can represent the gest that a reduction in the PIV can occur by two interaction of light with a FA cluster since scattering mechanisms: (1) materials that act as efﬁcient absorbers of describes the effect of an electromagnetic plane wave the resonant wavelength (such as gold nanoparticles) propagating through a dielectric particle. To predict the Zhuo et al. Light: Science & Applications (2018) 7:9 Page 7 of 15 effects on the resonant reﬂection spectrum from a PC that A difference of PWS and PIS images for the same cell at can be induced by a FA on its surface, we compared the each time point is clearly observed in the spectral data computed reﬂection spectra with and without a small acquired by PROM. In Fig. 3b, the dashed lines represent region of dielectric contrast on a PC surface. In a FDTD the background spectra for a representative pixel acquired electromagnetic computer model of a FA on a PC surface before cell attachment (0 min), and the dotted lines with (Fig. 2e–j), we represent the FA as a locus of a material dot markers represent the spectra of the same pixel after cell attachment (~16 min). PWS and PIS images are with a designated radius (50–500 nm) and designated refractive index elevation (n = ~1.46) in contrast to the simultaneously extracted from the spectra data at the FA surrounding medium (estimated to be n = ~1.333). The PWV and PIV, respectively (at every ~0.6 × 0.6 µm pixel simulation results demonstrate that, when the FA cluster area on the PC surface). In Fig. 3a, the regions with the is not present, the PWS in the resonant reﬂection spec- greatest values in the PWS and PIS images show distinct trum increases (Fig. 2g) as the refractive index contrast of distribution patterns, which suggests that these regions the surrounding media increases, whereas the PIS remains may represent two different physical mechanisms. For the same (Fig. 2f). However, when the FA cluster is pre- instance, the PWS image of the cell marked by the white sent, as the radius of the FA increases (Fig. 2h), the arrow (e.g., ~16 min) has a high PWS on the top and maximum in the resonant reﬂection spectrum decreases bottom of the cell body, whereas the PIS image of the (Fig. 2i, Inset), indicating that more energy is outcoupled same cell has a “ring” of a high PIS along the cell from the PC resonant standing wave. Thus the PWSs to a boundary. The zoomed-in images of the PWS and PIS higher wavelength (Fig. 2j), as expected, and the PIS taken 16 min after introduction of a single cell are shown (compared with the original PIS without a FA cluster) in Fig. 4a and S-Fig. 1, respectively, and the overlaid PWS increases as the FA cluster size increases (Fig. 2i) due to and PIS images (high intensity values only, PWS—green, the scattering-induced outcoupling (for more details, PIS—red, overlap—yellow) over the BF image of the same see Supplementary Materials S-2). cells show the distinct distribution patterns between these two physical quantities. The cross-sectional curves (L1 Dynamic PIS images of live cells and L2) along the diameter of the cell are plotted in Dynamic PIS images can be acquired by PROM during Fig. 4b, and the corresponding statistical results are live cell adhesion over an extended time period to create shown in Fig. 4c(N = 5 cells). The high intensity of the movies of FA development, which are difﬁcult to acquire PWS (in green at the bottom of Fig. 4a) represents a via ﬂuorescence imaging due to photobleaching. The PIS higher mass density of cellular materials associated with here is deﬁned such that greater reductions in the the cell membrane. The high intensity of the PIS (in red at reﬂection efﬁciency are displayed as higher intensity the bottom of Fig. 4a) along the cell boundary represents values for a simpler visual comparison with other imaging the scattering outcoupling effect from the locally gener- modalities. As shown in Fig. 3a, the resulting PIS image ated FA clusters. Typically, a larger cluster size of protein sequence reveals that the cell periphery has a greater aggregates corresponds to a greater PIV reduction com- degree of scattering than the cell center. This “ring” effect pared with the background. Therefore, these measure- demonstrates that the PIS intensity is not homogeneously ments of local PWS and PIS can quantify the surface- distributed throughout the cell membrane. Fig. 3b shows attached cellular mass density and dimension of the FA spectra from three sample points marked in Fig. 3a(A— clusters dynamically and simultaneously. red, B—black, C—green) at different times. Initially, all three points are located outside of the cell (~0 min), and Comparison of PIS and ﬂuorescence images all of the points show high resonant PIVs as highlighted Fluorescence images were acquired for the same cells to by the dashed line in the spectra (Fig. 3b). During cell further investigate the FA areas detected in the PIS ima- adhesion, the attachment perimeter expands and sur- ges. In Fig. 5a, the top row shows BF, PWS, and PIS passes the three points as the cell extends its attachment images and the bottom row displays ﬂuorescence images area. Once the cell ﬁrmly attaches and adheres to the PC of the same cell with ﬂuorescence tags applied to three surface (~16 min), points A’,B’, and C’ represent locations different cellular components (nucleus, actin, and vincu- inside, near, and outside of the cell boundary, respectively. lin). There is no obvious pattern similarity between the The solid lines in Fig. 3b demonstrate that the spectra of PIS image and actin (indicating the presence of cytoske- point A’ shifts to a lower resonance peak intensity, point leton components) or nucleus images. However, the B’ shifts to the lowest resonance peak intensity, and point ﬂuorescent image of vinculin (a type of FA molecule) in C’ remains at the original resonance peak intensity. the bottom row of Fig. 5a indicates that ﬁlopodia reside in Considering all of the pixels within the cell, we observe a the FA area along the stem cell boundary. As shown in ring of enhanced scattering that encompasses much of the Fig. 5a in the right column, the PIS image shows a nearly cell periphery. identical distribution pattern along the cell peripheral Zhuo et al. Light: Science & Applications (2018) 7:9 Page 8 of 15 0 min 2 min 4 min 6 min 8 min 10 min 12 min 0.5 BF A 0.8 (AU) PIS 0.5 2.5 (nm) PWS 1.5 14 min 16 min 18 min 20 min 22 min 24 min 26 min 0.5 BF C’ B’ A’ 0.8 (AU) PIS 0.5 C’ B’ 2.5 (nm) A’ PWS 1.5 C’ 0.95 A BG-outside BG-inner cell A’ BG-cell boundary Outside Inner cell 0.8 Cell boundary B’ 0.6 0.7 0.4 624 626 628 630 Wavelength (nm) 0.2 600 620 640 660 Wavelength (nm) Fig. 3 PIV images and spectra during live stem cell (mHAT) attachment. a Sequence of PROM-acquired images at several time points (0–26 min) during the cell adhesion process. Top row: brightﬁeld (BF) images; middle row: peak intensity shift (PIS) images; bottom row: peak wavelength shift (PWS) images. b PROM-measured spectra at three locations on the PC surface before (A, B, C, in dashed lines) and after (A’,B’,C’, in dotted lines) cell adhesion. Point A’ (red) represents the inner cell area; point B’ (black) represents the cell boundary; point C’ (green) represents the outside of the cell boundary. Scale bar: 20 μm region to that obtained by ﬂuorescence microscopy with a (highlighted in the light-yellow regions), which indicates labeled vinculin where the FA areas are concentrated that the high intensity in the PIS images are probably co- along the cell boundary. Fig. 5b shows two cross-sections localized with the FA areas. Therefore, the dimensional along different radial directions (L3 and L4, as shown in change of the FA cluster can also be detected with PIS Fig. 5a) sampled across the cell diameter for the PWS images using PROM. (blue curves), PIS (red curves), and ﬂuorescence-tagged Statistical analyses are shown in Fig. 5c(N = 5 cells) for images (nucleus—green, actin—magenta, vinculin— the ﬂuorescent, PIS, and PWS images along the cell black). Both the PIS (in red) and vinculin (in black) curves boundary (marked as “Edge”) and within the nucleus area exhibit a similar “ring” effect along the cellular boundary (marked as “Inner”). Fig. 5c (Left) displays ﬂuorescence Reflectance (AU) Reflectance (AU) Zhuo et al. Light: Science & Applications (2018) 7:9 Page 9 of 15 PWS (nm) PIS (AU) BF (AU) 2.5 0.8 1 L1 L1 L2 L2 0.5 1 0.5 Overlaid image Overlaid image (PWS+PIS) (PWS+PIS+BF) Cross-section L1 Cross-section L2 1 4 1 PWS PWS PIS PIS 0.5 2 0.5 0 0 0 0 10 20 30 40 50 10 20 30 40 50 Length (μm) Length (μm) 0.8 2.5 0.75 0.7 1.5 0.65 0.6 Edge Inner Edge Inner Fig. 4 Zoomed-in PROM images and cross-section (before and after mHAT cell adhesion). a PROM-acquired images (including PWS, PIS, and BF images) and their overlap images at ~16 min. b Comparison of cross-sectional curves (L1 and L2) of the PWS and PIS images. c Bar graph of the statistical comparison between the edge and the center of the cells. Scale bar: 20 μm images (including actin, nucleus, and vinculin) that scaffold protein, the distribution of actin is relatively demonstrate three different patterns of distribution along uniform, and thus the difference of the ﬂuorescence the cell edge and center. The nucleus image only shows intensity between the cell edge and center in the actin high intensity within the area of the nucleus because the image is small. Vinculin is a membrane-cytoskeletal pro- ﬂuorescent molecular probes only tag nucleic acid mate- tein that is often localized in the FA area because it par- rial, such as chromosomes. Actin mainly functions as a ticipates in the linkage between the transmembrane cytoskeleton molecule that is rapidly remodeled by protein (e.g., integrin) and cytoskeletal protein (e.g., actin). dynamically forming microﬁlaments to support the cell Therefore, a ﬂuorescence dye for vinculin is often used to structure or participating in many important cellular visualize the locations of FAs. Fig. 5c clearly shows that processes, including cell division or cell signaling. As a the distribution of vinculin is mainly along the cell PWS (nm) Normalized PIS (AU) Normalized PIS (AU) PWS (nm) PWS (nm) Normalized PIS (AU) Zhuo et al. Light: Science & Applications (2018) 7:9 Page 10 of 15 PIS BF PWS L3 L3 L4 L4 1 2.5 (nm) 0.2 1 (AU) Nucleus Actin Vinculin L3 L3 L3 L4 L4 L4 Cross-section L3 Cross-section L4 Nucleus Nucleus Actin Actin Vinculin Vinculin 1 1 0.5 0.5 0 0 50 100 150 50 100 150 Length (μm) Length (μm) PWS PWS PIS PIS 0.5 0.5 50 100 150 50 100 150 Length (μm) Length (μm) Edge Inner 2.5 0.6 0.4 1.5 0.5 0.2 0.5 0 0 0 Actin Nucleus Vinculin Edge Inner Edge Inner Fig. 5 (See legend on next page.) Normalized intensity (AU) Normalized intensity (AU) Normalized intensity (AU) Label-free images Fluorescence images Normalized intensity (AU) Normalized intensity (AU) PWS (nm) Normalized PIS (AU) Zhuo et al. Light: Science & Applications (2018) 7:9 Page 11 of 15 (see ﬁgure on previous page) Fig. 5 Comparison of label-free images and ﬂuorescence images with cross-section. a Top row: brightﬁeld (BF), PWS, and PIS images; bottom row: ﬂuorescence images, including dyes that selectively stain the nucleus, actin, and vinculin. b Comparison of the image cross-sections through lines (L3 and L4) for the ﬂuorescent (nucleus—green, actin—magenta, vinculin—black), PWS (blue), and PIS (red) images. Light-yellow regions represent the regions near the cell edges, where the vinculin (black curves) and PIS (red curves) both have high intensities. c Statistical comparison among the ﬂuorescence images (actin, nucleus, and vinculin) and label-free images (normalized PWS and PIS value comparison between cell edges and centers). Scale bar: 20 μm periphery for a surface-attached cell, which is highly (Fig. 6a). The mechanisms of the temporal curves for cell consistent with the distribution pattern of the PIS (high in adhesion for the PWS and PIS are shown in Fig. 6d, e (N “Edge”, low in “Inner”) (Fig. 5c Right), which is not similar = 5 cells). Directly comparing the dynamics between the to that of the PWS (Fig. 5c Middle). There is a small PWS and PIS is difﬁcult because they have different difference between the PIS image and vinculin ﬂuores- dynamic ranges. However, different slopes indicating cence image around (outside of) the nucleus. This is likely different increasing ratios along the temporal dimension because the vinculin ﬂuorescence image is a transmission are observed if the PWS and normalized PIS are plotted image in the axial direction across the entire cell body together, as shown in Fig. 6f. (with a thickness of several microns to several tens of The PROM images show cell borders and intra-cell microns) including the nucleus area. By contrast, the PIS features that are approximately in the order of the pixel image is only measured through the evanescent ﬁeld, size limit in the axial direction, which is near the dif- which has a thickness that is several hundred times fraction limit of the resonant wavelength. It is important thinner (several tens of nanometers) in the axial direction to put these images in the context of PCEM images that starting from the bottom of the cell body (before reaching were gathered previously from high-contrast objects. In nucleus). the lateral directions, although the effect of a point To highlight how information from PROM images dielectric object on the reﬂected wavelength from a PC complements those obtained by orthogonal imaging can extend to the surrounding pixels (because the electric modalities, ﬁve selected stem cells are shown in Fig. 6a ﬁeld standing wave “samples” a greater lateral dimension imaged by PWS images, PIS images, confocal images with than one period), the outcoupling from a surface adsorbed ﬂuorescence dyes (FL), phase-contrast images (PH), and scatterer (or absorber) is observed to be more highly SEM images. PROM images obtained using PWS and PIS localized. The full-widths at half-maximums of the point information reveal different features of cell attachment, spread function of TiO nanoparticles (e.g., diameter of and both show clear details of the cell attachment ~100 nm) were measured as ~1.20–1.56 µm for PWS boundary. PWS and PIS images highlight only behavior images and as ~0.95 µm for PIS images . Our previous associated with the cell–ECM interface and thus do not study shows that the spatial resolution of the dielectric show material in the upper cell body. Unlike SEM and objects in PWV-based PCEM images is directly correlated ﬂuorescence images, PWS and PIS images yield dynamic with the refractive index contrast of the object. Although and highly quantitative information that can be visualized high-contrast objects (such as a TiO dielectric nano- graphically. As shown in Fig. 6b, c, the stem cell boundary particle or the edge of a photoresist pattern) can extend can be tracked along the local normal direction frame-by- their “inﬂuence” on the measured PWV by as much as a frame, enabling the PIS to be sampled along the cell couple of microns (e.g., 2–3 μm) in any direction, lower- boundary spatially and temporally at the same time. contrast objects have a much more limited perturbation. Associated dynamic analyses with different sampling In PROM images of attached cells, there is an extremely bands (S-Fig. 2, black for band 1—near cell boundary, low refractive index contrast between the attached cell white for band 2—inner region of the cell) in cells were membrane and surrounding cell media. Within the foot- performed on PWS images and PIS images. The resulting print of an attached cell, the refractive index contrast 2D maps shown in Fig. 6b, c represent the PWS and PIS between a FA and the neighboring cell membrane regions with spatiotemporal information along the cell boundary is even lower. These hypotheses were formed by the and time frames. Comparing the different bands between contrast observed at the cell attachment border and the both maps, the PIS increase is much higher in band 1 contrasting regions of attachment within a cell. Instead compared with that in band 2 (Fig. 6c), whereas the PWS of observing smeared borders that extend for several shows the opposite trend (Fig. 6b). The dramatic increase microns, we observed a contrast in PIS and PWS images of the PIS may be due to the aggregations of FAs along the with micron-scale features. These observations are con- cell boundaries, which is conﬁrmed in the FL images sistent with our earlier measurements but have been Zhuo et al. Light: Science & Applications (2018) 7:9 Page 12 of 15 PWS PIS FL PH SEM SEM (zoom) Cell 1 Cell 2 5 μm Cell 3 Cell 4 Cell 5 0.6 0.4 0.8 (AU) 1.5 (nm) 20 μm bc PWS band 1 PWS band 2 PIS band 1 PIS band 2 (nm) (AU) 1.5 0.8 0.6 0.4 12 40 12 40 12 40 12 40 Time (min) Time (min) Time (min) Time (min) de 1.5 f PWS PIS 1.1 1 0.8 0.8 0.6 0.9 0.5 0.4 0.6 0.8 Band 1 0.2 Band 1 Band 2 Band 2 0 0 0.4 10 20 30 40 10 20 30 40 10 20 30 40 Time (min) Time (min) Time (min) Fig. 6 Image modality comparison and dynamic analysis of PROM images during cell adhesion. a Five selected cells imaged by peak wavelength shift (PWS), peak intensity shift (PIS), confocal ﬂuorescence microscopy (FL) (red—actin, green—vinculin, blue—nucleus), phase-contrast microscopy (PH), and scanning electron microscopy (SEM). Scale bar: 20 μm. (Top-right inset: zoom-in SEM image for cell 1. Scale bar: 5 μm). 2D spatiotemporal maps are tracked from different bands (band 1—near cell boundary, band 2—inner region of cell) in stem cells with b PWS images and c normalized PIS images. Mean and standard deviation of the temporal curves of cell adhesion for d PWS, e normalized PIS, and f the comparison between PWS and normalized PIS Sampling window (AU) PWS (nm) Normalized PIS (AU) Sampling window (AU) PWS (nm) Normalized PIS (AU) Zhuo et al. Light: Science & Applications (2018) 7:9 Page 13 of 15 applied here for the ﬁrst time in the context of attached University of Illinois Research Park, 1800 South Oak Street, Champaign, IL 61820, USA. Department of Electrical and Computer Engineering, University of cell images of the PIS. Illinois at Urbana-Champaign, Urbana, IL 61801, USA Conclusions Authors' contributions Y.Z. designed the experiments; Y.Z. and J.S.C. performed experiments and This study describes a label-free microscopic approach wrote the manuscript; Y.Z. and T.M. developed the analysis software and that quantitatively measures the scatter-induced changes performed data analysis; Y.Z. and H.Y. fabricated the PC sensors; B.T.C. and B.A. in the reﬂected intensity from a PC biosensor surface to H. provided guidance and edited the manuscript. reveal the kinetic evolution and spatial features of FAs Conﬂict of interest that form at the cell–surface interface. Compared to a The authors declare that they have no conﬂict of interest. sensing approach in which image contrast is generated by the dielectric permittivity of attached cell components, Supplementary information accompanies this paper at https://doi.org/ 10.1038/s41377-018-0001-5. PROM provides contrast in the reﬂected resonant inten- sity that is induced by the refractive index contrast of the Received: 11 May 2017 Revised: 13 February 2018 Accepted: 14 February localized protein clusters that occur at the cell–surface 2018 Accepted article preview online: 23 February 2018 interface, which comprise FA sites. Our hypothesis is supported by electromagnetic computer simulations that have modeled small and low refractive index contrast regions on a PC that induce measurable reductions in the References 1. Davies, P. F. & Tripathi, S. C. Mechanical stress mechanisms and the cell. An resonant reﬂection efﬁciency. Our hypothesis is also endothelial paradigm. Circ. Res. 72,239–245 (1993). supported by ﬂuorescence microscopy of cells in which 2. Schaller, M. D. & Parsons, J. T. Focal adhesion kinase and associated proteins. the patterns of FA regions are similarly distributed as Curr. Opin. Cell Biol. 6,705–710 (1994). 3. 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