Meta-amplified dark-field interferometric scattering microscopy

Domain: neuroscience
Published: 2026-01-23
Editorial outcome: major_revision

Abstract
Label-free optical detection of nanometer-scale bioparticles is highly desirable for noninvasive biological studies but challenging due to the weak scattering signals that are difficult to distinguish from the illumination background. Interferometric scattering microscopy (iSCAT) has enabled high-sensitivity imaging by detecting the interference between the particle’s scattered light and a reference beam. However, enhancing the detection sensitivity and the image contrast for small particles continues to be a challenge in iSCAT. Here, we introduce meta-amplified dark-field interferometric scattering microscopy (MAD-iSCAT), which leverages a plasmonic metasurface to drastically enhance nanoparticle detection sensitivity in iSCAT. By employing a metasurface comprising sub-diffraction plasmonic meta-atom arrays, MAD-iSCAT generates bright radiation modes that intensely scatter light toward the far field in the presence of a detection nanoparticle, substantially amplifying the sensitivity. In the absence of a nanoparticle, the metasurface produces minimal background due to the dark collective mode, resulting in improved image contrast. We present a theoretical analysis of amplified interferometric imaging using designed metasurfaces and experimentally demonstrate enhancements in contrast and signal-to-noise ratio for detecting dielectric nanoparticles, exosomes, and proteins. Our approach offers broad applications in label-free biosensing and optical mass spectrometry, enabling significantly improved throughput and sensitivity.

Introduction
The identification and analysis of biomolecular activities and interactions are crucial for understanding physiological and pathological processes. Direct observation of nanometer-scale bioparticles using light microscopy without fluorescence labeling enables noninvasive investigation with excellent biocompatibility1. However, as the light scattering cross-section (σsc) scales with the sixth power of the particle’s diameter, improving the detection sensitivity and contrast between the weak signal and the background noise has been a key challenge in optical nanoparticle imaging2. Interferometric imaging addresses this challenge by leveraging the interference of scattered light from nanoscale objects with reference light, yielding label-free images with high sensitivity. Recent advancements in interferometric scattering microscopy (iSCAT) have demonstrated the exceptional sensitivity in quantifying small particles, with applications ranging from sensing and tracking nanoparticles3–6, proteins7,8, and viruses9, as well as enabling label-free intracellular imaging10–12 and mass spectroscopy13–15.

In iSCAT, the image contrast C can be written as
C=2 E r E s cos(α), (1)
where Es is the scattered electric field from the detection particle, Er is the electric field of the reference light, and α is the phase difference between Es and Er16. It can be readily seen that Er needs to be minimized to ensure a sufficiently large image contrast for practical detection dynamic range. However, decreasing Er is accompanied by a reduction in total detected photon counts. For detecting nanometer-scale dielectric objects, such a reduction leads to a low signal-to-noise ratio (SNR) or indistinguishable signals due to the shot-noise limit13. In a typical iSCAT, a partial reflector is deployed to reduce the reference beam intensity before detection10,12,14,17. Recently, substantial efforts have been dedicated to engineering Es and Er to attain optimal detection sensitivity, including pupil engineering17–19 and optimizing the interfacing structures such as dielectric layers18, plasmonic nanostructures20, photonic resonators21, and nanofluidic channels22. To obtain high SNR and large detection dynamic range, differential analysis and other algorithmic approaches have been developed for single protein detection and detailed subcellular imaging9,12–14,23. Meanwhile, the field of plasmonics has been developed over the past two decades, enabling numerous applications. Plasmonic nanostructures can produce highly intensified electric fields within deep-subwavelength regions, enhancing light interaction with adjacent objects. Such localized optical modes are extremely sensitive to surrounding environments and have found wide applications in biosensing technology24,25, optical microscopy26,27, photocatalyst28, and more29. Recently, metasurfaces comprising subwavelength arrays of plasmonic nanostructures have emerged as a prominent research area due to their unique optical properties resulting from their collective response30,31. They have been adapted to many optical imaging techniques such as holography32,33, edge detection34,35, and others36,37, demonstrating substantial improvements compared to conventional optics alone.

Here, we propose an interferometric imaging technique, called meta-amplified dark-field interferometric scattering microscopy (MAD-iSCAT), for label-free imaging. MAD-iSCAT employs a plasmonic metasurface as the reference substrate instead of a conventional glass substrate, providing a uniform dark background. As a nanoparticle approaches, the plasmonic metasurface transitions to a radiation mode, amplifying the scattered signal and thus enhancing the nanoparticle detection. This approach can improve the contrast and sensitivity of traditional iSCAT.

Results
Principles of contrast and SNR amplification in interferometric images
Conventional iSCAT typically employs a bare glass substrate, where the reflected light generates an interference image of the scatterers to be detected. Similar label-free imaging methods have been introduced using dark-field illumination, which offers excellent background attenuation38–41. It has been shown that interferometric dark-field images are also generated through the interference between scattered lights from the object and surface roughness (static background)39,40,42. Fig. 1a, b illustrates a schematic of MAD-iSCAT using a plasmonic metasurface with a conventional dark-field illumination. When a scatterer to be detected is located at (0, 0, z) on the metasurface comprising an array of meta-atom elements, the detected signal is composed of the superposition of propagating electromagnetic (EM) fields from the meta-atom elements at (xi, yj, -z) (-∞ ≤ i ≤ ∞ and -∞ ≤ j ≤ ∞) and the scatterer as
|P E|2 = |Σ (i,j) Eij|2 + 2 s Σ (i,j) |Eij| |Es| cos(αij) + |Es|2 (2)
where Eij and Es are the electric field distribution generated by the meta-atom elements at (xi, yj, -z) and the scatterer at (0, 0, z), respectively. αij denotes the difference in radiation phases between the meta-atom elements and the scatterer. For deep-subwavelength scatterers, the detected scattering intensity |Es|2 scales with the sixth power of the particle size in conventional dark-field imaging (Fig. 1c). In previous and dark-field iSCAT schemes, Σ (i,j) Eij in Eq. (2) are regarded as a static random background. Especially, |Σ (i,j) Eij|2 is not involved in the scattered light detection and is removed by subtracting consecutive frames.

In this study, we introduce a metasurface comprising sub-wavelength meta-atom arrays to improve the detection sensitivity in iSCAT. Each metallic meta-atom possesses a large σsc compared to the dielectric nanoparticle (Fig. 1d). Without a nanoscatterer, the meta-atom array behaves as a sub-diffraction grating, primarily reflecting incident light (constructive interference). In contrast, the excited scattering modes of the meta-atoms collectively create destructive interference towards the free space within the detection numerical aperture (NA), resulting in an imaginary Poynting vector (Fig. 1e). This destructive interference, termed dark mode of the metasurface, is represented by |Σ (i,j) Eij|2 term in Eq. (2) in the absence of a nanoscatterer. When a nanoscatterer approaches, it disturbs the local resonance mode of the meta-atom elements and alters the phase profile of the sub-diffraction grating (Fig. 1f). The design of the meta-atom is chosen at a resonance mode, such that the amplitude Aij and phase ϕij of the resonance mode are sensitive to the nearby environmental change due to the presence of the nanoscatterer, and the perturbed mode within the sub-diffraction array radiates towards the detection optics. Considering the perturbed electric fields of meta-atom elements, Eq. (2) is modified as
|Σ (i,j) E'ij|2 + 2 Σ (i,j) |E'ij| |Es| cos(α'ij) + |Es|2 (3)
The bright mode of the metasurface |Σ (i,j) E'ij|2 and thereby generates an interferometric image with significantly enhanced contrast and SNR. Accordingly, the image contrast can be written as
C0 = |Σ (i,j) E'ij + Es|2 - |Σ (i,j) E'ij|2 / |Σ (i,j) E'ij|2 (4)
We implement such a plasmonic metasurface with sub-diffraction hexagonal meta-atom arrays, where degenerate plasmonic hybridized modes achieve a strong scattering condition (see details in Supplementary Section 1).

Metasurface characterization
The plasmonic hexagonal meta-atom arrays have dimensions of h=60 nm in height and d=70 nm in diameter. As shown in Fig. 2a, meta-atom arrays are embedded in the polymethylmethacrylate (PMMA) on the silica substrate. The incident direction is set to be just one direction from the top medium (air) with azimuthal angle φ=180° and altitude angle θin=75° (see details in Supplementary Section 2,3). Figure 2b presents the absorption cross-section (σab) spectra of a meta-atom element in an array with different periodicities Λ under p-polarization. The eigenmode analysis results are depicted by gray lines. In the visible wavelength regime, a meta-atom has mainly three transverse plasmon (TP1, TP2, and TP3) modes and one longitudinal plasmon (LP1) mode43. The charge distribution patterns for each plasmon mode are presented in Fig. 2c. When Λ reduces, wavelength shifts of longitudinal modes and transverse modes behaved differently along their coupling direction of the plasmonic hybridization44. The theoretical analysis confirms the existence of degenerate plasmon modes arising from the antibonding longitudinal modes and bonding transverse modes at the sub-diffraction Λ. Here, we refer to such degenerate plasmon modes, which result in higher σab and σsc as super-scattering plasmon (SSP) modes, as shown in Fig. 2b,d45. In contrast, for an isolated silver nanoantenna of the same dimension, TP2 and LP1 modes are spectrally separated (see details in Supplementary Section 4).

We design Λ to be 145 nm such that the meta-atoms achieve the degenerate mode of TP2 and LP1 modes. The σsc of SSP mode with Λ=145 nm and λ=450 nm is calculated to be 2.3 × 10−14 m2, surpassing those of non-degenerate dipolar and quadrupole modes which are typically broadened and weakened in sub-diffraction arrays compared to the isolated case46. This implies that the dense sub-diffraction meta-atom array can exhibit a strong scattering reference signal to form an interferometric image when SSP mode is coupled into free space by the detection target. However, without a nanoparticle to be detected, the deep subwavelength Λ ensures minimal background scattering under dark-field illumination. In addition, a shorter periodicity with a larger fill factor is preferred for a higher detection yield of the nanoparticles. It is also noteworthy that SSP mode is useful for particle detection since we can induce denser charge distribution on the interfacing surface where the detection target will be placed.

Since in real experiments, we use unpolarized light with a dark-field illumination with 0° ≤ φ ≤ 360°, we also study σab and σsc properties with different incident directions φ and polarizations, as detailed in Supplementary Section 5. Under the same p-polarization, the spectral characteristics of the meta-atoms do not change significantly along the incident direction. In addition, when Λ=145 nm and λ=450 nm, σsc with p-polarization dominates over that with s-polarization which is 3.5 × 10−15 m2. These findings support that our metasurface can be applied to a simple optical setup, such as a dark-field objective lens, without the need for precise polarization and illumination direction controls, facilitating easy use. Figure 2e shows absorbance peaks of 84% and 38% for p- and s-polarizations at λ=450 nm, respectively. Those absorbance peaks from numerical calculations match the peak wavelength of the scattering background measurement from our metasurface.

The formation of a scattering image by a metasurface
The bright mode of the metasurface determines the total detectable intensity in a particle-sensing event. To understand the radiation mode of a meta-atom element inside the array, we obtain its far-field radiation pattern in the upper half-space by using electric polarization components extracted from the numerical simulation when φ=180° and θin=75°, as shown in Fig. 3a,b47. Note that the scattering radiation properties of the hybridized meta-atom in the sub-diffraction arrays would be different from a non-hybridized silver nanoantenna and cannot be directly measured in the experiment. Figure 3c shows a dark-field image of the effective single meta-atom element using NA=0.9, which corresponds to the collection angle θ=64°. Under dark-field illuminations, each meta-atom element within the array exhibits an identical radiation power (see Fig. 3b). However, they vary in oscillation phases, determined by their horizontal positions on the xy-plane with respect to the incoming light along the x-axis. The total radiation pattern from such meta-atom arrays in Fig. 3d is obtained using the phased array antenna theory. The constructive interference from the meta-atom arrays (19.4 × 19.3 µm2) forms a beam along the reflection of dark-field illumination (φ=0° and θ=75°) as shown in Fig. 3e. It should be emphasized that the element factor of the phased arrays determines the radiation properties of each meta-atom, while the array factor governs the constructive or destructive interference, thereby determining the direction of the Poynting vector into free space. Figure 3f shows that the collective scatterings from phased array meta-atoms do not exhibit energy propagation into the detection NA, leading to a significant reduction in the background signal, in contrast to Fig. 3c. For practical implementation with finite-sized metasurfaces, we also examine the effect of the array size on the background suppression with different numbers of meta-atom elements, as detailed in Supplementary Section 6.

As shown in Fig. 3g, when single or multiple meta-atom elements interact with the detection object, the dark mode can be turned into the radiation mode locally due to the introduced amplitude deviations ΔA and phase retardations Δϕ. These perturbations induce discontinuity in phased arrays, leading to radiation within the detection NA. Figure 3h depicts the far-field radiation difference between such local radiation induced by a 200-nm polystyrene (PS) nanosphere and the dark mode. The local radiation results from the interference of EM fields from meta-atom elements and the scatterer (see details in Supplementary Section 7). Figure 3i shows an interference image of a 200-nm PS nanosphere on top of the metasurface. For better visualization, the colorbars of Fig. 3f, i are linearly scaled to those of Fig. 3c. Here, the scatterer not only adds its own scattering to the total radiation pattern but also modulates the radiation power and phase of neighboring meta-atom elements. Supplementary Section 8 shows ΔA and Δϕ introduced by the nanosphere as a function of the sizes. The phase perturbation of a meta-atom near the nanosphere is obtained by performing the numerical simulation of the metasurface with and without the particle and calculating the electric field phase difference (see methods for numerical calculation details).

Consequently, the significant difference in amplitude and phase of the meta-atoms between the bright mode and the dark mode leads to a greatly improved image contrast compared to the case where a dielectric nanosphere is situated within a homogeneous medium or a planar interface made by conventional media39,40. Supplementary Section 8 shows that the scattering intensity in the image as a function of particle diameter scales much more slowly than pure Rayleigh scattering. This is because the dominant scattering intensity arises from the interference between the meta-atom arrays, the background, and the detection particle rather than from the detection particle itself (see details in Supplementary Section 9)39,40.

Experimental results
To experimentally demonstrate the scattering intensity and contrast enhancement of MAD-iSCAT, we fabricate hexagonal silver meta-atom arrays by nanoimprint lithography on a silica substrate with an additional PMMA protection layer and use PS nanobeads as scatterers. We utilize a broadband supercontinuum laser as the light source to produce interference images of PS nanobeads at different wavelengths ranging from 430 to 700 nm with a dark-field objective in an air environment (see details in Supplementary Section 10). The PS nanobeads have varied sizes of 200, 100, 62, and 45 nm. Figure 4a–d represents the enhanced scattering intensities of PS nanobeads on meta-atom arrays. Figure 4e, f compares those with the case of nanobeads on a bare PMMA layer for diameters of 200 and 100 nm, respectively. In Fig. 4g, h, we identify the same nanobeads with scanning electron microscopy (SEM) as the ground truth images (see details in Supplementary Section 11). In the case of 200- and 100-nm diameter nanobeads, the maximum enhancement factors (IMS/IPMMA) are found to be 11.44 ± 2.08 and 31.45 ± 5.41 at λ=580 and 490 nm, respectively. Meanwhile, the enhancement factors at λ=450 nm are 5.60 ± 1.22 and 20.83 ± 4.50, respectively. We experimentally confirm the scattering enhancement factors of 66.20 and 128.87 for nanobeads of 62- and 45-nm diameters at the resonant wavelength λ=450 nm, respectively. The MAD-iSCAT metasurface exhibits significant scattering enhancement factors over visible wavelengths, as shown in Fig. 4i. Figure 4j presents the normalized detected intensity of the PS nanobeads on the meta-atom arrays and PMMA as a function of nanobead size, with the cubic power of particle sizes shown for comparison (see details in Supplementary Section 12). We also conduct MAD-iSCAT imaging of small nanoparticles in aqueous environments, as shown in Fig. 5a. The imaging system is constructed with a custom axicon-based water-immersion dark-field microscopy48. Fig. 5b, c compares MAD-iSCAT and regular iSCAT using PS nanobeads with diameters of 22, 41, and 81 nm. Regular iSCAT measurements are performed using a standard microscope and a laterally modulating piezoelectric nano-positioning stage with lock-in image detection. The iSCAT images are obtained by integrating 330 frames (effective exposure time (EET) of 116 ms). For those particle sizes, regular iSCAT yields image contrasts of 0.6 ± 0.2%, 3.9 ± 1.7%, and 23.9 ± 3.1% (n=127, 137, and 38), respectively. In contrast, MAD-iSCAT demonstrates image contrasts of 14.9 ± 4.5%, 165.6 ± 32.7%, and 869.1 ± 124.9% (n=451, 283, and 20) for the same particle sizes, employing a single-frame differential process (EET of 1 ms × 2 = 2 ms). MAD-iSCAT shows a contrast enhancement of 26 ± 7.5 for the 22 nm beads compared to regular iSCAT. This comparison highlights the improved sensitivity and high-throughput capability of MAD-iSCAT.

To demonstrate the sensitivity of MAD-iSCAT in biological nanoparticle detection scenarios, we image individual exosomes extracted from MDA-MB-231 human breast cancer cells. Figure 6a, b shows exemplary MAD-iSCAT images of exosomes in an aqueous environment. The size of each exosome is determined through mean-squared displacement analysis4 of identified particles in subsequent frames (see details in Supplementary Section 13). Figure 6c shows the size-dependent image intensity of each detected exosome using MAD-iSCAT and Rayleigh scattering. The performance of MAD-iSCAT offers 1–2 orders of magnitude improvement for various sizes of exosomes.

To further validate the sensitivity of MAD-iSCAT, we conduct experiments using ferritin, a protein with a molecular weight of ~440 kDa. With an exposure time of 1 ms (EET of 2 ms), Fig. 6d demonstrates the MAD-iSCAT measurement for ferritin molecules, with an observed contrast of 7.7 ± 1.4% (n=805). Figure 6e presents an exemplary image showing individual ferritin molecules. The image SNR comparison shows that our approach demonstrates an SNR enhancement factor of 19.8 relative to conventional iSCAT (see details in Supplementary Section 14). This contrast and SNR enhancement for a protein illustrate the improved sensitivity of our method.

Methods
Metasurface fabrication
A plasmonic metasurface that consists of silver meta-atom arrays on a silica substrate is fabricated based on nanoimprint lithography (NIL), reactive ion etching (RIE), and metal e-beam evaporation. A mother mold of the designed meta-atom arrays for NIL is fabricated on a silicon wafer using standard e-beam lithography. The mother mold contains 360 copies of the meta-atom array to ensure a sufficiently large field-of-view during imaging. The silica substrate is prepared with two spin-coated resist layers. One is a PMMA underlayer as the lift-off layer and the other is a UV-curable resist layer at the top. NIL is performed to define the meta-atom structures on the UV resist layer. After curing the UV resist and releasing the mother mold, the residual UV resist and the underlying PMMA underlayer are both etched by RIE to expose the bottom silica substrate. 60 nm silver is evaporated onto the sample afterward with e-beam evaporation, followed by a lift-off process. After NIL, a PMMA reflow step is employed to planarize the metasurface as well as to protect the silver meta-atoms from oxidation and potential damage. A thin PMMA spin-coating process is repeated 3 times on the metasurface to produce a conformal coating that fully encloses the meta-atoms while maintaining a flat top surface. The total thickness of the PMMA planarization layer is around 75 nm. The 60 nm meta-atoms remain close to the substrate surface.

Experimental setup
A supercontinuum laser light source (SuperK Extreme EXB-6, NKT Photonics) is utilized to acquire MAD-iSCAT images at wavelengths ranging from 430 to 700 nm. The laser power of the dark-field ring at the center wavelength of λ=450 nm is measured to be 0.15 mW. The light source is delivered to the metasurface surface through a dark-field objective lens (UMPlanFl, ×100, 0.9 NA, Olympus). The incident angle of the dark-field objective lens is 75°. An electron-multiplying charge-coupled-device camera (iXon EM+ DU-897, Andor Technology) with a 100-ms exposure time and 160 accumulations is employed to obtain a large dynamic range for recording background noise and strong scattering signals from meta-atom arrays. The scattering spectrum of the metasurface is measured using a spectrometer (Shamrock 303i, Andor Technology) and a halogen lamp as the illumination source. To verify the amplified scattering signals from small nanoparticles, PS nanobeads with diameters of 200 (NanosphereTM Size Standards, 3200A, Thermo Fisher Scientific), 100 (FluoSpheresTM carboxylate, F8803, Invitrogen), 81 (NanosphereTM Size Standards, 3080A, Thermo Fisher Scientific), 62 (NanosphereTM Size Standards, 3060A, Thermo Fisher Scientific), 45 nm (FluoSpheresTM carboxylate, F8792, Invitrogen), 41 (NanosphereTM Size Standards, 3040A, Thermo Fisher Scientific), and 22 (NanosphereTM Size Standards, 3020A, Thermo Fisher Scientific) are utilized after dilution. The size deviation for each size is provided as 6, 7, 3, 3, 8, 4, and 2 nm by the manufacturer, respectively. Each PS bead solution is drop-casted onto the metasurface after cleaning the interfacing surface with deionized water. Image processing is conducted using home-built MATLAB R2023b codes and ImageJ functions for data analysis and presentation. In the case of 62-nm nanobeads, applying background subtraction results in a scattering image with enhanced intensity without requiring temporal differentiation (Fig. 4c). In the case of 45-nm nanobeads, we apply temporal differentiation (Fig. 4d). In Fig. 4j, each scattering intensity is normalized by that of an isolated silver nanoantenna. For the exosome detection, we use the water-immersion objective lens (UPlanSApo, ×60, 1.2 NA, Olympus). The laser power of the dark-field ring at λ=450 nm is measured to be 1.1 mW. An sCMOS camera (ORCA-Flash4.0, C13440-20CU, Hamamatsu) with an exposure time of 487.218 µs is employed. The dark-field ring at the back focal plane of the water-immersion objective is generated and its size adjusted using an axicon and two lenses in series, as illustrated in Fig. 5a. An example of the dark-field ring is shown in the inset of Fig. 5a. For MAD-iSCAT measurements for PS nanobeads and ferritin proteins (F6754-1VL, Sigma-Aldrich) in aqueous environment, we employ an identical optical setup as used for exosome detection with an additional ×1.6 magnifier and an exposure time of 1 ms. The PMMA surface is incubated with poly(allylamine hydrochloride) (PAH) to facilitate adhesion. No frame averaging is applied. Images are processed using stabilization, Gaussian filtering to reduce noise, and background correction. For the 80-nm PS nanobeads, the contrast is obtained using frames separated by 10 frames, whereas for the 20- and 40-nm beads, neighboring frames are used. Incident angles are selected to satisfy the same plasmon modes excitation with kx = k0 sin 75° = kwater sin 47° = kPMMA sin 40°.

For iSCAT measurement, we use a studied iSCAT setup incorporating a nano-positioning stage (Nano-Drive 85, Mad City Labs) to enable translational modulation of scatterers on a glass substrate7. This setup allows us to isolate the scattered signal from the background. An sCMOS camera (ORCA-Flash4.0, C13440-20CU, Hamamatsu) is employed. The experiments are conducted with an exposure time of 351 μs and stage modulation at 25 Hz. The five periods of translation modulation are utilized to acquire the scatter signal by eliminating the background. This is achieved by discarding the frames captured during stage translation, resulting in an integration of 330 frames. A high-NA objective lens (UAPON, 100×, 1.49 NA, Olympus) is used with an additional ×1.6 magnifier and the glass surface is treated with poly-D-lysine in PBS (1 mg/mL) to facilitate adhesion of negatively charged PS beads.

Sample preparation
Human breast cancer cell line MDA-MB-231 (HTB-26) is obtained from the American Type Culture Collection and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich). Small extracellular vesicles enriched in exosomes are purified from the conditioned medium (CM) using differential centrifugation. Briefly, CM is collected from cells incubated in serum-free DMEM for 24 h, precleared by centrifugation at 500 × g for 15 minutes, and then at 10,000 × g for 20 min. Extracellular vesicles are passed through a 0.22-μm filter, pelleted by ultracentrifugation at 110,000 × g for 120 min, and washed in PBS using the same ultracentrifugation conditions. The exosomes suspended in phosphate-buffered saline (PBS) solution are sealed in an aqueous chamber made of the MAD-iSCAT metasurface and a coverslip so that their diffusion dynamics can be captured.

Simulations
Finite-element analysis of EM fields for the metasurface is conducted using COMSOL 6.0 Multiphysics. Initially, a unit cell (Λ × √3Λ nm2) with a height of 1 µm is employed, applying periodic boundary conditions laterally and perfectly matched layer (PML) boundary conditions along the vertical direction. The near-field distributions are shown in Supplementary Section 16. The analysis aims to investigate σab and σsc spectra of meta-atom elements, the absorbance spectrum of meta-atom arrays, charge distribution, and eigenmode analysis. Field intensity normalization is performed using reference calculations conducted in the absence of meta-atom arrays. Incident light with different polarizations, directed from the top medium (air) at φ=180° and θin=75°, is considered. The mesh size ranges from 1 to 25 nm. Refractive indices of silver, PS, PMMA, and SiO2 are taken from the references55–58. In Fig. 2b, d, the wavelength and periodicity are swept with different step sizes to reduce the simulation time and interpolation is applied afterward. Subsequently, to examine the formation of interferometric scattering images by the metasurface, a larger array of unit cells, laterally spanning 10Λ along the x-axis and 5√3Λ along the y-axis is employed. The EM field distribution of meta-atom arrays without a detection nanosphere is calculated using Floquet periodic boundary conditions for in-plane directions. Scattered EM field distribution by a detection target is obtained by replacing Floquet periodic boundary conditions with PML boundary conditions21. Here, the phase perturbation of the nanoantenna caused by the particle was obtained by the phase difference of the electric component in the meta-atom between the first and second FEM calculations. The mesh size ranges from 1 to 50 nm. In numerical simulation, the edges of the nanoantenna are truncated to be round-shaped, considering practical experimental constraints. Electric polarization components extracted from the finite-element analysis are utilized to generate far-field properties of the detection target and neighboring meta-atom elements45. Here, far-field properties of the remaining meta-atom elements are confirmed to be almost identical as a result of the first simulation with the Floquet boundary condition. The far-field radiation pattern is calculated at a distance of 2 mm from the array center. For sufficiently large meta-atom arrays, edge scattering nearly interferes with light signals within the array. A home-built MATLAB code is utilized to generate a collective scattering of meta-atom arrays by adding more meta-atom elements for larger array sizes, whose far-field properties remain unmodified by the detection target.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript reports of an interferometric technique, MAD-iSCAT, to improve nanoparticle detection sensitivity. Conventional i-SCAT uses a glass slide to accommodate the sample and generate the reference beam, while in MAD-iSCAT this reference beam is generated using a plasmonic metasurface. By engineering the metasurface and the illumination, a dark-field detection scheme is demonstrated. The presence of a particle scatters the nearfield mode of the structure into the far-field. The authors report a modified contrast equation (Eqn. 5,6) because of an additional ‘bright mode’ arising from the meta-atoms in the presence of a scatterer. As a result, high-contrast images of nanoparticles with enhanced sensitivity over conventional iSCAT is quantified and demonstrated. The results are interesting and the proposed concept is similar to Nature Communications volume 13, 2298 (2022).\n(1) The following is not clear from the manuscript with regards to image processing and the contrast equation arising from it:\nThe detected signal in conventional iSCAT in the presence of a scatterer is I_s=|E_r |^2+|E_s |^2+2|E_s ||E_r | cos⁡α, where\n|E_r | is the reference, |E_s | is the scattered and α is the phase difference between these fields. After subtraction of two\nsuccessive images (with and without a particle) the equation for the detected signal is approximated as 2|E_s ||E_r | cos⁡α,\nassuming that |E_r | is constant between the different temporal frames. The contrast of the processed image is C≈(2|E_s\n||E_r | cos⁡α)/I_bg , where I_bg=|E_r |^2.\nIn MAD-iSCAT, because of the dark-field detection, ideally no light should reach the camera and hence, this\nsubtraction scheme isn’t possible, so C≈(|E_r |^2+2|E_s ||E_r | cos⁡α)/I_bg . For high-visibility, the magnitudes of the interfering fields\nmust be very close. But since for nanoparticles, |E_s | is low, then |E_r | must also be low if you are to get a good contrast\nimage. So if you assume that |E_r |≈|E_s | and neglect |E_r |^2 or you subtract the background, then the processed image\ncontrast C≈(2|E_s ||E_r | cos⁡α)/I_bg , which is the contrast achievable using a conventional iSCAT. However, experimentally\nthere will always be some light reaching the camera, also seen in Fig. 3f. But then again if you perform temporal subtraction,\nthe contrast of the processed image will still be C≈(2|E_s ||E_r | cos⁡α)/I_bg .\n(a) Please elaborate as to how this Eqn. (5,6) in the manuscript is arrived at and\n(b) also why is MAD-iSCAT different from Zhang et.al.s paper cited above? In that work also, light scattered from the\ncoverslip’s surface roughness interferes with the scattered light by the sample. It will be beneficial for the readers to clearly\nunderstand how you derived the contrast equation, what assumptions are made and how is it different from the above\nmentioned paper? This will bring the novelty of the paper evident.\n(c) When will this assumption break? For e.g., the plot in Fig. 3l, when will they intersect? Of course, iSCAT is for the\ndetection of nanoparticles. So this question is purely out of curiosity to see when these assumptions break.\n(2) Lines 54-55: ‘‘However, as the light scattering cross-section scales with the sixth power of the particle’s diameter…’’\n(a) Rayleigh scattering scales with sixth power of the particle radius and not diameter. Please check and correct.\n(b) Fig. 4l – black dashed line represents cubic power. Is this plotted as a function of radius or diameter?\n(c) Fig. 4j – gray dashed line represents sixth power dependence.\n(d) Explain Fig. 4l and Fig. 4j based on Rayleigh scattering scaling inversely as sixth power of radius. Please mention the\nvalues of the scattering intensity at 50 nm, 100 nm, upto 200 nm for both MAD-iSCAT and Rayleigh scattering. It is provided\nin the plot, but it is easier to compute the dependence on particle size from numbers directly given.\n(3) Fig. 4k --- can you explain why the particle locations have negative pixel values? Isn’t particle location supposed to be\naffecting the pixel value because of Gouy phase? Then why do they have positive values in the Mad-iSCAT?\n(4) Fig. 4f – it is mentioned that all particles are validated with ground truth fluorescence and SEM images.\n(a) Is this done for all the results shown in the manuscript?\n(b) How was it ensured that fluorescence signal didn’t leak into the iSCAT measurements?\n(5) Since MAD-iSCAT is a near-field technique, it will be good to have a plot illustrating the field strength as a function of\ndistance from the meta-atom surface.\n(6) It was not clear whether the SSP mode was utilized for all the experimental results demonstrated in the manuscript.\n(7) Fig. 2a, can you provide the simulation results with and without the nano scatterer. This will aid the reader to see how the\nscatterer affects the confined mode.\n(8) Fig.3 - What is the spatial extent covered by the polar plots with respect to the meta-atom array? Maybe add a scale bar\nto highlight it and also, where exactly on the meta-atom array is this measurement carried out? Is it at the intersection of the\ndotted lines in Fig. 3a?\n(9) Fig.1(c-f) – a suggestion is that the plots can be better represented. For example, wavelength range along the x-axis will\nbe useful as these meta-atoms do not operate at all wavelengths. Another point of confusion was the grey plot in the case of\nthe dark-mode. This can be understood after reading the manuscript, but a bit more detailed explanation in the figure caption\nwill also help.\n(10) Though the array spans a few microns, what is the field-of-view achievable (FoV)? Is it 9.6 х 9.3 μm2? Do you think you\ncan enhance the FoV? It will be useful to highlight these issues in the manuscript.\n(11) MAD-iSCAT is a near-field detection technique. Conventional iSCAT can help isolate particles within the focal volume\nof the detection objective. Therefore, though it is obvious, highlighting the limitations of MAD-iSCAT is important\nespecially for biologists.\n(12) Fig. 4j – please correct the legends in the plot.\n(13) Due to enhanced field strengths, can particles get trapped to the high field intensity?\n(14) Have you quantified any temperature changes due to plasmonic effects? Will this affect the biological samples under\nstudy?", "reviewer_2": "The manuscript entitled “Meta-amplified dark-field interferometric scattering microscopy” has undergone one round of peer\nreview, and the authors have revised the manuscript in response to the reviewers’ comments. After reviewing the revised\nversion and the response letter, I find that the authors have addressed the earlier concerns in a satisfactory manner.\nA notable strength of this work lies in the central concept of exploiting the dark mode of the plasmonic meta-atom array. By\nengineering the metasurface so that its collective radiation is suppressed in the absence of objects, the authors achieve a\nbackground-free baseline. Upon the attachment of nanoparticles, local perturbations induce a transition from the dark mode\nto a bright radiation mode, resulting in a strong and detectable signal. This mechanism represents an original idea that\nclearly differentiates MAD-iSCAT from previously reported iSCAT approaches. The physical picture is compelling and\nprovides a solid foundation for the reported contrast enhancements. I strongly recommend this work for publication after one\nfurther round of minor revisions.\n1. On page 3, line 57, the statement ‘Interferometric imaging addresses this challenge by leveraging the interference of\nscattered light from nanoscale objects with reference light, yielding label-free images with high sensitivity and resolution’\nseems to overstate the claim of ‘high resolution’. Interferometric imaging does not inherently surpass the diffraction limit;\nrather, it improves localization precision or effective resolution through enhanced signal-to-noise.\n2. In Fig. 1a, the schematic illustrates a reflection geometry where the illumination incidence angle is drawn larger than the\ndetection NA cone, suggesting complete separation between illumination and detection. However, for a 0.9 NA dark-field\nobjective, the illumination annulus specified by the manufacturer is typically NA = 0.8–0.9, corresponding to ~53°–64° in air,\nwhich partially overlaps with the collection cone (0–64°). This discrepancy between the schematic, the stated 75° incidence\nangle, and the actual objective specification should be clarified. In principle, one could increase the laser input incidence\nangle beyond the nominal annulus by back focal plane engineering while restricting the detection NA to a smaller cone. If\nsuch a strategy was employed, it would be important for the authors to explicitly state this in the Methods, as it would resolve\nthe apparent inconsistency.\n3. Despite the conceptual novelty, one potential weakness of the approach is that achieving a perfect dark mode in practice\nmay be difficult. Fabrication imperfections, array inhomogeneities, and finite-size effects could lead to residual scattering\nfrom the meta-atoms themselves, limiting the reproducibility and robustness of the method. It would be helpful if the authors\ncould briefly acknowledge this limitation in the manuscript, as it provides important context for the practical implementation of\nMAD-iSCAT. Although the authors emphasize that the dark mode of the metasurface prevents radiation into the detection\nNA, the fact that a measurable scattering spectrum of the bare metasurface has been obtained indicates that residual\nscattering does exist. This residual signal, even if weak, could act as background noise. Reviewer #2 already noted that the\nlimited sensitivity is likely due to fabrication imperfections, resulting in nonspecific and nonuniform background signals. In\ntheir response, the authors acknowledged that improving fabrication quality and array uniformity could further reduce\nbackground scattering, which indirectly admits that perfect dark mode suppression is difficult to achieve in practice.\nHowever, this limitation is not explicitly described in the main text. It would strengthen the manuscript if the authors could\nbriefly acknowledge this point as a limitation, or provide a short discussion to clarify to what extent intrinsic meta-atom\nscattering contributes to the background under the dark mode condition.\n4. On page 7, line 177, the statement ‘In addition, a shorter periodicity with a larger fill factor is preferred for a higher\ndetection yield of the nanoparticles’ appears. This raises the question of how such a design fundamentally differs from using\na continuous metallic film. Since a bare film would not support the array-induced dark mode that underpins the proposed\nmechanism, it would be helpful if the authors could clarify this distinction more explicitly in the manuscript, particularly\nin terms of background suppression, scattering enhancement, and overall detection sensitivity.\n5. Fig. 3l shows that the scattering intensity decreases much more slowly with particle size than expected from Rayleigh\nscattering. The authors attribute this to interference between the particle and the meta-atom array, suggesting an\napproximate ∼d³ scaling. It remains unclear, however, whether this cubic dependence arises primarily from the general\ninterference mechanism in iSCAT or from a specific particle–meta-atom interaction effect unique to the MAD-iSCAT\ngeometry. Since the particle–meta-atom interaction itself should also be size-dependent, it would strengthen the discussion\nif the authors could elaborate on how this interaction contributes to the observed scaling behavior. While Figs. 3l and 4j\ndemonstrate weaker-than-Rayleigh scaling down to ~20 nm particles, it remains uncertain how far this trend can be\nmaintained for even smaller diameters. In particular, it would be valuable for the authors to discuss whether the scaling\nslope is expected to remain shallow below 20 nm, and what sets the ultimate detection limit for MAD-iSCAT.\n6. In Fig. 5a,b the authors state that exosome sizes are determined through mean-squared displacement (MSD) analysis,\nwith trajectories shown in Supplementary Section 9. However, only sample trajectories are provided, and it is not clear how\nthese lead to MSD values and size estimates. A brief explanation of how MSD is calculated from the trajectories and\nconverted into particle size (e.g., via the diffusion coefficient and Stokes–Einstein relation) would improve clarity.\n7. On page 10, line 301, the manuscript states: “This leads to a higher dark mode background level as well as a degraded\nTP2 mode in silver meta-atoms, which is reflected in the broader wavelength response shown in Fig. 4i compared to the\nideal meta-atom resonance.” However, Fig. 4i presents the scattering enhancement factor over the visible range, not a\nresonance broadening effect. This suggests that the figure reference may be incorrect. The authors should verify the figure\nlabeling and clarify which figure actually demonstrates the resonance broadening due to fabrication imperfections— perhaps\nFig. 2e?\nOverall, the manuscript presents a novel and compelling approach with clear potential impact. I would strongly support its\npublication once the above clarifications and minor revisions are addressed.", "reviewer_3": "Review Report:\nThe article titled, “Meta-amplified dark-field interferometric scattering microscopy” by Lee et al proposes a technique (MAD-\niSCAT) that combines dark field microscopy and interference to detect nanoparticles on a plasmonic metasurface. In this\nreport, the authors demonstrated the technique by detecting polymer-nanoparticles and exosomes. They claimed the method\nto be better than the existing iSCAT technique in terms of sensitivity and contrast. I noted that the first three figures are\nlargely schematic and simulation. The actual results are mainly limited to Fig. 4 and Fig. 5. Moreover, the results are shown\non nanoparticles, rather than real systems such as cells, making the work somewhat limited in scope. Here are my detailed\ncomments :\n1. One of the major concerns is the lack of validation and poor understanding of what transpires at the metasurface related to\nfield-particle interaction.\n2. The particles used by authors are comparatively large. The technique (MAD-iSCAT) needs to demonstrate for small\nparticle (<10 nm nanoparticles and < 300 kDa proteins) detection. This will strengthen the claim experimentally that MAD-\niSCAT is better than iSCAT.\n3. As claimed, high throughput and sensitivity needs further justification.\n4. From the present results, the proposed technique does not appear to offer significant advance compared to iSCAT.\n5. The abstract lacks to mention key details / outputs of the MAD-iSCAT technique. This include, (i) radiation modes and\nwhy they are special, (2) contrast enhancement, and (3) the level of suppression of the background. These information\nshould have been mentioned in the abstract.\n6. The manuscript lacks schematic of detailed optical setup in the main manuscript. This is essential to understand the\nproposed technique, instrumentation, and system development.\n7. The authors mention, “By careful engineering meta-atom arrays, ………. the presence of nanoscatterer.” The statement is\nvague, needs elaborate explanation, and must be substantiated.\n8. In the results section, the authors claim high contrast. This is not substantiated and far from what is evident from the\nimages shown in Fig. 4 and Fig. 5.\n9. One of the shortcomings of the present paper is the lack of experimental validation and application on real systems such\nas cell. Most of the results appear to be simulation based (Fig. 1-3). Actual experimental results are largely limited to\nFig. 4 and Fig. 5. It should be other way round i.e., experimental validation must have a larger footprint.\n10. In Fig. 5, direct comparison of MAD-iSCAT and iSCAT image is missing.\n11. There are many such points that needs to be take care-off in the manuscript.\nWhile the technique appears to be somewhat new, but it does little to advance the field and lacks experimental validation &\napplication. This is in addition to the detailed comments by other two reviewers. The investigation appears to be in its initial\nstages and requires a lot more conclusive validation to show that MAD-iSCAT is better that iSCAT. Specifically, the images\nare not convincing and appears to be mild enhancement (visually). Overall, the work may be premature for publication at this\nstage.", "reviewer_4": "This manuscript reports the use of a plasmonic metasurface to perform interferometric scattering microscopy (iSCAT). The\nauthors claim that the radiation mode supported by the metasurface interferes with the scattered light from nanoparticles,\nthereby producing high-contrast interferometric images. Reviewer #1 and Reviewer #2, both experts in iSCAT, have already\nraised critical questions regarding the novelty and performance of the work, to which the authors have responded. As a\nspecialist in plasmonics and metasurfaces, I will not repeat the iSCAT-related issues already discussed, but instead provide\nthree comments from the perspective of employing metasurfaces for iSCAT.\n1. The primary novelty of this work lies in the conceptual innovation of employing a metasurface for iSCAT for the first time.\nHowever, the manuscript lacks clarity in its presentation and fails to provide a concise physical picture. As a result, the\nauthors introduce a section, “Principles of contrast amplification in interferometric images,” supported by eight equations; this\nstyle is more reminiscent of Physical Review rather than Nature Communications. Similarly, the section “The formation of\nscattering image by metasurface” relies heavily on numerical simulations to justify the mechanism behind, which is not a\nparticularly strong form of evidence.\n2. It is well established that plasmonic metasurfaces can enhance light–matter interactions and consequently boost\nscattering signals. Therefore, the reported contrast enhancement by one to two orders of magnitude (Fig. 4) is in fact\nexpected. However, such enhancement comes at the cost of local heating due to metallic nanostructures. This heating may\nseverely limit the applicability of iSCAT for biologically relevant tasks such as single-molecule tracking. The manuscript\ndoes not discuss this critical issue, and such a discussion should be included.\n3. Comparative analysis is essential to place the method into context. For example,\nTable R1-2 in the response letter provides a very useful benchmark. I strongly recommend that the authors incorporate this\ntable into the main text and expand the discussion accordingly.\nIn summary, the manuscript presents an interesting concept and demonstrates promising experimental results, but the\nnovelty and implications are not clearly articulated. I recommend a major revision before the work can be considered further."}, "author_response": "Color codes used in this response letter:\nBlue: original reviewer comments;\nBlack: our response;\nRed: Modification in the manuscript.\n══════════════════════════════════════════════════\nReviewer #1 (Remarks to the Author):\nThe manuscript reports of an interferometric technique, MAD-iSCAT, to improve\nnanoparticle detection sensitivity. Conventional i-SCAT uses a glass slide to\naccommodate the sample and generate the reference beam, while in MAD-iSCAT\nthis reference beam is generated using a plasmonic metasurface. By engineering\nthe metasurface and the illumination, a dark-field detection scheme is\ndemonstrated. The presence of a particle scatters the nearfield mode of the\nstructure into the far-field. The authors report a modified contrast equation (Eqn.\n5,6) because of an additional ‘bright mode’ arising from the meta-atoms in the\npresence of a scatterer. As a result, high-contrast images of nanoparticles with\nenhanced sensitivity over conventional iSCAT is quantified and demonstrated. The\nresults are interesting and the proposed concept is similar to Nature\nCommunications volume 13, 2298 (2022).\n(1) The following is not clear from the manuscript with regards to image processing\nand the contrast equation arising from it: The detected signal in conventional\niSCAT in the presence of a scatterer is I = |E |2+|E |2+2|E ||E |cosα, where |E | is the\ns r s s r r\nreference, |E | is the scattered and α is the phase difference between these fields.\ns\nAfter subtraction of two successive images (with and without a particle) the\nequation for the detected signal is approximated as 2|E ||E | cosα, assuming that\ns r\n|E | is constant between the different temporal frames. The contrast of the\nr\nprocessed image is C≈(2|E ||E | cosα)/I , where I =|E |2. In MAD-iSCAT, because\ns r bg bg r\nof the dark-field detection, ideally no light should reach the camera and hence, this\nsubtraction scheme isn’t possible, so C≈(|E |2+2|E ||E |cosα)/I . For high-visibility,\nr s r bg\nthe magnitudes of the interfering fields must be very close. But since for\nnanoparticles, |E | is low, then |E | must also be low if you are to get a good contrast\ns r\nimage. So if you assume that |E |≈|E | and neglect |E |2 or you subtract the\nr s r\nbackground, then the processed image contrast C≈(2|E ||E |cosα)/I , which is the\ns r bg\ncontrast achievable using a conventional iSCAT. However, experimentally there\nwill always be some light reaching the camera, also seen in Fig. 3f. But then again\nif you perform temporal subtraction, the contrast of the processed image will still\nbe C≈(2|E ||E |cosα)/I .\ns r bg\nconventional iSCAT, the image contrast remains C≈(2|E ||E|cosα)/I whether E is weak\ns r bg r\ncompared to E or not. However, the contrast mechanism of MAD-iSCAT is not\ns\ndetermined by a constant E; the locally varied E’ due to the presence of the nanoparticle\nr r\nproduces an enhanced scattering mechanism, as we will clarify below.\nIn both conventional and dark-field iSCAT, subtracting consecutive frames\nremoves the |E|2 term and isolates the interference term 2|E ||E|cosα, giving rise to a\nr s r\nhigh contrast detection of the weak scattered field of |E |, which is dependent on E. As a\ns r\nstrong |E|2 can saturate photon detectors, a dark-field iSCAT has an advantage of using\nr\nthe detection dynamic range effectively. While the ideal background intensity I is zero in\nbg\ndark-field detection, in practice, any substrate that provides the reference electric field,\nwhether regular glass (Nat. Commun. 13, 2298 (2022)) or the sub-diffraction grating as\nour plasmonic metasurface, introduces a nonzero I that needs to be subtracted.\nbg\nHowever, despite the better utilization of detection dynamic range, we agree with the\nreviewer that iSCAT with conventional dark-field processes the same image contrast as\nstandard iSCAT in the form of C≈(2|E ||E|cosα)/I . What makes MAD-iSCAT unique is\ns r bg\nthat the static E changes into a bright plasmonic mode E’ locally due to the perturbation\nr r\nof a nanoparticle nearby and interferes with the scattered field of the nanoparticle.\nSubtraction of dark-mode frames (without a particle present) removes residual\nbackground, leaving only the combined bright-mode and interference signals. Here, the\nbackground signal for MAD-iSCAT in a practical experiment is largely governed by the\narray size and thereby the leakage edge scattering from the boundary of the metasurface,\nas detailed in the Supplementary Sections 6 and 15. Unlike earlier dark-field iSCAT,\nwhere |E | is regarded as constant (always a dark-mode with or without the\nr\ndetection particle), MAD-iSCAT leverages the bright-mode (only with the detection\nparticle) of |E ’|to boost the scattering signal.\nr\n(a) Please elaborate as to how this Eqn. (5,6) in the manuscript is arrived at and\nThank you for the valuable comments. We have clarified the equations as follows.\nThe original Equation (5)\n(Eq. R1-1)\n𝐼𝑆 2∑(𝑖,𝑗)|𝐸𝑖𝑗||𝐸𝑠|cos(𝛼𝑖𝑗)\n2\nprovides a generalized formul\n𝐶\nat\n=\nion\n𝐼𝑏 𝑔\no\n≈\nf iSCA\n|∑\nT\n( 𝑖,𝑗\ni\n) m𝐸𝑖𝑗\na\n|\nge contrast by decomposing the\nreference light as a superposition of all fields originating from every position of the\ndetection area. This superposition is crucial to incorporate the local field variation of MAD-\niSCAT. In the case of a regular or dark-field iSCAT, the relative phase and amplitude from\na glass substrate are fixed, and the superposition can be simplified to a static\nfield profile , leading to the well-known simplified form of\n∑(𝑖,𝑗)𝐸𝑖𝑗\n𝐸𝑟\n2\n(Eq. R1-2)\n𝐼𝑆 2|𝐸𝑟||𝐸𝑠|cos(𝛼) 2|𝐸𝑠|cos(𝛼)\n𝐶\nan\n= eo𝐼𝑏\nu\\𝑔\ns\n≈\nly enh\n| a𝐸𝑟\nn\n|\nces th\n=\ne num\n|𝐸𝑟\ne\n|\nrator as is varying locally\ndepending on the scatter. Exploiting the bright super scattering plasmon (SSP) modes of\n𝐸𝑖𝑗\nour plasmonic meta-atoms, the original Equation (5) is modified for MAD-iSCAT to the\n‐\noriginal Equation (7) of the manuscript as,\n(Eq. R1-3)\n′ 2 2\n|∑(𝑖,𝑗)𝐸𝑖𝑗 +𝐸𝑠| −|∑(𝑖,𝑗)𝐸𝑖𝑗|\n2\n𝐶′ = |∑(𝑖,𝑗)𝐸𝑖𝑗| .\nHere, represents the residual background intensity associated with the dark-\n2\nmode, while signifies the perturbed field from the bright SSP mode induced by the\n|∑(𝑖,𝑗) 𝐸𝑖𝑗|\npresence of a p′article. The revised MAD-iSCAT contrast formula thus improves both the\n𝐸𝑖𝑗\nnumerator and denominator. Compared to (Nat. Commun. 13, 2298 (2022)), the\nnumerator gains an altered bright mode term, instead of relying solely on interfering with\nthe same reference light. Unlike previous approaches that treat the background as a static,\n‐\nnoncontributing term, MAD iSCAT leverages amplitude and phase perturbations in the\nsub diffraction grating metasurface to collect extra photons from the bright mode,\n-\nresulting in a substantial boost in overall detection signal.\n-\n-\nThe original Equation (6)\n(Eq. R1-4)\n′\ndefines the signal to noise ratio\n𝑆\n(\n𝑁\nS\n𝑅\nNR\n=\n)\n|\n∑b(a𝑖,s𝑗)e\n𝐸\nd𝑖 𝑗on\n+\nt\n𝐸\nh𝑠e\n| \nshot noise limited optical detection\nmodel, where the noise scales as the square root of the detected photon count, as\n‐ ‐ ‐ ‐\n. (Eq. R1-5)\n𝐼\nIn prior iSCAT work (Nat. Commun𝑆. 𝑁13𝑅, =22√9𝐼8= (2√0𝐼22) and others), the SNR is expressed\nas\n. (Eq. R1-6)\n2|𝐸𝑏||𝐸𝑠|cos(𝜃)\nFor MAD iSCAT, we 𝑆 a 𝑁 cc 𝑅 o = rdi√n|g𝐸𝑏| 2 +d2e|𝐸𝑏|f|𝐸i𝑛|e|𝐸d𝑠| cito sa(𝜃s) +th|𝐸e𝑠 |o 2 r ≈ igi 2 n | a 𝐸 l 𝑠 E |c q o u s a (𝜃 tio ) n (6)\n‐\n. (Eq. R1-7)\n′ 2\n|∑(𝑖,𝑗)𝐸𝑖𝑗 +𝐸𝑠|\n′\n𝑆𝑁𝑅 = ′ = |∑(𝑖,𝑗) 𝐸𝑖𝑗 +𝐸𝑠|\n√|∑(𝑖,𝑗)𝐸𝑖𝑗 +𝐸𝑠|\nWe thank the reviewer for the valuable comment. In addressing other reviewer concerns,\nwe have substantially revised those original Equations (5-7) in the manuscript by\nincorporating a more concrete physical background. Please refer to Revision 1-2 for the\ndetailed updates.\n3\n(b) also why is MAD-iSCAT different from Zhang et.al.s paper cited above? In that\nwork also, light scattered from the coverslip’s surface roughness interferes with\nthe scattered light by the sample. It will be beneficial for the readers to clearly\nunderstand how you derived the contrast equation, what assumptions are made\nand how is it different from the above mentioned paper? This will bring the novelty\nof the paper evident.\nThank you for your insightful question. Here, we clarify the novelty of our work compared\nto Zhang et al. (Nat. Commun. 13, 2298 (2022)), in which the random surface roughness\nacts as a reference field that interferes with light scattered by the target particle. In MAD-\niSCAT, we demonstrate a novel detection mechanism in which the reference field itself\ngoes through significant perturbation during the detection process by using an artificially\nengineered metasurface rather than the random roughness to enhance scattering\ndetection. To validate this concept, we have undertaken the following steps.\n(1) First, we provide a comprehensive theoretical framework by introducing the sub-\ndiffraction phased array antenna concept to explain how engineered surface features\nenhance iSCAT detection. This model elucidates why surface roughness serves as an\neffective reference source and guides the rational design of optimized scattering\nsubstrates.\n(2) Second, we demonstrate that the proposed super-scattering plasmon (SSP) mode of\nplasmonic meta-atoms produces much stronger interaction with the nanoparticle\ncompared to a rough glass surface.\nAs a result, we experimentally confirm that our plasmonic metasurface intensively\ninteracts with the detection object more than the rough surface on the glass. In general,\nthe detection intensity of iSCAT is described as\n(Eq. R1-8)\n2 2\nWhen the nanoparticle is lar𝐼g=e (|𝐸𝑟| + 2|𝐸)𝑟, |t|h𝐸e𝑠 |dcoeste(𝛼ct)e+d |in𝐸t𝑠e|nsity scales with the sixth\npower of the particle diameter as the third term dominates. For smaller particles, where\n|𝐸𝑟| ≪ |𝐸𝑠|\n, the second term dominates, and the detection intensity has a cubic power\ndependence on the particle size. In (Nat. Commun. 13, 2298 (2022)), the cubic power\n|𝐸𝑟| ≫ |𝐸𝑠|\ndependence starts to appear for particle sizes smaller than 22 nm. In contrast, MAD-\niSCAT shows the approximate cubic power dependence at the particle size over 100 nm\nand the sixth power dependence is difficult to observe even for the 200-nm particle size.\nThis represents that the plasmonic meta-atom element has much larger scattering cross-\nsections than the roughness of the bare glass substrate that can interact with the\ndetection object.\nWe appreciate this insightful comment. We have revised the manuscript accordingly to\naddress the reviewer's suggestion.\nMain text:\n“… The perturbed electric field from meta-atoms generates strong interferometric signals, which\nexhibit an approximate cubic power law dependence that persists even for comparably larger\nparticle sizes where Rayleigh scattering dominates. …”\nTheoretical framework for contrast and SNR enhancement in MAD-iSCAT\nIn response to the reviewer’s comment, which was also raised by other reviewers, we\npresent a more comprehensive theoretical framework and elucidate the precise\nmechanism underlying MAD-iSCAT as below.\nAs noted above, our plasmonic metasurface operates as a phased-array antenna\nsupporting a bright mode. For an ideal metasurface comprising many meta-atoms, the\narray factor (AF) in the null directions where destructive interference occurs is given by\n. (Eq. R1-9)\n𝑁 𝑗Ψ𝑛\nWhen a detection particle re\n𝐴\ns\n𝐹\nid𝑖𝑑e𝑒s𝑎 𝑙n(𝜃 e\n,\n\n𝜑\n,\n\n𝜙\n)\nsi\n=\n=∑𝑛 =1\n𝑒\n=\n0\n, it introduces an additional\nphase shift Δɸ. For a small particle, assuming this perturbation affects only the k-th\nelement in two-dimensional arrays, the modified array factor becomes\n𝑗Ψ𝑛 𝑗(Ψ𝑘+𝛥ɸ)\n𝐴𝐹𝑝𝑒𝑟𝑡𝑢𝑟𝑏𝑒𝑑(𝜃,𝜑,𝜙) = ∑𝑒 +𝑒\n𝑛≠𝑘 . (Eq. R1-10)\n𝑗Ψ𝑘 𝑗(Ψ𝑘+𝛥ɸ) 𝑗Ψ𝑘 𝑗𝛥ɸ\nFor a given inciden\n=\nt i\n𝐴\nn\n𝐹\nte𝑖𝑑n𝑒s𝑎i𝑙ty\n−\nI\n0𝑒\n, the\n+\nsc\n𝑒\nattered p\n=\now\n𝑒\ner in\n( 𝑒\nthe b\n−\nrig\n1\nh\n)\nt mode from the phased\narray, formed by plasmonic meta-atoms each characterized by a single-element\nscattering cross section σ ,is\nSSP\n2\n𝑃𝐵𝑀 = 𝜎𝑆𝑆𝑃𝐼0|𝐴𝐹𝑝𝑒𝑟𝑡𝑢𝑟𝑏𝑒𝑑| . (Eq. R1-11)\n𝑗𝛥ɸ 2\nFor smallΔɸ, applyin=g t𝜎h𝑆e𝑆𝑃 s𝐼0m|𝑒all-an−g1le −ap1pr|ox=im2a𝜎t𝑆io𝑆n𝑃 𝐼10(1− 1-c-ocso(s𝛥(ɸɸ)) )≈ Δɸ2/2,\n. (Eq. R1-12)\n𝑃\nt𝐵te𝑀re\n=\nd\n𝜎\nt\n𝑆\np\n𝑆𝑃\no\n𝐼\nw\n0𝛥\ne\nɸ\nresulting from the perturbed meta-atom\nelement. In practice, the detected interferometric signal arises from the perturbed electric\nfield (from the meta-atom), the scattered field of the particle, and the static background\nfield.1, 2 Thus,\n5\n2\n𝑃MAD−iSCAT ∝ |𝐸𝐵𝑀 +𝐸𝑠 +𝐸𝐵𝐺|\n2 2 2\n= |𝐸𝐵𝑀| +|𝐸𝑠| +|𝐸𝐵𝐺| +2|𝐸𝐵𝑀||𝐸𝑠|𝑐𝑜𝑠𝛽1 +2|𝐸𝑆||𝐸𝐵𝐺|𝑐𝑜𝑠𝛽2 +2|𝐸𝐵𝐺||𝐸𝐵(𝑀E | q 𝑐 . 𝑜 R 𝑠𝛽 13-13)\nwhere , , and represent the difference in radiation phases.\nIn conventional iSCAT schemes, the SNR is defined as\n𝛽1 𝛽2 𝛽3\n. (Eq. R1-14)\n2|𝐸𝑆||𝐸𝐵𝐺|cos𝛽2\nSimilarly, in MAD-iS 𝑆 C 𝑁 A 𝑅 T 𝑖𝑆 , 𝐶 𝐴 w 𝑇 it = h g√i|v𝐸𝑆e|n 2 + 2s|t𝐸a𝑆t|i|c𝐸 𝐵b𝐺|accoks𝛽g2r+o|u𝐸𝐵n𝐺| 2 e ≈ le 2 c | t 𝐸 ri 𝑠 c | c fi o e s ld 𝛽 s 2 , the SNR can be\nexpressed as\n.\n2 2\n|𝐸𝐵𝑀| +|𝐸𝑠| +2|𝐸𝐵𝑀||𝐸𝑠|𝑐𝑜𝑠𝛽1+2|𝐸𝑆||𝐸𝐵𝐺|𝑐𝑜𝑠𝛽2+2|𝐸𝐵𝐺||𝐸𝐵𝑀|𝑐𝑜𝑠𝛽3\n𝑆𝑁𝑅𝑀𝐴𝐷−𝑖𝑆𝐶𝐴𝑇 = |𝐸𝐵𝐺| . (Eq. R1-15)\n2\n|𝐸𝐵𝑀| +2|𝐸𝐵𝑀||𝐸𝑠|𝑐𝑜𝑠𝛽1+2|𝐸𝐵𝐺||𝐸𝐵𝑀|𝑐𝑜𝑠𝛽3\nThe contrast of MAD≈-iS𝑆C𝑁A𝑅T 𝑖𝑆 c 𝐶 a 𝐴 n 𝑇 b+e described as|𝐸𝐵𝐺|\n.\n2 2\n|𝐸𝐵𝑀| +|𝐸𝑠| +2|𝐸𝐵𝑀||𝐸𝑠|𝑐𝑜𝑠𝛽1+2|𝐸𝑆||𝐸𝐵𝐺|𝑐𝑜𝑠𝛽2+2|𝐸𝐵𝐺||𝐸𝐵𝑀|𝑐𝑜𝑠𝛽3\n𝐶𝑀𝐴𝐷−𝑖𝑆𝐶𝐴𝑇 = |𝐸𝐵𝐺| 2 .. (Eq. R1-16)\n2\n|𝐸𝐵𝑀| +2|𝐸𝐵𝑀||𝐸𝑠|𝑐𝑜𝑠𝛽1+2|𝐸𝐵𝐺||𝐸𝐵𝑀|𝑐𝑜𝑠𝛽3\n≈ 𝐶𝑖𝑆𝐶𝐴𝑇 + |𝐸𝐵𝐺| 2\nThis demonstrates that compared to previous dark-field iSCAT configurations, where the\ninterfacing glass surface often presents random features, MAD-iSCAT enables\nsignificantly enhanced light signals for detection.\nWe greatly appreciate the reviewer's insightful comments, which have enabled us\nto deepen the theoretical foundation of MAD-iSCAT.\nRevision 1-2\nIn line with another reviewer’s suggestion for a more concise presentation, we now\nprovide detailed explanations together with equations in the Supplementary Information.\nMain text:\n“… It has been shown that dark-field images are also generated through the interference between\nscattered lights from the object and surface roughness (static background). …”\n“…where E and E are the electric field distribution generated by the meta-atom elements at (x ,\nij s ij\ny , -z ) and the scatterer at (0, 0, z ), respectively. α denotes the difference in radiation phases\nij a s ij\nbetween the meta-atom elements and the scatterer. For deep-subwavelength scatterers, the detected\nscattering intensity |E |2 scales with the sixth power of the particle size in conventional dark-field\ns\nimaging (Fig. 1c). In previous regular and dark-field iSCAT schemes, E terms in Equation (2) are\nij\nregarded as a static random background. Especially, is not involved in the scattered\n2\nlight detection and is removed by subtracting consecutive frames.\n|∑(𝑖,𝑗) 𝐸𝑖𝑗|\nimprove the detection sensitivity in iSCAT. Each metallic meta-atom possesses a large σ\nsc\ncompared to the dielectric nanoparticle (Fig. 1d). Without a nanoscatterer, the meta-atom array\nbehaves as a sub-diffraction grating, primarily reflecting incident light (constructive interference).\nIn contrast, the excited scattering modes of the meta-atoms collectively create destructive\ninterference towards the free space within the detection numerical aperture (NA), resulting in an\nimaginary Poynting vector (Fig. 1e). This destructive interference, termed dark mode of the\nmetasurface, is represented by term in Equation (2) in the absence of a nanoscatterer.\n|∑(𝑖,𝑗) 𝐸𝑖𝑗|\nand alters the phase profile of the sub-diffraction grating (Fig. 1f). The design of the meta-atom is\nchosen at a resonance mode, such that the amplitude A and phase of the resonance mode are\nij\nsensitive to the nearby environmental change due to the presence of the nanoscatterer, and the\nij\n𝜙\nperturbed mode within the sub-diffraction array radiates towards the detection optics. Considering\nthe perturbed electric fields of meta-atom elements, Equation (2)is modified as\n. (3)\n′ 2 ′ 2\nThe bright mode of the m |∑ et(a𝑖,s𝑗u) 𝐸 rf𝑖a𝑗c | e +2∑(𝑖,𝑗) |𝐸 𝑖a𝑗n | d | 𝐸𝑠|cos t ( h 𝛼 e𝑖r𝑗e ) b + y g |𝐸 e𝑠n | erates an interferometric\nimage with significantly enhanced contrast and′ S 2 NR. Acco′rdingly, the image contrast for MAD-\n|∑(𝑖,𝑗) 𝐸𝑖𝑗 | |𝐸𝑖𝑗 |\niSCAT can be written as\n(4)\n′ 2 2\n|∑(𝑖,𝑗)𝐸𝑖𝑗 +𝐸𝑠| −|∑(𝑖,𝑗)𝐸𝑖𝑗|\n|∑\ni\n(\n𝑖\n,\n𝑗) 𝐸\n𝑖𝑗\n|\nt\nh\nsu\nb\n-diffract.ion hexagonal meta-atom arrays,\nwhere degenerate plasmonic hybridized modes achieve a strong scattering condition (see details\nin Supplementary Section 1). …”\nSupplementary information:\n“Supplementary Section 1. Detailed explanation of the working mechanism of MAD-iSCAT.\nOur plasmonic metasurface operates as a phased-array antenna supporting a bright mode. For an\nideal metasurface comprising many meta-atoms, the array factor (AF) in the null directions where\ndestructive interference occurs is given by\n(Eq. S1)\n𝑁 𝑗Ψ𝑛\nWhen a detection particle resid\n𝐴\nes\n𝐹\n𝑖n𝑑e𝑒a𝑎r𝑙 (a\n𝜃\ns\n,\n\ni\n𝜑\nn\n,\ng\n𝜙\nle) m\n=\n=∑𝑛 =1\n𝑒\n=\n0\n. \nIt i\nntr\no\n.\nduces an additional phase shift\nΔɸ. Assuming this perturbation affects only the k-th element in two-dimensional arrays, the\nmodified array factor becomes\n𝑗Ψ𝑛 𝑗(Ψ𝑘+𝛥ɸ)\n𝐴𝐹perturbed(𝜃,𝜑,𝜙) = ∑𝑒 +𝑒\n𝑛≠𝑘 (Eq. S2)\n𝑗Ψ𝑘 𝑗(Ψ𝑘+𝛥ɸ) 𝑗Ψ𝑘 𝑗𝛥ɸ\n= 𝐴𝐹ideal −𝑒 +𝑒 = 𝑒 (𝑒 −1).\n0\nformed by plasmonic meta-atoms each characterized by a single-element scattering cross section\nσ ,is\nSSP\n2\n𝑃𝐵𝑀 = 𝜎𝑆𝑆𝑃𝐼0|𝐴𝐹𝑝𝑒𝑟𝑡𝑢𝑟𝑏𝑒𝑑| (Eq. S3)\n𝑗𝛥ɸ 2\nFor smallΔɸ, applying th=e 𝜎sm𝑆𝑆a𝑃l𝐼l0-|a𝑒ngle −ap1pr|ox=im2a𝜎t𝑆io𝑆n𝑃 𝐼10-(c1o−s(ɸc)o ≈s( Δ𝛥ɸɸ 2 )/2).,\n. (Eq. S4)\n𝑃\nl𝐵t𝑀ing\n=\nfr\n𝜎\no𝑆m𝑆𝑃\n𝐼\nt0h\n𝛥\ne\nɸ\nperturbed meta-atom element. In practice,\nthe detected interferometric signal arises from the superposition of the perturbed electric field\n(meta-atom), the scattered field of the particle, and the static background field.1, 2 Thus,\n2\n𝑃MAD−iSCAT ∝ |𝐸𝐵𝑀 +𝐸𝑠 +𝐸𝐵𝐺|\n2 2 2\nwhe=re |𝐸𝐵,𝑀 | , +an|d𝐸 𝑠| r+ep|r𝐸e𝐵se𝐺n|t t+he2 d|𝐸if𝐵fe𝑀r|e|n𝐸c𝑠e| 𝑐i𝑜n 𝑠r𝜷a1di+ati2o|n𝐸 p𝑆|h|a𝐸s𝐵e𝐺s|. 𝑐T𝑜h𝑠e𝜷 s2iz+e 2d|e𝐸p𝐵e𝐺n|d|e𝐸n𝐵c𝑀y| 𝑐o𝑜f 𝑠𝜷3\nis more complex than the simple cubic or sixth-power scaling characteristic of conventiona2l\n𝜷1 𝜷2 𝜷3 |𝐸𝐵𝑀|\niSCAT or pure Rayleigh scattering. The maximum power dependence of follows a sixth-\npower law. Thus, for small nanoparticles, the power dependency tends to app2roximate a near-\n|𝐸𝐵𝑀|\ncubic scaling when the interference terms are dominant.\nIn conventional iSCAT schemes, the SNR is defined as\n. (Eq. S5)\n2|𝐸𝑆||𝐸𝐵𝐺|cos𝜷2\nSimilarly, in MAD-iS 𝑆 C 𝑁 A 𝑅 T 𝑖𝑆 , 𝐶 𝐴 w 𝑇 it = h g√i|v𝐸𝑆e|n 2 + 2s|t𝐸a𝑆t|i|c𝐸 𝐵b𝐺|accoks𝜷g2r+o|u𝐸𝐵n𝐺| 2 e ≈ le 2 c | t 𝐸 ri 𝑠 c | c fi o e s ld 𝜷 s 2 , the SNR can be\nexpressed as\n.\n2 2\n|𝐸𝐵𝑀| +|𝐸𝑠| +2|𝐸𝐵𝑀||𝐸𝑠|𝑐𝑜𝑠𝜷1+2|𝐸𝑆||𝐸𝐵𝐺|𝑐𝑜𝑠𝜷2+2|𝐸𝐵𝐺||𝐸𝐵𝑀|𝑐𝑜𝑠𝜷3\n𝑆𝑁𝑅𝑀𝐴𝐷−𝑖𝑆𝐶𝐴𝑇 = |𝐸𝐵𝐺| . (Eq. S6)\n2\n|𝐸𝐵𝑀| +2|𝐸𝐵𝑀||𝐸𝑠|𝑐𝑜𝑠𝜷1+2|𝐸𝐵𝐺||𝐸𝐵𝑀|𝑐𝑜𝑠𝜷3\nThe contrast of MAD≈-iS𝑆C𝑁A𝑅T 𝑖𝑆 c 𝐶 a 𝐴 n 𝑇 b+e described as |𝐸𝐵𝐺|\n.\n2 2\n|𝐸𝐵𝑀| +|𝐸𝑠| +2|𝐸𝐵𝑀||𝐸𝑠|𝑐𝑜𝑠𝜷1+2|𝐸𝑆||𝐸𝐵𝐺|𝑐𝑜𝑠𝜷2+2|𝐸𝐵𝐺||𝐸𝐵𝑀|𝑐𝑜𝑠𝜷3\n𝐶𝑀𝐴𝐷−𝑖𝑆𝐶𝐴𝑇 = |𝐸𝐵𝐺| 2 . (Eq. S7)\n2\n|𝐸𝐵𝑀| +2|𝐸𝐵𝑀||𝐸𝑠|𝑐𝑜𝑠𝜷1+2|𝐸𝐵𝐺||𝐸𝐵𝑀|𝑐𝑜𝑠𝜷3\nThis demonstrates tha≈t 𝐶c 𝑖 o 𝑆 m 𝐶𝐴 p 𝑇 a+red to previous d|𝐸a𝐵r𝐺k|- 2 field iSCAT configurations, where the\ninterfacing glass surface often presents random features, MAD-iSCAT enables significantly\nenhanced light signals for detection.”\n8\n(c) When will this assumption break? For e.g., the plot in Fig. 3l, when will they intersect? Of course, iSCAT is for the\ndetection of nanoparticles. So this question is purely out of curiosity to see when these assumptions break.\nIn reality, once particle diameters exceed ~200 nm, the pure Rayleigh scattering\ntransitions toward the more complex Mie scattering regime. Consequently, scattering\nintensity no longer follows a strict sixth power law for larger particles. MAD-iSCAT,\nhowever, combines contributions of direct particle scattering, the bright mode, and the\n-\nstatic background. So its signal curve does not intersect that of either pure Rayleigh or\nMie scattering. To illustrate this, we have replaced the Rayleigh scattering cross-section\nin our analysis with the appropriate Mie scattering cross-section for particles up to 800\nnm. Within this size range, Rayleigh and Mie predictions remain similar, but above 200\nnm the size exponent falls below six, as shown in Figure R1-1. Thus, even though bright-\nmode enhancement diminishes for very large particles, MAD-iSCAT still provides an\nelevated signal relative to pure scattering, and no intersection appears in Fig. 3l.\nFigure R1-1. Mie scattering cross-section of a polystyrene bead.\nRevision 1-3\nWe appreciate this comment to clarify and enhance the manuscript. In line with another\nreviewer’s suggestion for footprint, we revised the Fig. 3l to Supplementary Information.\nSupplementary information:\n“Supplementary Section 8. Amplitude and phase perturbations of the meta-atom in the presence\nof a detection target.\nsc\npolystyrene (PS) nanospheres having different diameters when the array size is 9.6 × 9.3 µm2. The\ninset shows the meta-atom element interfacing with the detection target atop. c, The comparison\nof the scattering intensities from MAD-iSCAT and Mie scattering cross-section.”\n(2) Lines 54-55: ‘‘However, as the light scattering cross-section scales with the sixth power of the particle’s diameter…’’\n(a) Rayleigh scattering scales with sixth power of the particle radius and not diameter. Please check and correct.\nThank you for this observation. Rayleigh scattering scales strictly with the sixth power of\nthe particle’s radius r, i.e., σ r6, or when using diameters, σ d6 with a constant\nscaling factor of 1/64. The scattering size dependency remains the same for either particle\n∝ ∝ 1/64\nsize metric. We have chosen to express size in terms of diameter throughout the\nmanuscript for consistency with other iSCAT studies.\n(b) Fig. 4l – black dashed line represents cubic power. Is this plotted as a function of radius or diameter?\nThank you for pointing this out. The black dashed line in Fig. 4l is plotted against diameter.\nFor Rayleigh scattering, intensity scales as the sixth power of the diameter, whereas in\niSCAT the detected signal follows a cubic dependence on diameter, so we have used\ndiameter consistently for both cases. In revision, Fig. 4l was moved to Fig. 5c.\n(c) Fig. 4j – gray dashed line represents sixth power dependence.\nOn the PMMA substrate in air, the scattering intensity of particles follows a sixth power\ndependence on diameter. The gray dashed line in Fig. 4j is plotted accordingly for\n‐\ncomparison.\n10\n(d) Explain Fig. 4l and Fig. 4j based on Rayleigh scattering scaling inversely as sixth power of radius. Please mention the\nvalues of the scattering intensity at 50 nm, 100 nm, upto 200 nm for both MAD-iSCAT and Rayleigh scattering. It is\nprovided in the plot, but it is easier to compute the dependence on particle size from numbers directly given.\nThank you for this insightful suggestion. On a PMMA substrate in air, particle scattering\nintensity follows a sixth power scaling with diameter, which the gray dashed line in Fig. 4j\nconfirms. Because absolute cross-sections are commonly challenging to measure, we\n‐\ninstead report relative scattering intensities using standard polystyrene beads. As\nrequested, we have added values of scattering intensity at diameters in Fig. 4j in the\nSupplementary Information. The comparative values for MAD-iSCAT and regular iSCAT\nare already provided in the main text (first paragraph of the Experimental Results section).\nIn Fig. 4l, iSCAT is dominated by the interference term 2|E ||E|, which scales as the cube\ns r\nof particle diameter, rather than the Rayleigh |E |2 term. The iSCAT data follow this cubic\ns\ndependence, and MAD-iSCAT likewise exhibits a power dependence near the cubic law\npower. In revision, Fig. 4l was moved to Fig. 5c.\nRevision 1-4\nWe appreciate this comment to clarify and enhance the manuscript. We have added\nvalues of scattering intensity at diameters in Fig. 4j to Supplementary Information.\nSupplementary information:\n“Supplementary Section 12. The comparison of MAD-iSCAT and Rayleigh scattering.\nSupplementary Table 1. Scattering intensities of polystyrene beads used in Fig. 4j.\nMAD-iSCAT Rayleigh scattering\nSize (nm)\nMean (a.u.) Standard deviation (a.u.) Mean (a.u.) Standard deviation (a.u.)\n46 0.109 0.0312 0.000759 N/A\n60 0.235 0.0534 0.00317 N/A\n100 1.036 0.3216 0.0495 0.0100\n200 12.74 2.1802 2.302 0.2517\nFor the Rayleigh scattering plot of 46- and 60-nm polystyrene beads, a size-dependent power law\nof 5.38 was applied.2 In this size regime, background signals dominate and direct difference\nimaging in an air environment with drop-cast beads on bare surfaces is not practical.”\n(3) Fig. 4k --- can you explain why the particle locations have negative pixel values? Isn’t particle location supposed to be\naffecting the pixel value because of Gouy phase? Then why do they have positive values in the Mad-iSCAT?\nIn conventional iSCAT, only the scattered field from the particle acquires this Gouy phase\nshift, whereas the reference reflection does not. This phase difference underlies the axial\ncontrast in iSCAT. When the particle’s scattered light is π out of phase with the reflected\nfield, destructive interference produces a darker signal, yielding negative pixel values\nrelative to the background. In contrast, MAD-iSCAT employs dark-field interferometric\nillumination rather than relying on a plane-wave reflection. Because the reference field is\ngenerated by the bright super-scattering plasmon mode and not by a focused reflection,\nthe scattered signal appears brighter than the surrounding background when the particle\nis within the focal plane. Consequently, particle locations in MAD-iSCAT manifest as\npositive pixel values.\n(4) Fig. 4f – it is mentioned that all particles are validated with ground truth fluorescence and SEM images.\n(a) Is this done for all the results shown in the manuscript?\nThe ground-truth fluorescence and SEM validations were performed primarily for the data\nin Fig. 3a–j to confirm that each optical scattering signal corresponds to an individual\nnanoparticle rather than an aggregate and to verify its spectral response at a known\nlocation under air conditions. For experiments conducted in aqueous environments, such\nas those involving 80, 40, and 20 nm polystyrene beads, exosomes, and ferritin,\nfluorescence bleaching and SEM fixation are impractical. In these cases, we rely on\nstatistical sampling, acquiring a larger number of detection events, consistent with\nstandard practice in iSCAT studies of particles in liquid.\n(b) How was it ensured that fluorescence signal didn’t leak into the iSCAT measurements?\nThank you for your valuable comments. Prior to the fluorescence bead experiments, we\napplied maximum laser power at the fluorophore excitation wavelength for several hours\n‐\nto bleach the beads’ fluorescence emission. We have also confirmed that the scattering\nintensity is much stronger than the fluorescence emission.\nRevision 1-5\nWe appreciate this comment to clarify and enhance the manuscript. We have revised the\nmanuscript accordinglyto address the reviewer's concerns.\nMethods:\n“…Fluorescence beads are bleached before the scattering measurements. …”\n(5) Since MAD-iSCAT is a near-field technique, it will be good to have a plot illustrating the field strength as a function of\ndistance from the meta-atom surface.\nalong the z-axis is shown in Fig. R1-2.\nFigure R1-2. a-c, Field intensity distribution within a rectangular unit cell along the z-axis\nat heights of (a) 5 nm, (b) 15 nm, and (c) 25 nm.\nRevision 1-6\nWe have added the suggested simulation results, as shown in Figure R1-2, in the\nSupplementary Information.\nMethods:\n“…The near-field distributions are shown in Supplementary Section 16. …”\nSupplementary information:\n“Supplementary Section 16. Near-field distribution.\nSupplementary Fig. 13.a-c,Field intensity distribution within a rectangular unit cell along the z-\naxis at heights of(a) 5 nm, (b) 15 nm, and (c) 25 nm.”\n(6) It was not clear whether the SSP mode was utilized for all the experimental\nresults demonstrated in the manuscript.\nThank you for this insightful comment. We employed the SSP mode of our plasmonic\nmetasurface in all experimental measurements. Embedding the meta-atoms within a\nPMMA layer ensures that the SSP resonance remains unchanged despite variations in\n13\nthe overlying medium. Because the refractive index of PMMA is constant, we adjust the\nincident angle to satisfy the same plasmon modes excitation, 75° in air and 46° in\naqueous environments. In both cases, the in-plane wavevector in PMMA satisfies k =\nx\nk sin75° = k sin46° = k sin40°. The SSP mode’s field profiles and scattering cross-\n0 water PMMA\nsections are approximately identical under these conditions.\nRevision 1-7\nWe appreciate this comment to clarify and enhance the manuscript. We have revised the\nmanuscript accordinglyto address the reviewer's concerns.\nMethods:\n“…Incident angles are selected to satisfy the same plasmon modes excitation with k = k sin75° =\nx 0\nk sin47° = k sin40°.…”\nwater PMMA\n(7) Fig. 2a, can you provide the simulation results with and without the nano scatterer. This will aid the reader to see how the\nscatterer affects the confined mode.\nWe appreciate this comment to clarify and enhance the manuscript. The simulation results\nwith and without the nanoscatterer are shown in Fig. R1-3.\nFigure R1-3. a,b, Exemplary scattered electric field distributions (a) without a 200-nm\npolystyrene nanoscatterer and (b) with a polystyrene nanoscatterer.\nWe appreciate this comment to clarify and enhance the manuscript. We have added the\nsuggested simulation results, both with and without the nanoscale scatterer, as shown in\nFigure R1-3, in the Supplementary Information.\nSupplementary information:\n“Supplementary Section 3. Exemplary scattered electric field distributions with a polystyrene\nnanoscatterer.\nSupplementary Fig. 2.a,b, Exemplary scattered electric field distributions (a) without a 200-nm\npolystyrene nanoscatterer and (b) with a polystyrene nanoscatterer.”\n(8) Fig.3 - What is the spatial extent covered by the polar plots with respect to the\nmeta-atom array? Maybe add a scale bar to highlight it and also, where exactly on\nthe meta-atom array is this measurement carried out? Is it at the intersection of the\ndotted lines in Fig. 3a?\nThank you for your detailed review. Using multipole decomposition and the Green’s\ntensor formalism, we computed the far-field radiation patterns. The polar plots in Fig. 3\nspan an arbitrary angular range in the far field. Specifically, Fig. 3b,e,h depict the upper\nhemisphere (reflection) of the far field, with a hemisphere radius set to 2 mm. The\nmetasurface array is positioned at the center of the hemisphere’s base plane.A polar plot\nof the far-field radiation pattern was generated from meta-atom arrays (19.4 × 19.3 µm2).\nWe appreciate this comment to clarify and enhance the manuscript. We have revised the\nmanuscript accordinglyto address the reviewer's concerns.\nMethods:\n“…The far-field radiation pattern is calculated at a distance of 2 mm from the array center. …”\n(9) Fig.1(c-f) – a suggestion is that the plots can be better represented. For example,\nwavelength range along the x-axis will be useful as these meta-atoms do not\noperate at all wavelengths. Another point of confusion was the grey plot in the case\nof the dark-mode. This can be understood after reading the manuscript, but a bit\nmore detailed explanation in the figure caption will also help.\nRevision 1-10\nWe appreciate this comment to clarify and enhance the manuscript. We have revised Fig.\n1 accordinglyto address the reviewer's concerns.\nMain text:\n“ ”\n16\n(10) Though the array spans a few microns, what is the field-of-view achievable\n(FoV)? Is it 9.6 х 9.3 μm2? Do you think you\ncan enhance the FoV? It will be useful to highlight these issues in the manuscript.\nThank you for your detailed review. The achievable field of view is primarily limited by\nfabrication capabilities, but larger areas are technically accessible. We chose a 40 × 40\ngrid of 9.6 × 9.3 µm² meta-atom arrays to facilitate SEM correlation by identifying the\nspecific optical array before locating it under SEM. For future work such as cellular\nimaging, which requires areas larger than 10 × 10 µm², we anticipate scaling the FOV to\nat least 19.4 × 19.3 µm² or even up to 48 × 46.15 µm². Larger arrays will accommodate\ntypical cell sizes and further suppress background intensity, as demonstrated in our\nSupplementary Section 6.\nRevision 1-11\nWe appreciate this comment to clarify and enhance the manuscript. We have revised the\nmanuscript accordinglyto address the reviewer's concerns.\nMain text:\n“… MAD-iSCAT has the potential to dramatically improve single-molecule mass imaging\nthroughput by orders of magnitude through enhanced fabrication approaches such as self-assembly\nfor better metasurface quality51 and large array geometries to enable high-throughput nanoparticle\nimaging with expanded field-of-view and suppressed background noise. …”\n(11) MAD-iSCAT is a near-field detection technique. Conventional iSCAT can help\nisolate particles within the focal volume of the detection objective. Therefore,\nthough it is obvious, highlighting the limitations of MAD-iSCAT is important\nespecially for biologists.\nWe appreciate you highlighting this important consideration. Many iSCAT applications\nachieve exceptional sensitivity for small-nanoparticle detection using wide-field imaging,\nwithout the need to adjust the focal plane extensively above the glass–air interface. MAD-\niSCAT, however, operates within the evanescent-field regime (≤200 nm from the surface).\nThus, for routine nanoparticle sensing where long-range 3D tracking is unnecessary,\nMAD-iSCAT is fully compatible with conventional iSCAT methods. In contrast, biological\ncell imaging will be limited to a very thin axial region near the substrate.\nRevision 1-12\nWe appreciate this comment to clarify and enhance the manuscript. We have revised the\nmanuscript accordinglyto address the reviewer's concerns.\nMain text:\n“… It is important to note that the current MAD-iSCAT has a detection volume more constrained\nthan conventional iSCAT and suffers from fabrication imperfections, array inhomogeneities, and\nfinite-size effects. Nonetheless, MAD-iSCAT combines the advantages of iSCAT and meta-atom\narrays, opening new opportunities for optimization in label-free sensing applications. …”\n(12) Fig. 4j – please correct the legends in the plot.\nRevision 1-13\nWe appreciate this comment to clarify and enhance the manuscript. We have revised the\nmanuscript accordinglyto address the reviewer's concerns.\nMain text:\n“ ”\n(13) Due to enhanced field strengths, can particles get trapped to the high field\nintensity?\nWe appreciate this observation. Optical trapping relies on the gradient of the\nelectromagnetic field intensity, drawing particles toward regions of highest field\nenhancement. Achieving sufficient trapping stiffness for nanoparticles against Brownian\n18\nmotion typically requires either very high laser power densities or tightly confined fields\nwithin nanoscale gaps. In our design, the plasmonic meta-atoms are embedded beneath\na PMMA layer, which attenuates the field enhancement at the surface where particles\nreside. With a 145 nm lattice period and no sharp gap structures, the field enhancement\nat the PMMA interface remains moderate (under ~20×), and MAD-iSCAT contrast arises\nprimarily from interferometric detection rather than extreme field confinement.\nConsequently, we do not expect significant optical trapping or thermophoretic effects\nunder our operating conditions.3\n(14) Have you quantified any temperature changes due to plasmonic effects? Will\nthis affect the biological samples under study?\nThank you for highlighting this important factor. We used a relatively low laser power\ndensity of 42 W/cm². In previous studies of silver nanospheres (10–50 nm radius) at 4,000\nW/cm², where scattering cross-sections peaked as 1×10 ¹⁴ m², temperature rises of 0.5–\n3 °C were confirmed. Since our SSP mode features cross-sections in the same order of\n⁻\nmagnitude but operates at 100× lower power density, we estimate a temperature increase\nof less than 1 °C. Under these conditions, biological samples are not affected by\nplasmonic heating, alleviating concerns about thermal damage.4\nRevision 1-14\nWe appreciate this observation. We have revised the manuscript accordinglyto address\nthe reviewer's concerns.\nMain text:\n“…The laser power employed is sufficiently low to avoid significant photothermal effects and our\nprotective layer also minimizes the heating effect on detection targets and prevents photodamage\nto silver meta-atoms.51…”\nIn conclusion, we sincerely appreciate the reviewer's detailed evaluation and valuable\ncomments that have enhanced our manuscript.\nThe manuscript entitled “Meta-amplified dark-field interferometric scattering\nmicroscopy” has undergone one round of peer review, and the authors have\nrevised the manuscript in response to the reviewers’ comments. After reviewing\nthe revised version and the response letter, I find that the authors have addressed\nthe earlier concerns in a satisfactory manner.\nA notable strength of this work lies in the central concept of exploiting the dark\nmode of the plasmonic meta-atom array. By engineering the metasurface so that\nits collective radiation is suppressed in the absence of objects, the authors achieve\na background-free baseline. Upon the attachment of nanoparticles, local\nperturbations induce a transition from the dark mode to a bright radiation mode,\nresulting in a strong and detectable signal. This mechanism represents an original\nidea that clearly differentiates MAD-iSCAT from previously reported iSCAT\napproaches. The physical picture is compelling and provides a solid foundation for\nthe reported contrast enhancements. I strongly recommend this work for\npublication after one further round of minor revisions.\n1. On page 3, line 57, the statement ‘Interferometric imaging addresses this\nchallenge by leveraging the interference of scattered light from nanoscale objects\nwith reference light, yielding label-free images with high sensitivity and resolution’\nseems to overstate the claim of ‘high resolution’. Interferometric imaging does not\ninherently surpass the diffraction limit; rather, it improves localization precision or\neffective resolution through enhanced signal-to-noise.\nRevision 2-1\nWe appreciate this comment to clarify and enhance the manuscript. We have revised the\nmanuscript accordinglyto address the reviewer's concerns.\nMain text:\n“… Interferometric imaging addresses this challenge by leveraging the interference of scattered\nlight from nanoscale objects with reference light, yielding label-free images with high\nsensitivity. …”\n2. In Fig. 1a, the schematic illustrates a reflection geometry where the illumination\nincidence angle is drawn larger than the detection NA cone, suggesting complete\nseparation between illumination and detection. However, for a 0.9 NA dark-field\nobjective, the illumination annulus specified by the manufacturer is typically NA =\n0.8–0.9, corresponding to ~53°–64° in air, which partially overlaps with the\ncollection cone (0–64°). This discrepancy between the schematic, the stated 75°\nincidence angle, and the actual objective specification should be clarified. In\n20\nprinciple, one could increase the laser input incidence angle beyond the nominal\nannulus by back focal plane engineering while restricting the detection NA to a\nsmaller cone. If such a strategy was employed, it would be important for the\nauthors to explicitly state this in the Methods, as it would resolve the apparent\ninconsistency.\nThank you for your detailed review. As shown in Figure R2-1, the dark-field objective lens\n(UMPlanFl, 100×, 0.9 NA, Olympus) supports an oblique hollow light cone illumination.\nThe incident angle of the dark-field objective lens is measured to be 75°.\nFigure R2-1. An oblique hollow light cone illumination of a dark-field objective.\nRevision 2-2\nWe have added the information related to Figure R2-1 in the Supplementary Information.\nSupplementary Information:\n“Supplementary Section 10. MAD-iSCAT imaging in air environments.\n3. Despite the conceptual novelty, one potential weakness of the approach is that\nachieving a perfect dark mode in practice may be difficult. Fabrication\nimperfections, array inhomogeneities, and finite-size effects could lead to residual\nscattering from the meta-atoms themselves, limiting the reproducibility and\nrobustness of the method. It would be helpful if the authors could briefly\nacknowledge this limitation in the manuscript, as it provides important context for\nthe practical implementation of MAD-iSCAT. Although the authors emphasize that\nthe dark mode of the metasurface prevents radiation into the detection\nNA, the fact that a measurable scattering spectrum of the bare metasurface has been obtained\nindicates that residual scattering does exist. This residual signal, even if weak,\ncould act as background noise. Reviewer #2 already noted that the limited\nsensitivity is likely due to fabrication imperfections, resulting in nonspecific and\nnonuniform background signals. In their response, the authors acknowledged that\nimproving fabrication quality and array uniformity could further reduce\nbackground scattering, which indirectly admits that perfect dark mode\nsuppression is difficult to achieve in practice. However, this limitation is not\nexplicitly described in the main text. It would strengthen the manuscript if the\nauthors could briefly acknowledge this point as a limitation, or provide a short\ndiscussion to clarify to what extent intrinsic meta-atom scattering contributes to\nthe background under the dark mode condition.\nthe residual scattering helps to obtain an interferometric image,1, 2 we acknowledge that\ncurrent MAD-iSCAT obviously shows a larger background level than the dark-field iSCAT\nwith continuous substrate.\nRevision 2-3\nWe appreciate this comment to clarify and enhance the manuscript. We have revised the\nmanuscript accordinglyto address the reviewer's concerns.\nMain text:\n“… It is important to note that the current MAD-iSCAT has a detection volume more constrained\nthan conventional iSCAT and suffers from fabrication imperfections, array inhomogeneities, and\nfinite-size effects. Nonetheless, MAD-iSCAT combines the advantages of iSCAT and meta-atom\narrays, opening new opportunities for optimization in label-free sensing applications. MAD-\niSCAT has the potential to dramatically improve single-molecule mass imaging throughput by\norders of magnitude through enhanced fabrication approaches such as self-assembly for better\nmetasurface quality and large array geometries to enable high-throughput nanoparticle imaging\nwith expanded field-of-view and suppressed background noise. …”\n4. On page 7, line 177, the statement ‘In addition, a shorter periodicity with a larger\nfill factor is preferred for a higher detection yield of the nanoparticles’ appears.\nThis raises the question of how such a design fundamentally differs from using\na continuous metallic film. Since a bare film would not support the array-induced\ndark mode that underpins the proposed mechanism, it would be helpful if the\nauthors could clarify this distinction more explicitly in the manuscript, particularly\nin terms of background suppression, scattering enhancement, and overall\ndetection sensitivity.\nThank you for your insightful question. A similar approach using bare gold films was\ndemonstrated by Zhang et al. (Nat. Methods 17, 1010 (2020)). While continuous films rely\non surface roughness to provide a reference field for interferometric imaging, we\ndemonstrate that these naturally occurring irregularities can be artificially engineered into\nperiodic meta-atoms to enhance scattering detection. A key difference between\ncontinuous metallic film and the MAD-iSCAT is that the metasurface is designed to take\nadvantage of the SSP mode of the meta-atoms, and therefore creates much stronger\nscattering interactions. The periodicity of the metasurface is chosen so that the resonance\nmode provides maximized coverage of the entire patterned area, while still maintaining\nthe SSP mode for the meta-atoms, as shown in Figure 2d.\nAs the reviewer correctly noted, continuous metallic films offer superior background\nsuppression. In MAD-iSCAT, background intensity arises from two primary factors: finite\narray size and fabrication imperfections. As shown in our Supplementary Section 6, larger\narrays minimize background scattering at the center by reducing edge diffraction, while\n23\nfabrication errors also contribute unwanted signals. These results demonstrate that array\nsize significantly affects background suppression. We anticipate scaling our arrays to at\nleast 19.4 × 19.3 µm² or up to 48 × 46.15 µm² for future applications.\nIn general, the detection intensity of iSCAT is described as\n. (Eq. R2-1)\n2 2\nThe detection intensity has\n𝐼\na\n= \nc |u\n𝐸\nb𝑟i|c\n+\npo\n2\nw|e\n𝐸\nr𝑟 |d|e\n𝐸\n𝑠|c\no\ns\n(\n𝛼\n)\n+\n|𝐸𝑠|2\nthe particle size since the\nsecond term dominates over the third term. However, when | | < | | the intensity\nbecomes proportional to the sixth power of the particle size (the third term becomes\n𝐸𝑟 𝐸𝑠\ndominant). In Zhang et al. (Nat. Methods 17, 1010 (2020), the cubic power dependence\nstarts to appear for particle sizes smaller than 100 nm. In contrast, MAD-iSCAT shows\napproximate cubic dependence for particles over 100 nm, with sixth-power scaling difficult\nto observe even at 200 nm diameter. This demonstrates that our plasmonic meta-atoms\nprovide much larger scattering cross-sections (which can transition between dark and\nbright modes) than bare gold film roughness, offering enhanced sensitivity for smaller\nnanoparticles. This represents the key novelty of our approach.\nRevision 2-4\nWe appreciate this insightful comment. We have revised the manuscript accordingly to\naddress the reviewer's suggestion.\nMain text:\n“… The perturbed electric field from meta-atoms generates strong interferometric signals, which\nexhibit an approximate cubic power law dependence that persists even for comparably larger\nparticle sizes where Rayleigh scattering dominates. …”\n5. Fig. 3l shows that the scattering intensity decreases much more slowly with\nparticle size than expected from Rayleigh scattering. The authors attribute this to\ninterference between the particle and the meta-atom array, suggesting an\napproximate d³ scaling. It remains unclear, however, whether this cubic\ndependence arises primarily from the general interference mechanism in iSCAT or\n∼\nfrom a specific particle–meta-atom interaction effect unique to the MAD-iSCAT\ngeometry. Since the particle–meta-atom interaction itself should also be size-\ndependent, it would strengthen the discussion if the authors could elaborate on\nhow this interaction contributes to the observed scaling behavior. While Figs. 3l\nand 4j demonstrate weaker-than-Rayleigh scaling down to ~20 nm particles, it\nremains uncertain how far this trend can be maintained for even smaller diameters.\nIn particular, it would be valuable for the authors to discuss whether the scaling\nslope is expected to remain shallow below 20 nm, and what sets the ultimate\ndetection limit for MAD-iSCAT.\nacknowledge that explicit information regarding the size dependence of MAD-iSCAT was\nnot clearly provided in the original manuscript. To address this point, we have developed\na more comprehensive theoretical framework to clarify the underlying mechanism of\nMAD-iSCAT. Since this issue was also raised by other reviewers and discussed in detail\nover four pages, we kindly refer the reviewer to our previous response in the section\nTheoretical framework for contrast and SNR enhancement in MAD-iSCAT on page\n5. In brief, the size dependence of light scattering mediated by the bright mode in our\nmetasurface is more complex than the simple cubic or sixth-power scaling typically\nobserved in conventional iSCAT or Rayleigh scattering. For small nanoparticles, the\npower dependence approaches a near-cubic scaling regime when interference terms\ndominate the response.\nRevision 2-5\nWe have revised the manuscript accordingly to address the reviewer's concern. Please\nkindly refer to the Revision 1-2for detailed updates.\n6. In Fig. 5a,b the authors state that exosome sizes are determined through mean-\nsquared displacement (MSD) analysis, with trajectories shown in Supplementary\nSection 9. However, only sample trajectories are provided, and it is not clear how\nthese lead to MSD values and size estimates. A brief explanation of how MSD is\ncalculated from the trajectories and converted into particle size (e.g., via the\ndiffusion coefficient and Stokes–Einstein relation) would improve clarity.\nThank you for your detailed review. To perform MSD analysis, particle trajectories r(t) =\n(x(t), y(t)) are constructed by recording positions across N frames with an exposure time\nof Δt. At each time lag τ, the MSD is calculated by averaging the squared displacements\nbetween all position pairs separated by this specific lag interval as\n(Eq. R2-2)\n2\nwhere the angular brackets de\n𝑀\nn\n𝑆\no\n𝐷\nte(\n𝜏\n)\n=\n〈|𝑟(\nt\n+\n𝜏\n)\n−\n𝑟\n(\nt\n)\n|\n〉\nte\nem\nb\nle\nav\ner\nag\nin\ng\nov\ner\nall applicable time points t.\nFor exosome characterization, high-speed imaging is conducted with an exposure time\nof 487.218 µs to capture rapid Brownian motion, with individual exosome trajectories\nextracted as shown in the Supplementary Fig. 11. When analyzing MSD as a function of\ntime lag τ, freely diffusing particles exhibiting Brownian motion demonstrate a linear\nrelationship described by\n(Eq. R2-3)\nwhere the intercept term corrects 𝑀fo𝑆r 𝐷lo(c𝜏a)li=za𝑒t𝑟io𝑟n𝑜 𝑟er+ro𝑏r𝜏s and the slope directly scales with\nthe diffusion coefficient. In the case of three-dimensional Brownian motion projected onto\n25\na two-dimensional imaging plane, the coefficient is given by D = b/4. Finally, the\nexosome’s radius r is calculated using the Stokes–Einstein equation for a spherical\nparticle\n(Eq. R2-4)\n𝑘𝐵𝑇\nwhere is Boltzmann's constant, T th𝑟e =ab6s𝜋o𝜂𝐷lute temperature, and η the fluid viscosity.\nThis procedure transforms particle trajectories into estimates of exosome size.5, 6\n𝑘B\nRevision 2-6\nWe appreciate this comment to clarify and enhance the manuscript. We have revised the\nmanuscript accordingly to address the reviewer's concerns in the Supplementary\nInformation.\nSupplementary Information:\n“Supplementary Section 14. Sample trajectories of exosomes with different sizes.\nTo perform mean-squared displacement (MSD) analysis, particle trajectories r(t) = (x(t), y(t)) are\nconstructed by recording positions across N frames with an exposure time of Δt. At each time lag\nτ, the MSD is calculated by averaging the squared displacements between all position pairs\nseparated by this specific lag interval as\n(Eq. S8)\nwhere the angular brackets denote ensemble averaging over2 all applicable time points t. For\n𝑀𝑆𝐷(𝜏) = 〈|𝑟(𝑡+𝜏)−𝑟(𝑡)| 〉\nexosome characterization, high-speed imaging is conducted with an exposure time of 487.218 µs\nto capture rapid Brownian motion, with individual exosome trajectories extracted as shown in\nSupplementary Fig.11. When analyzing MSD as a function of time lag τ, freely diffusing particles\nexhibiting Brownian motion demonstrate a linear relationship described by\n(Eq. S9)\nwhere the intercept term corrects for localization errors and the slope directly scales with the\n𝑀𝑆𝐷(𝜏) = 𝑒𝑟𝑟𝑜𝑟+𝑏𝜏\ndiffusion coefficient. In the case of three-dimensional Brownian motion projected onto a two-\ndimensional imaging plane, the coefficient is given by D = b/4. Finally, the\nexosome’s radius r is calculated using the Stokes–Einstein equation for a spherical\nparticle\n(Eq. S10)\n𝑘𝐵𝑇\nwhere is Boltzmann's constant, T the a𝑟bs=olu6𝜋te𝜂 𝐷temperature, and η the fluid viscosity. This\nprocedure transforms particle trajectories into estimates of exosome size.4,5”\n𝑘B\n7. On page 10, line 301, the manuscript states: “This leads to a higher dark mode background level as well as a degraded\nTP2 mode in silver meta-atoms, which is reflected in the broader wavelength response shown in Fig. 4i compared to the\nideal meta-atom resonance.” However, Fig. 4i presents the scattering enhancement factor over the visible range, not a\nresonance broadening effect. This suggests that the figure reference may be incorrect. The authors should verify the figure\nlabeling and clarify which figure actually demonstrates the resonance broadening due to fabrication imperfections— perhaps\nFig.2e?\nRevision 2-7\nThank you for your detailed review. We find the reviewer’s comment is correct. We have\nupdated the manuscript to clarify this.\nMain text:\n“… This leads to a higher dark mode background level as well as a degraded TP2 mode\nin silver meta-atoms, which is reflected in the broader wavelength response shown in Fig.\n2e compared to the ideal meta-atom resonance. …”\nOverall, the manuscript presents a novel and compelling approach with clear\npotential impact. I would strongly support its publication once the above\nclarifications and minor revisions are addressed.\nIn conclusion, we sincerely thank the reviewer for the valuable comments, which have\ngreatly helped improve our manuscript.\nReview Report:\nThe article titled, “Meta-amplified dark-field interferometric scattering microscopy”\nby Lee et al proposes a technique (MAD-iSCAT) that combines dark field\nmicroscopy and interference to detect nanoparticles on a plasmonic metasurface.\nIn this report, the authors demonstrated the technique by detecting polymer-\nnanoparticles and exosomes. They claimed the method to be better than the\nexisting iSCAT technique in terms of sensitivity and contrast. I noted that the first\nthree figures are largely schematic and simulation. The actual results are mainly\nlimited to Fig. 4 and Fig. 5. Moreover, the results are shown on nanoparticles, rather\nthan real systems such as cells, making the work somewhat limited in scope. Here\nare my detailed comments :\n1. One of the major concerns is the lack of validation and poor understanding of\nwhat transpires at the metasurface related to field-particle interaction.\nWe thank the reviewer for the valuable comment. We acknowledge that clear theoretical\nevidence was not provided in the original manuscript. To address this point, we have\ndeveloped a more comprehensive theoretical framework to elucidate the underlying\nmechanism of MAD-iSCAT. As this issue was also raised by other reviewers and\ndiscussed in detail over four pages, we kindly refer the reviewer to our previous response\nin the section Theoretical framework for contrast and SNR enhancement in MAD-\niSCAT on page 5. To summarize, the underlying working principles of MAD-iSCAT have\nnow been thoroughly investigated to explain the enhancement of contrast and SNR in\niSCAT. We hope this satisfactorily addresses the reviewer’s concerns.\nRevision 3-1\nWe have incorporated a comprehensive theoretical discussion and comparison of\nimaging contrast and SNR with conventional iSCAT. The manuscript has been revised\naccordingly to the reviewer’s concern. Please refer to Revision 1-2 for detailed updates.\n2. The particles used by authors are comparatively large. The technique (MAD-\niSCAT) needs to demonstrate for small particle (<10 nm nanoparticles and < 300\nkDa proteins) detection. This will strengthen the claim experimentally that MAD-\niSCAT is better than iSCAT.\nWe thank the reviewer for this insightful comment. We respectfully argue that our work\ndemonstrates significantly enhanced sensitivity and contrast compared to conventional\niSCAT imaging.\nwe employ a standardized metric that accounts for varying experimental conditions. In\nscattering-based detection, the measured signal from a given particle scales linearly with\ntotal photon dosage, the product of effective exposure time (EET) and incident power\ndensity P . Assuming a shot noise-limited system, the square root of the EET and P\nD D\nproduct(EET×P )0.5, enables a relative comparison of the detection SNR.\nD\nThe theoretical study (Becker et al., ACS Photonics 10, 2699-2710 (2023)) predicts\nthat conventional iSCAT on bare glass can achieve SNR ~ 21 for 66 kDa proteins under\noptimized conditions (illumination P of 0.1 MW/cm2, EET = 100 ms, and λ = 445 nm).\nD\nMany prior experimental studies do not report image SNRs for iSCAT but plasmonic\nscattering microscopy (PSM), where a single rough metallic film is used (Zhang et al., Nat.\nMethods 17, 1010-1017 (2020)), reaches an image SNR of ~11 for 385 kDa IgA protein\nwith EET = 100 ms. The comparison is summarized below.\nTable R3-1. A comparison of MAD-iSCAT against conventional iSCAT and plasmonic\nscattering microscopy (PSM, plasmonic iSCAT).\nEET λ P (EET×P )0.5\nMethod Targets D D SNR Reference\n(ms) (nm) (W/cm2) ((W·ms/cm2)0.5)\nMAD-iSCAT 440 kDa 1 × 2 450 42 9.2 9 This study\nTheoretical ACS Photonics10,\n66 kDA 100 445 100,000 3,162 21\nstudy(iSCAT) 2699-2710 (2023)\nNat. Methods17,\nPSM 385 kDA 50 × 2 670 2,000 447 11\n1010-1017 (2020)\nIn comparison, MAD-iSCAT achieves SNR ~ 9 for 440 kDa ferritin under dramatically\nreduced photon dosage. By normalizing the SNR values by target molecular weight,\n(EET×P )0.5, and λ4, the projected SNR for theoretical iSCAT and PSM are 0.91 and 1.55\nD\nunder the same illumination conditions of MAD-iSCAT. Our approach demonstrates an\nSNR enhancement factor of 23.2 relative to conventional iSCAT and a 7.1-fold\nimprovement over PSM. While PSM's enhancement originates solely from the field\nenhancement provided by the uniform gold film, MAD-iSCAT's superior performance\nstems from the perturbed bright modes of the plasmonic metasurface, consistent with our\ntheoretical predictions.\nAs noted by the reviewer, further measurements with smaller molecules would\nrobustly establish MAD-iSCAT’s advantage. This measurement was an objective\nemphasized by two reviewers in the initial review round, and we accomplished the 20-nm\npolystyrene beads and ferritin measurement. Our current experimental configuration,\nbased on a supercontinuum laser with narrow transmission linewidth, already operates at\nmaximum available laser power. Theoretical analysis (Becker et al., ACS Photonics 10,\n2699–2710 (2023)) indicates that the detection threshold of SNR = 3 in our current system\n29\nwith SNR enhancement corresponds to a molecular weight of approximately 141 kDa\n(representing a substantial improvement compared to required 3,279 kDa without SNR\nenhancement). Extending detection to even smaller proteins below this threshold would\nrequire higher-power illumination sources, which we plan to implement in future work.\nThrough both theoretical analysis (revised theoretical framework) and\nexperimental validation (SNR comparison presented above), we have demonstrated that\nMAD-iSCAT achieves substantial SNR enhancement over existing technologies. We\nhope this comprehensive comparison adequately addresses the reviewer's concerns\nregarding sensitivity and future prospects for smaller particle detection.\nRevision 3-2\nTo demonstrate the superior performance of MAD-iSCAT, we have revised the main text\nand expanded comparisons in the Supplementary Information.\nMain text:\n“… The image SNR shows that our approach demonstrates an SNR enhancement factor of 23.2\nrelative to conventional iSCAT (see details in Supplementary Section 13).”\nSupplementary Information:\n“Supplementary Section 13. The comparison of MAD-iSCAT and iSCAT.\nSupplementary Table 2. Imaging performances of different interferometric scattering imaging\ntechniques. (EET: effective exposure time, PSNR: peak signal-to-noise ratio, and P : incident\nD\npower density). R\nEET λ P (EET×P )0.5\nMethod Targets D D PSNR Reference\n(ms) (nm) (W/cm2) ((W·ms/cm2)0.5)\n20-nm PS 51 (PS\nMAD-iSCAT bead and 1 × 2 450 42 9.2 bead) This study\n440 kDa 9 (protein)\nACS Photonics\nTheoretical\n66 kDA 100 445 100,000 3,162 21 10, 2699-2710\nstudy (iSCAT)\n(2023)\nPhotonic\nresonator Nat. Commun.\n16 ×\ninterferometric 440 kDa 633 25 28.3 N/A 12, 1744\n2\nscattering (2021)\nmicroscopy\nQuantitative 53–803 300\n445 420,000 15,874.5 N/A 423-427\nmass imaging kDa × 2\n(2018)\nSingle-protein Nat. Photon.\n90-540 6.25\noptical 450 50,000 790.6 N/A 18, 388\nkDa × 2\nholography (2024)\nEvanescent 28-nm PS Nat. Commun.\n20 ×\nscattering bead and 450 60,000 1,549.2 N/A 13, 2298\n2\nmicroscopy 66 kDA (2022)\n200 (PS\nPlasmonic 26-nm PS Nat. Methods\n50 × bead) 141.4 (PS bead)\nscattering bead and 670 11 (protein) 17, 1010-1017\n2 2,000 447.2 (Protein)\nmicroscopy 385 kDA (2020)\n(protein)\nWe compare MAD-iSCAT with other advanced interferometric scattering-based imaging\ntechniques. … Although these comparisons are indirect (since variations in imaging components\ncan affect η and PSNRs are not well reported), Supplementary Table 2 summarizes the reported\nperformance metrics. The exposure time is calculated from their frame rates. By normalizing the\nPSNR values by target molecular weight, (EET×P )0.5, and λ4, the projected PSNR for theoretical\nD\niSCAT and plasmonic scattering microscopy are 0.91 and 1.55 under the same illumination\nconditions of MAD-iSCAT. Our approach demonstrates an SNR enhancement factor of 23.2\nrelative to conventional iSCAT and a 7.1-fold improvement over plasmonic scattering microscopy.\nBeyond this enhanced sensitivity, MAD-iSCAT achieves the highest imaging throughput reported\nfor interferometric scattering microscopy. This performance derives from our single-frame\ndifferential imaging approach, where acquisition speed is limited only by the camera readout rate\nrather than requiring temporal averaging or post-processing integration, while detection sensitivity\ncan be further enhanced by increasing laser power.”\n3. As claimed, high throughput and sensitivity needs further justification.\nAs discussed in Revision 3-2, the scattering enhancement mechanism of MAD-iSCAT is\nstrong enough that we do not need to perform data averaging to achieve a good SNR\nand therefore leads to more than one order imaging speed improvement compared to\nother iSCAT methods. The scaling of the metasurface has also been considered during\nthe design process for future large field-of-view high-throughput imaging. Should a larger\npatterned area be required, only one master mold is needed from e-beam lithography,\nand the metasurfaces can be mass-produced through nanoimprinting.\nIn addition to the reduced data acquisition time, we would also like to point out the\nimportance of the reduced incident light intensity requirement. For practical biological\nimaging, especially in a cellular environment, a lower illumination intensity is preferred to\navoid photodamage to the sample. The required intensity of MAD-iSCAT is less than that\nof a typical fluorescence wide-field microscope (100 W/cm2), providing access to\nprolonged high-throughput bioimaging.\nRevision 3-3\n4. From the present results, the proposed technique does not appear to offer\nsignificant advance compared to iSCAT.\nWe thank the reviewer for this comment. We respectfully argue that our responses to the\nreviewer’s Comments 1 and 2 provide comprehensive evidence of MAD-iSCAT's\nsignificant advancement over conventional iSCAT, including: (1) a detailed theoretical\nframework with equations describing the SNR and contrast enhancement mechanisms,\nand (2) experimental validation demonstrating a 23.2-fold SNR enhancement compared\nto conventional iSCAT when normalized for experimental conditions. Please refer to our\nresponses to the reviewer’s Comments 1 and 2 for the complete analysis.\nAlso, in general, we envision the unique scattering imaging mechanism of MAD-\niSCAT will represent the future of state-of-the-art iSCAT. With conventional iSCAT\ntechniques, particle sensitivity improvement has plateaued as we are reaching the\npractical limit of imaging dynamic range and shot noise limit using existing hardware. The\nconcept of using super-scattering meta-atom mode and switching between dark-mode\nand bright-mode of a nanoantenna array opens up a new degree of freedom in designing\na scattering imaging system.\nRevision 3-4\nWe appreciate the reviewer for the comments. We have updated the discussion section\nto emphasize the importance of MAD-iSCAT.\nMain text:\n“… It is important to note that the current MAD-iSCAT has a detection volume more constrained\nthan conventional iSCAT and suffers from fabrication imperfections, array inhomogeneities, and\nfinite-size effects. Nonetheless, MAD-iSCAT combines the advantages of iSCAT and meta-atom\narrays, opening new opportunities for optimization in label-free sensing applications. MAD-\niSCAT has the potential to dramatically improve single-molecule mass imaging throughput by\nenhanced fabrication approaches, such as self-assembly53 and large array geometries for expanded\nfield-of-view and suppressed background noise. …”\n5. The abstract lacks to mention key details / outputs of the MAD-iSCAT technique.\nThis include, (i) radiation modes and why they are special, (2) contrast\nenhancement, and (3) the level of suppression of the background. These\ninformation should have been mentioned in the abstract.\nRevision 3-5\nWe appreciate the reviewer for the comments. We have updated the abstract to\nemphasize the key aspects of MAD-iSCAT.\n“… By employing a metasurface comprising sub-diffraction plasmonic meta-atom arrays, MAD-\niSCAT generates bright radiation modes that intensely scatter light toward the far field in the\npresence of a detection nanoparticle, substantially amplifying the sensitivity. In the absence of a\nnanoparticle, the metasurface produces minimal background due to the dark collective mode,\nresulting in improved image contrast.…”\n6. The manuscript lacks schematic of detailed optical setup in the main manuscript.\nThis is essential to understand the proposed technique, instrumentation, and\nsystem development.\nWe appreciate the reviewer for the comments. We have added the schematics of the\noptical setup in the main text.\nRevision 3-6\nMain text:\n“ ”\niSCAT in aqueous environments (A: axicon, L: lens, BM: beam block, BS: beam splitter, and BFP: back-\nfocal plane). The right inset represents the measured BFP image of the incident beam and the detection NA.\nSolid arrows along the yellow beam path indicate the incident and reflected beams, while the dashed arrow\nrepresents the scattered light.…”\nWe have revised the manuscript accordinglyto address the reviewer's concerns.\nMethods:\n“… The dark-field ring at the back focal plane of the water-immersion objective is generated and\nits size adjusted using an axicon and two lenses in series, as illustrated in Fig. 5a. An example is\nshown in the inset of Fig. 5a. … Incident angles are selected to satisfy the same plasmon modes\nexcitation with k = k sin75° = k sin47° = k sin40°.…”\nx 0 water PMMA\n7. The authors mention, “By careful engineering meta-atom arrays, ………. the\npresence of nanoscatterer.” The statement is vague, needs elaborate explanation,\nand must be substantiated.\nRevision 3-7\nWe appreciate the reviewer for the comments. The detailed design of the metasurface is\ndescribed in the metasurface characterization section of the main text. We have updated\nthe manuscript to avoid confusion.\nMain text:\n“… When a nanoscatterer approaches, it disturbs the local resonance mode of the meta-atom\nelements and alters the phase profile of the sub-diffraction grating (Fig. 1f). The design of the\nmeta-atom is chosen at a super-scattering resonance mode, such that the amplitude A and phase\nij\nof the resonance mode are sensitive to the nearby environmental change due to the presence of\nij\nthe nanoscatterer, and the perturbed mode within the sub-diffraction array radiates towards the\n𝜙\ndetection optics. …”\n8. In the results section, the authors claim high contrast. This is not substantiated\nand far from what is evident from the images shown in Fig. 4 and Fig. 5.\nWe thank the reviewer for the comments. We would like to argue that our method in fact\nproduces scattering images with excellent contrast compared to conventional iSCAT,\nas shown in Fig. 5. For PS beads with sizes of 22, 41, and 81 nm, regular iSCAT yields\nimage contrasts of 0.6 ± 0.1%, 3.9 ± 1.7%, and 23.9 ± 3.1% (n = 127, 137, and 38),\nrespectively, with 116 ms EET. In contrast, MAD-iSCAT demonstrates image contrasts\nof 14.9 ± 4.5%, 156.5 ± 46.6%, and 869.1 ± 254.6% (n = 451, 246, and 20) for the same\nparticle sizes. MAD-iSCAT shows an enhancement factor of 26 ± 7.5 for the 22 nm beads\ncompared to regular iSCAT, employing a single-frame differential process with 2 ms EET.\ncomparison of conventional iSCAT with MAD-iSCAT in the revised figure with a\ncorresponding color bar. The contrast improvement can be clearly seen in the color bar\nscale.\n9. One of the shortcomings of the present paper is the lack of experimental\nvalidation and application on real systems such as cell. Most of the results appear\nto be simulation based (Fig. 1-3). Actual experimental results are largely limited to\nFig. 4 and Fig. 5. It should be other way round i.e., experimental validation must\nhave a larger footprint.\nWe thank the reviewer for the comments. MAD-iSCAT provides a new scattering imaging\nmechanism that has not been explored before. It is crucial to present new formulations\nthat are required to describe the physical process. Therefore, a significant portion of the\nmanuscript is devoted to the theoretical analysis and numerical calculation of the\nproposed technique.\nRevision 3-8\nWe appreciate the reviewer for the comments. To address the reviewer's concern, we\nhave significantly revised the figure layout and content. Specifically, we have streamlined\nand reduced Fig. 3, which contains numerical calculations. Also, we have rearranged the\nmain texts and experimental figures (now Fig. 4, 5, 6), and expanded their corresponding\ndescriptions to provide a more detailed and larger footprint for the experimental\nvalidations and applications.\nMain text:\n“… We also conduct MAD-iSCAT imaging of small nanoparticles in aqueous environments, as\nshown in Fig. 5a.The imaging system is constructed with a custom axicon-based water-immersion\ndark-field microscopy.48Fig. 5 b,c compares MAD-iSCAT and regular iSCAT using PS nanobeads\nwith diameters of 22, 41, and 81 nm. …”\n10. In Fig. 5, direct comparison of MAD-iSCAT and iSCAT image is missing.\nWe thank the reviewer for the suggestion. We have demonstrated ferritin MAD-iSCAT\nimaging with a significantly higher contrast of 0.027 compared to the literature value of\n0.001 using conventional iSCAT.7 The 27-fold contrast improvement is in line with our\ndemonstration of 25-fold contrast improvement using 20 nm PS beads shown in Fig. 5.\nIn addition, it is noteworthy that the reference paper has a much higher illumination\nrequirement of 3381, (EET×P )0.5 product (EET: effective exposure time and P : power\nD D\ndensity). In contrast, MAD-iSCAT requires only 9.2, (EET×P )0.5 product to obtain the 27-\nD\nfold stronger contrast image.\n35\n11. There are many such points that needs to be take care-off in the manuscript.\nWhile the technique appears to be somewhat new, but it does little to advance the\nfield and lacks experimental validation & application. This is in addition to the\ndetailed comments by other two reviewers. The investigation appears to be in its\ninitial stages and requires a lot more conclusive validation to show that MAD-\niSCAT is better that iSCAT. Specifically, the images are not convincing and appears\nto be mild enhancement (visually). Overall, the work may be premature for\npublication at this stage.\nWe appreciate the reviewers’ detailed feedback. To summarize this study, we want to\nemphasize the following points.\n(1) Conventional iSCAT sensitivity is constrained by shot noise and the practical limits\nof imaging dynamic range, requiring either high illumination intensities (risking\nphotodamage to biological samples) or extensive temporal averaging. MAD-iSCAT\novercomes these limitations by leveraging super-scattering meta-atom modes and\nswitching between dark and bright modes of a meta-atom array. This introduces\nadditional interference terms with enhanced scattering fields, amplifying detection\nsignals without increasing photon flux or integration times. As mentioned in the\nresponse, the new contrast and SNR of MAD-iSCAT are represented as\n.\n𝟐 𝟐\n|𝑬𝑩𝑴| +|𝑬𝒔| +𝟐|𝑬𝑩𝑴||𝑬𝒔|𝒄𝒐𝒔𝜷𝟏+𝟐|𝑬𝑺||𝑬𝑩𝑮|𝒄𝒐𝒔𝜷𝟐+𝟐|𝑬𝑩𝑮||𝑬𝑩𝑴|𝒄𝒐𝒔𝜷𝟑\n𝑪𝑴𝑨𝑫−𝒊𝑺𝑪𝑨𝑻 = |𝑬𝑩𝑮| 𝟐 ..\n𝟐\n|𝑬𝑩𝑴| +𝟐|𝑬𝑩𝑴||𝑬𝒔|𝒄𝒐𝒔𝜷𝟏+𝟐|𝑬𝑩𝑮||𝑬𝑩𝑴|𝒄𝒐𝒔𝜷𝟑\nand ≈ 𝑪𝒊𝑺𝑪𝑨𝑻 + |𝑬𝑩𝑮| 𝟐\n.\n𝟐 𝟐\n|𝑬𝑩𝑴| +|𝑬𝒔| +𝟐|𝑬𝑩𝑴||𝑬𝒔|𝒄𝒐𝒔𝜷𝟏+𝟐|𝑬𝑺||𝑬𝑩𝑮|𝒄𝒐𝒔𝜷𝟐+𝟐|𝑬𝑩𝑮||𝑬𝑩𝑴|𝒄𝒐𝒔𝜷𝟑\n𝑺𝑵𝑹𝑴𝑨𝑫−𝒊𝑺𝑪𝑨𝑻 = |𝑬𝑩𝑮| .\n𝟐\n|𝑬𝑩𝑴| +𝟐|𝑬𝑩𝑴||𝑬𝒔|𝒄𝒐𝒔𝜷𝟏+𝟐|𝑬𝑩𝑮||𝑬𝑩𝑴|𝒄𝒐𝒔𝜷𝟑\nrespectively. T≈his𝑺 𝑵a𝑹p 𝒊 p 𝑺 r 𝑪 o 𝑨 a 𝑻 c+h marks a leap |𝑬b𝑩e𝑮|yond incremental improvements,\nenabling enhanced label-free scattering detection.\n(2) MAD-iSCAT is the first demonstration of exploiting both amplitude and phase\nresponses of the metasurface’s collective modes for far-field scattering detection.\nUnlike many other near-field techniques, such as surface-enhanced Raman\nscattering or plasmonic hot-spot-based imaging, MAD-iSCAT utilizes the radiative\nproperties of meta-atom arrays to detect nanoparticles. We demonstrate this\nconcept with a robust theoretical framework, supported by numerical simulations\nand experiments using various nanoparticles, including polystyrene beads,\nexosomes, and protein molecules. Experimentally, we have shown 23.2-fold\nSNR enhancement compared to previous iSCAT studies. These results pave\nthe way for a new paradigm in scattering detection and optimized metasurface-\nbased particle detection.\nTo address the reviewer's concern, in addition to Revision 3-1 and 3-2, we have revised\nthe discussion section.\nMain text:\n“… MAD-iSCAT underscores that the enhanced image contrast arises not only from the field\nenhancement effects near the silver surfaces but also from the additional interference with the\nradiation modes of silver meta-atom arrays. …”\nIn conclusion, we sincerely thank the reviewer for the valuable feedback, which has\ngreatly strengthened the manuscript. We believe these revisions address the concerns\nraised and highlight the transformative potential of MAD-iSCAT in advancing scattering-\nbased imaging.\nThis manuscript reports the use of a plasmonic metasurface to perform\ninterferometric scattering microscopy (iSCAT). The authors claim that the radiation\nmode supported by the metasurface interferes with the scattered light from\nnanoparticles, thereby producing high-contrast interferometric images. Reviewer\n#1 and Reviewer #2, both experts in iSCAT, have already raised critical questions\nregarding the novelty and performance of the work, to which the authors have\nresponded. As a specialist in plasmonics and metasurfaces, I will not repeat the\niSCAT-related issues already discussed, but instead provide three comments from\nthe perspective of employing metasurfaces for iSCAT.\n1. The primary novelty of this work lies in the conceptual innovation of employing\na metasurface for iSCAT for the first time. However, the manuscript lacks clarity in\nits presentation and fails to provide a concise physical picture. As a result, the\nauthors introduce a section, “Principles of contrast amplification in interferometric\nimages,” supported by eight equations; this style is more reminiscent of Physical\nReview rather than Nature Communications. Similarly, the section “The formation\nof scattering image by metasurface” relies heavily on numerical simulations to\njustify the mechanism behind, which is not a particularly strong form of evidence.\nWe thank the reviewer for this constructive feedback regarding the clarity and\npresentation of our theoretical framework. In response to these concerns, we have\nsubstantially revised the \"Principles of contrast amplification in interferometric images\"\nsection. We have also developed a more comprehensive theoretical framework to\nelucidate the underlying mechanism of MAD-iSCAT. As this issue was also raised by\nother reviewers and discussed in detail over four pages, we kindly refer the reviewer to\nour previous response in the section Theoretical framework for contrast and SNR\nenhancement in MAD-iSCAT on page 5.\nRevision 4-1\nThe manuscript has been revised accordingly to the reviewer’s concern. Please refer to\nRevision 1-2 for detailed updates. We have also streamlined Fig. 3 related to \"The\nformation of a scattering image by a metasurface\" section to present a more concise\nvisual representation that emphasizes the essential physical mechanisms.\nMain text:\n38\n“ ”\n2. It is well established that plasmonic metasurfaces can enhance light–matter\ninteractions and consequently boost scattering signals. Therefore, the reported\ncontrast enhancement by one to two orders of magnitude (Fig. 4) is in fact\nexpected. However, such enhancement comes at the cost of local heating due to\nmetallic nanostructures. This heating may severely limit the applicability of iSCAT\nfor biologically relevant tasks such as single-molecule tracking. The manuscript\ndoes not discuss this critical issue, and such a discussion should be included.\nWe appreciate this observation. Thank you for highlighting this important factor. We used\na relatively low laser power density of 42 W/cm². In previous studies of silver nanospheres\n(10–50 nm radius) at 4,000 W/cm², where scattering cross-sections peaked as 1×10 ¹⁴\nm², temperature rises of 0.5–3 °C were confirmed. Since our SSP mode features cross-\n⁻\nsections in the same order of magnitude but operates at 100× lower power density, we\nestimate a temperature increase of less than 1 °C. Under these conditions, biological\nsamples are not affected by plasmonic heating, alleviating concerns about thermal\ndamage.4\nWe appreciate this observation. We have revised the manuscript accordinglyto address\nthe reviewer's concerns.\nMain text:\n“…The laser power employed is sufficiently low to avoid significant photothermal effects and our\nprotective layer also minimizes the heating effect on detection targets and prevents photodamage\nto silver meta-atoms.51…”\n3. Comparative analysis is essential to place the method into context. For example,\nTable R1-2 in the response letter provides a very useful benchmark. I strongly\nrecommend that the authors incorporate this table into the main text and expand\nthe discussion accordingly.\nRevision 4-3\nWe appreciate this observation. We have revised the manuscript accordinglyto address\nthe reviewer's concerns.\nMain text:\n“… MAD-iSCAT has the potential to dramatically improve single-molecule mass imaging\nthroughput by orders of magnitude through enhanced fabrication approaches such as self-assembly\nfor better metasurface quality53 and large array geometries to enable high-throughput nanoparticle\nimaging with expanded field-of-view and suppressed background noise. …”\nSupplementary information:\n“Supplementary Section 13. The comparison of MAD-iSCAT and iSCAT.\nSupplementary Table 2. Imaging performances of different interferometric scattering imaging\ntechniques. (EET: effective exposure time, PSNR: peak signal-to-noise ratio, and P : incident\nD\npower density). R\nEET λ P (EET×P )0.5\nMethod Targets D D PSNR Reference\n(ms) (nm) (W/cm2) ((W·ms/cm2)0.5)\n20-nm PS 51 (PS\nMAD-iSCAT bead and 1 × 2 450 42 9.2 bead) This study\n440 kDa 9 (protein)\nACS Photonics\nTheoretical\n66 kDA 100 445 100,000 3,162 21 10, 2699-2710\nstudy (iSCAT)\n(2023)\nPhotonic\nresonator Nat. Commun.\n16 ×\ninterferometric 440 kDa 633 25 28.3 N/A 12, 1744\n2\nscattering (2021)\nmicroscopy\nQuantitative 53–803 300\n445 420,000 15,874.5 N/A 423-427\nmass imaging kDa × 2\n(2018)\nSingle-protein Nat. Photon.\n90-540 6.25\noptical 450 50,000 790.6 N/A 18, 388\nkDa × 2\nholography (2024)\nEvanescent 28-nm PS Nat. Commun.\n20 ×\nscattering bead and 450 60,000 1,549.2 N/A 13, 2298\n2\nmicroscopy 66 kDA (2022)\n200 (PS\nPlasmonic 26-nm PS Nat. Methods\n50 × bead) 141.4 (PS bead)\nscattering bead and 670 11 (protein) 17, 1010-1017\n2 2,000 447.2 (Protein)\nmicroscopy 385 kDA (2020)\n(protein)\nWe compare MAD-iSCAT with other advanced interferometric scattering-based imaging\ntechniques. … Although these comparisons are indirect (since variations in imaging components\ncan affect η and PSNRs are not well reported), Supplementary Table 2 summarizes the reported\nperformance metrics. The exposure time is calculated from their frame rates. By normalizing the\nPSNR values by target molecular weight, (EET×P )0.5, and λ4, the projected PSNR for theoretical\nD\niSCAT and plasmonic scattering microscopy are 0.91 and 1.55 under the same illumination\nconditions of MAD-iSCAT. Our approach demonstrates an SNR enhancement factor of 23.2\nrelative to conventional iSCAT and a 7.1-fold improvement over plasmonic scattering microscopy.\nBeyond this enhanced sensitivity, MAD-iSCAT achieves the highest imaging throughput reported\nfor interferometric scattering microscopy. This performance derives from our single-frame\ndifferential imaging approach, where acquisition speed is limited only by the camera readout rate\nrather than requiring temporal averaging or post-processing integration, while detection sensitivity\ncan be further enhanced by increasing laser power.”\nIn summary, the manuscript presents an interesting concept and demonstrates\npromising experimental results, but the novelty and implications are not clearly\narticulated. I recommend a major revision before the work can be considered\nfurther.\nIn conclusion, we sincerely appreciate the reviewer’s insightful feedback, which has\nsignificantly enhanced the quality of our manuscript.\n1. Zhang, P. F. et al. Plasmonic scattering imaging of single proteins and binding kinetics. Nat.\nMethods17, 1010-1017 (2020).\n2. Zhang, P. F. et al. Evanescent scattering imaging of single protein binding kinetics and DNA\nconformation changes. Nat. Commun.13, 2298 (2022).\n3. Tanaka, Y., Kaneda, S. & Sasaki, K. Nanostructured potential of optical trapping using a plasmonic\nnanoblock pair. Nano Lett.13, 2146-2150 (2013).\n4. Kailash & Verma, S. S. Exploring thermoplasmonic profiles of some noble metallic nanospheres.\nMater. Today Commun.33, 104795 (2022).\n5. Michalet, X. Mean square displacement analysis of single-particle trajectories with localization\nerror: Brownian motion in an isotropic medium. Phys. Rev. E Stat. Nonlin. Soft Matter Phys.82,\n041914 (2010).\n6. Qian, H., Sheetz, M. P. & Elson, E. L. Single particle tracking. Analysis of diffusion and flow in two-\ndimensional systems. Biophys J.60, 910-921 (1991).\n7. Piliarik, M. & Sandoghdar, V. Direct optical sensing of single unlabelled proteins and super-\nresolution imaging of their binding sites. Nat. Commun.5, 4495 (2014).\n42\n══════════════════════════════════════════════════\nReviewer #1 (Remarks to the Author):\nThank you for addressing all the comments satisfactorily.\nResponse: We sincerely thank the reviewer for the positive evaluation. We deeply\nappreciate the reviewer’s time and constructive input, which helped us improve the\nmanuscript.\nReviewer #2 (Remarks to the Author):\nThe manuscript has been significantly improved, and the authors have clearly\naddressed all of the points I originally raised. I believe it is now of sufficient quality\nfor publication in Nature Communications.\nResponse: We sincerely appreciate the insightful feedback provided, which contributed\nsignificantly to refining the quality of our work. We thank the reviewer for the positive\nrecommendation.\nReviewer #4 (Remarks to the Author):\nThe authors responded very positively and effectively to the issues I mentioned\nearlier. The current version is satisfactory, and I recommend publication.\nResponse: We sincerely thank the reviewer for the thoughtful feedback that guided these\nrevisions. We thank the reviewer for the positive recommendation.\nWe would like to express our sincere gratitude to all reviewers and the editorial office for\ntheir time and insightful feedback, which greatly improved the quality of our manuscript.\nIn addition, we also fixed a mistake in one of the figures and corrected a few numbers\naccordingly.\n1"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for addressing all the comments satisfactorily.", "reviewer_2": "The manuscript has been significantly improved, and the authors have clearly addressed all of the points I originally raised. I believe it is now of sufficient quality for publication in Nature Communications.", "reviewer_3": "I prefer to remain anonymous.", "reviewer_4": "The authors responded very positively and effectively to the issues I mentioned earlier. The current version is satisfactory, and I recommend publication."}, "author_response": "Response: We sincerely thank the reviewer for the positive evaluation. We deeply appreciate the reviewer’s time and constructive input, which helped us improve the manuscript.\nResponse: We sincerely appreciate the insightful feedback provided, which contributed significantly to refining the quality of our work. We thank the reviewer for the positive recommendation.\nResponse: We sincerely thank the reviewer for the thoughtful feedback that guided these revisions. We thank the reviewer for the positive recommendation.\nWe would like to express our sincere gratitude to all reviewers and the editorial office for their time and insightful feedback, which greatly improved the quality of our manuscript.\nIn addition, we also fixed a mistake in one of the figures and corrected a few numbers accordingly."}]

Decision letter