† Corresponding author. E-mail:
Project supported by the National Natural Science Foundation of China (Grant Nos. 61735017 and 51672245), the Zhejiang Provincial Natural Science Foundation of China (Grant No. R17F050003), the National Key Basic Research Program of China (Grant No. 2015CB352003), the Fundamental Research Funds for the Central Universities, China, the Program for Zhejiang Leading Team of S&T Innovation, China, the Cao Guangbiao Advanced Technology Program, China, and First-class Universities and Academic Programs, China.
Super resolution imaging capable of resolving details beyond the diffraction limit is highly desired in many scientific and application fields, including bio-medicine, nanomaterial science, and opto-electronic integration. Up to now, many different methods have been proposed, among which wide-field, label-free super resolution microscopy is indispensable due to its good applicability to diverse sample types, large field of view (FOV), and high imaging speed. In recent years, nanostructures have made a crucial contribution to the wide-field, label-free subdiffraction microscopy, with various working mechanisms and configuration designs. This review summarizes the recent applications of the nanostructures in the wide-field, label-free super resolution microscopy, with the emphasis on the designs of hyperlens with hyperbolic dispersion, microsphere with “nano-jets”, and nanowire ring illumination microscopy based on spatial frequency shift effect. The bottlenecks of the current techniques and possible solutions are also discussed.
Optical microscopy techniques with high resolution play a primary role in the development or even revolution in many important areas, such as biomedicine, life science, and material science, by expanding our view of objects into micro or even nanoscale, which is too small to be seen with the naked eye. However, in classical microcopy systems, the physical resolution is limited to
To realize label-free, wide-field super resolution imaging, it is essential to realize light–matter interaction in the near-field within a wide area, and to then transfer the evanescent waves carrying subdiffraction spatial frequency information to propagating waves that can be detected by a conventional wide-field imaging system.
Nanostructures have shown great potential in generating or interacting with the evanescent waves with high efficiency due to their unique optical properties, strong spatial light localization,[17,18] high aspect ratio,[19,20] high refractive index,[21] and small footprint, thus becoming a good candidate for achieving the wide-field, label-free super resolution microscopy. Recent studies of the applications of nanostructures (e.g., hyperlens, microspheres, and fluorescent nanowire ring (NWR) illumination) in the wide-field, label-free super resolution microscopy have demonstrated great improvement in the available FOV, resolution, and versatility. In this paper, we will review the research progress in nanostructures based super resolution microscopy.
The dispersion relation of propagating electromagnetic (EM) wave in an ordinary medium with positive refractive index can be represented by a curve of circle or ellipse (the discussion is conducted in two-dimensional case for simplification), in which the increase of one component of the wave vector causes the other component to decrease. In particular, when one component exceeds the range covered by the dispersion curve, the other component would become a purely imaginary number, resulting in exponentially decaying EM wave along the corresponding direction; that is, evanescent wave. Consequently, the high spatial frequency information which represents the subdiffraction details of an object is carried by the evanescent wave and cannot be delivered to the far field, which leads to the diffraction limited resolution.
To overcome this limitation, researchers resort to the anisotropic medium that possesses dielectric permittivities of different signs along different directions (metamaterial). In this condition, the dispersion relation curve changes from elliptical to hyperbolic shape and allows the propagating of the waves with much larger wave vectors to carry subdiffraction spatial frequency information of the object. A cylindrical structure with a hollow core was proposed to enable the waves carrying subdiffraction spatial information to propagate after they have departed from this special medium and thus achieve a real far-field imaging.[22] Its dispersion relation is given as follows:
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This cylindrical medium with hyperbolic dispersion is called a hyperlens. Since metals tend to have negative dielectric permittivity and dielectrics have positive dielectric permittivity in the visible frequency range, metamaterials which contain both of the materials can be designed to exhibit the expected opposite signs of permittivity in two orthogonal directions.[22] Metals and dielectrics are fabricated as deep subwavelength layers for effective medium approximation. In 2007, Liu et al. first experimentally demonstrated a magnified optical hyperlens consisting of a curved periodic stack of Ag (35 nm) and Al2O3 (35 nm) deposited on half a cylindrical cavity fabricated on a quartz substrate[23] as shown in Fig.
![]() | Fig. 1. (color online) Multilayered cylindrical hyperlens. (a) Schematic of hyperlens and numerical simulation of imaging of sub-diffraction-limited object. (b) Hyperlens imaging of an object (a pair of lines with spacing of 150 nm). From left to right, scanning electron microscope (SEM) image of a line-pair object fabricated near inner side of hyperlens, magnified hyperlens image showing that 150-nm-spaced line-pair object can be clearly resolved, and resulting diffraction-limited image from control experiment without hyperlens. The hyperlens that is used is comprised of 16 layers of Ag/Al2O3. Reproduced with permission from Ref. [23]. |
The concentric multilayered hyperlens can only work in the UV spectrum range because the wavelengths are strictly limited within the UV range to achieve a negative permittivity along the layer (
![]() | Fig. 2. (color online) Optical fan-shaped hyperlens with radial layered structures. (a) Schematic of waveguide-integrated fan-shaped hyperlens, with inset showing hyperlens and blocking layer with two nano-slits that serve as object to be imaged. (b) SEM image of entire sample with hyperlens. On the left-hand is a grating coupler that is used as input port, and on the right-hand is the output port, which is an arc-shaped slit. The hyperlens is shown to be between the grating coupler and the curved slit. The capsule shaped protuberant ridge is excess gold that has expanded out of PMMA structure during electroplating. (c) Direct visualization at output port taken by using CCD camera. Panel on the right side shows profile of output beams. Reproduced with permission from Ref. [24]. |
The cylindrical hyperlens has previously been investigated and promoted, and the results can be found in Refs. [25]–[29], although most of those results were limited to one-dimensional imaging due to the cylindrical structure design of the hyperlens. In 2010, Rho et al. presented a spherical hyperlens for two-dimensional sub-diffraction-limited real-time imaging at visible frequencies.[30] Unlike the cylindrical hyperlens that uses the linearly p-polarized illumination, the spherical hyperlens uses the non-polarized illumination that stimulates transverse magnetic (TM) waves spanning the whole two-dimensional reciprocal space which follow the hyperbolic dispersion for two-dimensional super resolution imaging. In Rho et al.ʼs design, Ti3O5 (30 nm) with high dielectric permittivity was chosen to combine with Ag (as shown in Figs.
![]() | Fig. 3. (color online) Spherical hyperlens. (a) Schematic of spherical hyperlens comprised of 9 pairs of Ag and Ti3O5 layers. (b) Cross section of spherical hyperlens along green incident plane. The object with sub-wavelength features is carved in Cr layer atop Ag–Ti3O5 multilayer (also shown in light blue in (a)). TM component of unpolarized light relative to the plane is labelled by K. (c) Comparison between isofrequency contours for TM modes in hyperlens and isotropic medium made of silicon oxide. Arrows, which are of unit length and on ultra-flat curve, show that all the k components (including those much larger than the wave vectors available in dielectrics) propagate along the same radial direction, indicating the lack of diffraction. (d) SEM image of three dots positioned triangularly with gaps of 180 nm, 170 nm, and 160 nm. (e) Image and (f) intensity versus distance on cross section of the object along red line after being magnified. Reproduced with permission from Ref. [30]. |
The cylindrical or spherical geometry of a hyperlens is essential for magnification and the true far-field imaging. However, hyperlenses with cylindrical or spherical curved geometry usually face practical difficulties in placing the real sample on their inner surface and also in achieving a large FOV. Consequently, the metamaterial of planer structure but still possessing the ability to magnify has aroused considerable interest.[34–37] In 2010, Ma and Liu proposed a theoretical design by combining a metamaterial slab supporting the propagation of high wave vectors and a plasmonic waveguide couplers which can compensate for the phase, thus forming a magnified image.[36,37] According to this model, in 2018, Li et al.[34,35] experimentally demonstrated a planner super resolution lens by cascading a metallic meander cavity structure or distributed Bragg reflectors with a plasmonic waveguide coupler.[34,35] Lateral resolution at ∼180 nm can be achieved at λ = 640 nm with this kind of configuration.
Although a hyperlens allows the far-field super-resolution imaging at one snap shot, there are still some bottlenecks in this kind of device. First, the magnification factor of the hyperlens, which directly relates to the resolution improvement, can be calculated simply by the ratio between the outer cylindrical or spherical radius and the inner radius. However, a thicker hyperlens will lead to lower transmission level and will therefore prolong the exposure time, which is essential for real time imaging. The tradeoff between magnification and imaging speed is not very preferable; for example, a current hyperlens with 2×magnification would require an exposure time of up to tens of seconds.[22] Second, the working wavelengths for most of devices are limited to UV or short visible range, which is harmful to biological samples. Third, the metal and dielectric layers need to be deposited alternately for many circles. The requirement for the nanoscale thickness and the very concentric shape is a little harsh for current nano-fabrication techniques.
Due to the coupling of light to plasmons, there is always high intrinsic loss in the metallic-material-based hyperlens, which would limit their performance and practical applications. Compared with metallic devices, dielectric materials show much greater advantages due to their low loss and broadband transmission spectra which facilitate high-efficient and white light imaging. A macroscopic solid immersion dielectric lens (SIL) has been successfully demonstrated to improve the resolution of conventional optical imaging systems. However, the resolution improvement is limited to a factor of 2 in the visible spectrum due to the shortage of high-index lens materials and the large chromatic dispersion in the high-index materials.
The use of micro/nanoscale dielectric materials have brought new opportunities. Wavelength-scale SIL shows 25% improvement of the resolution compared with the macroscopic SIL.[38,39] Moreover in 2010, Wang et al.[40] presented a microsphere contacting technique for deep subdiffraction imaging.[40] They utilized ordinary glass to make microspheres (refractive index (RI) = 1.46;
![]() | Fig. 4. (color online) Microsphere contacting nanoscope. (a) Schematic of transmission mode microsphere superlens integrated with classical optical microscope. Spheres collect near-field object information and form virtual images that can be captured by conventional lens. (b)–(d) Intensity distributions calculated for (b) SIL with height ![]() ![]() ![]() ![]() |
In 2017, Fan et al.[41] proposed another method based on nano-sphere arrays to further increase the FOV without sacrificing the resolution.[41] They assembled visible-transparent, high-refractive-index, and deep-subwavelength-sized anatase TiO2 nanoparticles (D = 15 nm; RI = 2.55) into a hemisphere or super-hemisphere shape similar to that of a conventional SIL as illustrated in Fig.
![]() | Fig. 5. (color online) All-dielectric TiO2 mSIL. (a) Schematic of fabricated half-spherical and super-spherical mSIL. (b) Plane wave (λ = 550 nm) passing through the stacked TiO2 nanoparticles. Electric field hot spots are generated in gaps between contacting particles, which guide light to the underlying sample. (c) Large-area nanoscale evanescent wave illumination that can be focused onto the sample surface because of the excitation of nano-gap mode. (d) Size of illumination spots, which is equal to particle size and has FWHM resolution of ∼8 nm. (e) SEM image of a wafer pattern with 100 nm pitch size and its optical microscopy image with TiO2 mSIL with magnification factor 3.0. (f) SEM image of wafer pattern with 90 nm pitch size after being gold-coated and its optical microscopy image with TiO2 mSIL with magnification factor 3.1. Reproduced with permission from Ref. [41]. |
The mSIL method can work at a wide visible spectrum, form a two-dimensional image, and is applicable for metal and non-metal samples. Theoretically, the mSIL could be designed to be very large to achieve a satisfactory FOV. However, the reported mSILs have diameters limited to
Spatial frequency shift effect is very effective to detect the high spatial frequency information within a large area and thus achieve wide-field subdiffraction imaging. When the object is illuminated by light with spatially modulated intensity or phase, the spatial frequency spectrum of the object will shift with the incident spatial frequency, and this is expressed as
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Structured illumination microscopy (SIM) is a favorite wide-field microscopy and the resolution is improved by a factor of two.[42] In SIM, two propagating waves interfere at the object plane and produce an intensity modulated illumination pattern. The sample is fluorophore labelled, and the high-resolution spatial information can be observed in the form of Moiré fringes. Because the illumination pattern formed by propagating wave is still limited by the diffraction, the best resolution that can be achieved by conventional SIM is λ/4NA.
To further increase the resolution, nano-structures are resorted to stimulate and propagate plasmonic waves to construct structured illumination.[7] In 2014, Wei et al.[43] experimentally demonstrated a plasmonic SIM (PSIM). The object was illuminated by the interference pattern formed by counter propagating surface plasmonic (SP) waves at the interface between a thin silver layer and a silica substrate. Due to the high wave vector of the SP wave, a 2.6-times resolution improvement was achieved. In 2017, Ponsetto et al.[2] demonstrated another localized PSIM (LPSIM), which can triple in resolution. In the experiment, a nano silver antenna array was fabricated to generate a localized plasmonic field array, which works as a structured pattern to illuminate the object. The resolution improvement is directly determined by the pitch of the metal antenna array, which cannot be finer than λ/4NA otherwise an undetectable gap would occur between the low spatial frequency range and the shifted high spatial frequency range. To further increase the resolution, higher order SP illumination patterns and the dispersive property of the SP wave can be utilized in the future.
Although the conventional SIM has already shown potential in label-free microscopy,[44–46] all the reported super resolution PSIMs were only operated on the fluorescent-tagged samples. The reason can probably be attributed to the severe noise caused by the scattering from the nano-slits/nano-antennas, and to the deviation of the field distribution profile from a simple sine wave.
Evanescent wave illumination with pure and high transverse wave vector can be used to achieve label-free super resolution imaging. It is easy to excite large proportional evanescent waves around nano optical structures due to their natural small feature size on a wavelength scale and the devices based on nano-structures are usually more compact than the evanescent wave illumination based on total internal reflection.[47,48] In 2013, Hao et al.[49] used the surficial evanescent waves of a microfiber (diameter
In 2017, Liu et al. proposed a new on-chip evanescent illumination design to achieve label-free two-dimensional subdiffraction imaging with large FOV based on nano source active illumination.[50] This enables omnidirectional evanescent wave to propagate for two-dimensional imaging and its on-chip design makes it easily compatible with conventional microscope and other super resolution configurations. This method is called NWR illumination microscopy (NWRIM). Figure
![]() | Fig. 6. (color online) Schematic of the NWRIM. (a) Schematic of configuration and imaging process. Hexagon array is used as an example object to show effect of NWRIM. Under omnidirectional evanescent illumination from fluorescent NWR, subdiffraction nested features of central hexagon show up in far-field image. (b) Basic mechanism of NWRIM method. ![]() ![]() ![]() ![]() |
The NWRIM can detect the high spatial frequency information with large FOV and thus shows great potential on fast imaging. The FOV is directly related to the light propagation distance in the film waveguide. To improve the propagation distance and thus the FOV, Liu et al.[50] designed an Al2O3–SiO2 double layer waveguide as shown in Fig.
![]() | Fig. 7. (color online) FOV of NWRIM. (a) Simulated cross-sectional field distribution with fluorescent CdS nanowire on Al2O3–SiO2–Si double layer substrate. Intensity is shown on a log scale. (b) Simulated field magnitude in Al2O3 film along propagation direction. (c) CNR of images of fabricated slot structures at different distances from fluorescent CdS nanowire light source, achieved with film waveguide fabricated at 300 °C and room temperature, respectively. Inset shows two-dimensional “ZJU” pattern illuminated by NWR. The area circled by NWR is ![]() |
The main parameters of the reported label-free, wide-field super resolution imaging methods are listed in Table
![]() | Table 1. Main parameters of recently reported wide-field label-free subdiffraction imaging methods. . |
The NWRIM is also a versatile method that is applicable to diverse sample types. Figures
![]() | Fig. 8. (color online) NWRIM images of Bluray disk, integrated circuit, and “ZJU” pattern. (a) Conventional and (b) NWRIM images of Blu-ray disk. Boxed regions are magnified (top insets). (c) Conventional and (d) NWRIM images of integrated chip. Boxed regions are magnified (left bottom insets) and (e) SEM and (f) cross-sectional intensity distribution along the blue arrows. NWRIM and conventional illumination are denoted as NW and Conv., respectively. (g) SEM and (h) NWRIM images of arbitrary “ZJU” pattern etched on Al2O3 film. NA = 0.85. Reproduced with permission from Ref. [50]. |
In NWRIM, the detectable spatial frequency information is shifted outwards from the origin in the spatial frequency domain to center at the spatial frequency domain vector of the evanescent illumination. The highest detectable spatial frequency components are extended, while at the same time the low spatial frequency information around the origin is lost, especially when the NA of the imaging objective is small. The loss of low frequency information could cause blur and aberration of the image. As shown in Figs.
![]() | Fig. 9. (color online) Image reconstruction by multiple wavelength illumination. (a) Image of V-shaped structure (2° opening, ![]() ![]() |
In this review paper, we have summarized the applications of novel nanostructures in label-free, wide-field super-resolution microscopy, including hyperlens, microsphere, mSIL, and NWRIM. The corresponding mechanism and experimental results of how to improve the resolution, SNR, and FOV have been analyzed. The key point in all these methods is to detect the high spatial frequency information of the objects, which is carried by normally decayed evanescent waves under conventional illumination. To interact with these evanescent waves, the nanostructure-based-device has to be placed in the near field of the object, although the image can be projected to the far field. All these methods are limited to the surface detection without the ability to implement the deep three-dimensional imaging. Hyperlens, microspheres can couple the evanescent wave at the surface of the sample into propagating waves and form a super resolution image at one snap shot without needing any reconstruction. However, the SNR and FOV should be the main concern for hyperlens. Using gain materials but keeping the coherence in the hyperlens would be a potential solution. For the microsphere technique, it is difficult to observe a precise position and the FOV is also a tough problem. The NWRIM based on the frequency shift effect can detect the high spatial frequency information with a much wider FOV. However, for a complete spatial frequency components detection, many frames corresponding to different directions and shift values in the spatial frequency domain are required. The introduction of Galvo scanning system into the NW pumping system can increase the scanning speed and promise the real time wide-field label-free super resolution imaging.
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