Microsphere-assisted Microscopy Research Articles

Correlative super-resolution bright-field and fluorescence imaging by microsphere assisted microscopy.

The resolution of fluorescence imaging has been significantly enhanced with the development of super-resolution imaging techniques, surpassing the diffraction limit and reaching sub-diffraction scales of tens of nanometers. However, the resolution of the bright-field images of cells is restricted by the diffraction limit, leading to a significant gap between the resolutions of fluorescence and bright-field imaging, which hinders the research of the precise distribution of intracellular nanostructures. A microsphere superlens offers a promising solution by providing label-free super-resolution imaging capabilities compatible with fluorescence super-resolution imaging. In this study, we used microsphere superlenses to simultaneously enhance the resolution of bright-field and fluorescence imaging, achieving correlated super-resolution bright-field and fluorescence imaging. Compared to conventional bright-field images, we improved the imaging resolution from λ/1.3 to λ/4.2. A correlative super-resolution of mouse skeletal muscle cells was achieved, enabling the clear observation of the precise distribution of nanoparticles in mouse skeletal muscle cells. Furthermore, microsphere superlenses inherit the advantages of optical imaging, which is expected to enable the capturing of ultrafast biological activity within living cells with extremely high temporal resolutions.

Microsphere probe: combining microsphere-assisted microscopy with AFM.

In recent years, microsphere-assisted microscopy (MAM) and atomic force microscope (AFM) have been rapidly developed to meet the measurement needs of microstructures. However, the positioning of microspheres, the inability of AFM to touch the underlying sample through the transparent insulating layer, and the challenge of AFM fast positioning limit their use in practical measurements. In this paper, we propose a method that combines MAM with AFM by adhering the microsphere to the cantilever. This method allows MAM and AFM to work in parallel, and their imaging positions can correspond with each other. We use this method to measure memory devices, and the results show that MAM and AFM yield complementary advantages. This approach provides a new tool for analyzing complex structures in devices and has potential for wide application.

Miniaturized microsphere-assisted microscopy

Microsphere-assisted microscopy is a sub-diffraction-limited imaging technique, which uses dielectric microspheres to collect the near-field information of an object. In this work, the bulk of the classical microsphere-based system is reduced by the simple introduction of a ball lens. The miniaturized imaging system has been designed and vertically assembled. Performance of the proposed dual-sphere system is quantified through numerical simulations and experiments. A theoretical impulse response with a full width at half maximum smaller than half of the wavelength is reached in air. As a proof of concept, 500-nm-period standard gratings and gold nanoparticles are observed without a classical objective, validating a sub-wavelength resolution with a high imaging quality and low aberrations over the field of view.

Microsphere-assisted dark-field microscopy based on a fully immersed low refractive index microsphere.

Here we find that a fully immersed low refractive index SiO2 microsphere (or a microcylinder, a yeast cell) can clearly distinguish a sample with sub-diffraction features in dark-field illumination mode. The resolvable area of the sample by microsphere-assisted microscopy (MAM) is composed of two regions. One region locates below the microsphere, and a virtual image of this part of the sample is formed by the microsphere first and then the virtual image is received by the microscope. The other region is around the edge of the microsphere, and this part of the sample is directly imaged by the microscope. The simulated region of the enhanced electric field on the sample surface formed by the microsphere is consistent with the resolvable region in the experiment. Our studies show that the enhanced electric field on the sample surface generated by the fully immersed microsphere plays an important role in dark-field MAM imaging, and this finding will have a positive effect on exploring novel mechanisms in resolution improvement of MAM.

Surface plasmon-enhanced dark-field microsphere-assisted microscopy.

We present for the first time a surface plasmon-enhanced dark-field microsphere-assisted microscopy in imaging both low-contrast dielectric objects and metallic ones. We demonstrate, using an Al patch array as the substrate, the resolution and contrast in imaging low-contrast dielectric objects are improved compared to that of the metal plate substrate and a glass slide in dark-field microscopy (DFM). 365-nm-diameter hexagonally arranged SiO nanodots assembled on the three substrates can be resolved, with the contrast varied from 0.23 to 0.96, and the 300-nm-diameter hexagonally close-packed polystyrene nanoparticles can only be discerned on the Al patch array substrate. The resolution can be further improved by using the dark-field microsphere-assisted microscopy, and an Al nanodot array with a nanodot diameter of ∼65 nm and a center-to-center spacing of 125 nm can be just resolved, which cannot be distinguished in a conventional DFM. The focusing effect of the microsphere, as well as the excitation of the surface plasmons, provides evanescent illumination with enhanced local electric field (E-field) on an object. The enhanced local E-field acts as a near-field excitation source to enhance the scattering of the object, resulting in the improvement of imaging resolution.

Microsphere assistance in interference microscopy with high numerical aperture objective lenses

Various attempts have been discussed to overcome the lateral resolution limit and thus to enlarge the fields of application of optical interference microscopy. Microsphere-assisted microscopy and interferometry have proven that the imaging of structures well below Abbe’s resolution limit through near-field assistance is possible if microspheres are placed on the measured surface and utilized as near-field assisting imaging elements. The enhancement of the numerical aperture (NA) by the microspheres as well as photonic nanojets was identified to explain the resolution enhancement, but also whispering gallery modes and evanescent waves are assumed to have an influence. Up to now, to the best of our knowledge, there is no complete understanding of the underlying mechanisms and no model enabling to examine ideal imaging parameters. This contribution is intended to clarify how much the lateral resolution of an already highly resolving Linnik interferometer equipped with 100 × NA 0.9 objective lenses can be further improved by microspheres. Our simulation model developed so far is based on rigorous near-field calculations combined with the diffraction-limited illumination and imaging process in an interference microscope. Here, we extend the model with respect to microsphere-assisted interference microscopy providing a rigorous simulation of the scattered electric field directly above the sphere. Simulation and experimental results will be compared in the three-dimensional spatial frequency domain and discussed in context with ray-tracing computations to achieve an in-depth understanding of the underlying mechanism of resolution enhancement by the microsphere.

Microsphere-Assisted Imaging of Periodic and Non-Periodic Structures

In this work, the difference in imaging small clusters of nanoparticles and hexagonally close packed nanoparticle arrays by microsphere-assisted microscopy is studied. For both high-contrast samples and low-contrast ones, compared with small cluster samples, a better image quality and a larger lateral magnification are observed when imaging periodic array samples. We propose that in imaging periodic samples, the evanescent waves carrying high-frequency information can be converted into propagating waves by shifting the incident wavevectors into different diffraction orders. Therefore, more high-frequency information of the samples can be collected by the imaging system, which results in an improved image quality and a larger magnification when observing periodic array samples.

Improved performance in microsphere-assisted 2D and 3D imaging by polystyrene microspheres semi-immersed in SU-8 resist

In this work, we introduce a method to improve the performance of microsphere-assisted imaging by optimizing the refractive index of immersion media. In the method, polystyrene (PS) microspheres are semi-immersed in water, spin-on-glass, SU-8 resist, and S1805 resist. We experimentally find that the distortion of the image observed by a PS microsphere semi-immersed in SU-8 resist is the smallest while the resolution of the semi-immersed microsphere is the highest. ZEMAX software simulations show that PS microspheres semi-immersed in SU-8 resist have the smallest distortion and spherical aberration. Moreover, numerical simulations also reveal that they have the strongest peak intensity, the narrowest waist, and the shortest focal length of the photonic nanojet. When the semi-immersed PS microsphere is used in microsphere-assisted microscopy, a 200-nm-diameter hexagonally close-packed (hcp) silica nanoparticle array can be clearly distinguished. When they are used in microsphere-assisted interference microscopy, a 250-nm-diameter hcp silica nanoparticle array can be resolved.

Microsphere-assisted microscopy

Light microscopy is one of the most powerful techniques for nondestructive real-time imaging of specimens at a resolution beyond the reach of human eyes. However, the spatial resolution of any conventional microscope is fundamentally limited by the diffraction of light waves at the lens aperture. Microsphere-assisted microscopy (MAM) has emerged in the past decade as an interestingly simple yet efficient method to improve imaging resolution. In MAM, a micrometer-scale dielectric sphere is placed in the immediate vicinity of the specimen to enhance the imaging resolution and magnification. MAM is highly versatile and can be combined with various systems including wide-field, confocal, and fluorescent microscopes to name a few. The exact resolution enhancement mechanism in MAM is not yet clearly understood and is under intense investigation; however, fundamentally, it can be linked to the increase in the system's effective numerical aperture and evanescence wave collection, and possibly to contributions from photonic nanojet effect, resonance, and coherent effects. Various claims have been made in the literature about the resolution gain in MAM, most of which are due to using arbitrary criteria for quantifying the resolution and possible contributions from specimen-specific parameters in imaging metallic nanostructures. In this Tutorial, we discuss the progress in MAM with special scrutiny of the imaging resolution.

Microsphere-Assisted Digital Holographic Microscopy Integrated with On-Line Thermal Lens Spectrometry for 3d Imaging and Enhanced Detection

Microsphere-Assisted Digital Holographic Microscopy Integrated with On-Line Thermal Lens Spectrometry for 3d Imaging and Enhanced Detection

Super-resolution imaging of plasmonic nanostructures by microsphere-assisted microscopy.

We fabricate both triangularly and circularly shaped Au, Ag, and Cr nanoparticle arrays and observe the imaging properties of these plasmonic nanostructures by BaTiO3 glass (BTG) microsphere-assisted microscopy. We experimentally find that the resolution of triangularly shaped Ag nanoparticle arrays is higher than that of Au and Cr ones, and a gap resolution of ∼λ/7.7 is demonstrated for the circularly shaped Au, Ag, and Cr nanostructures. Numerical simulations show that when a fully immersed BTG microsphere is dispersed on the surface of a plasmonic nanostructure sample, an enhanced electric field is generated in the vicinity of the sample, especially at the gap of the microsphere and the sample, due to the focusing effect of the microsphere and the excitation of localized surface plasmon resonance in the plasmonic nanostructure. The enhanced electric field in Ag nanostructures is significantly stronger than that in Au and Cr ones. Besides, the microsphere collects, amplifies, and propagates the enhanced near-field information to the far field, resulting in the improvement of imaging resolution.

Optoplasmonic-Enhanced Imaging of Monolayer Polystyrene Nanoparticle Arrays by Barium Titanate Glass Microsphere-Assisted Microscopy: Implications for Nanoparticle Characterization

Low-contrast nanoparticles are difficult to detect optically because they weakly interact with photons. In this work, we have addressed this challenge by developing optoplasmonic hybrid structures composed of barium titanate glass (BTG) microspheres and Blu-ray disk (BD) substrates deposited with multilayer metal/dielectric coatings. A 300 nm and a 250 nm hexagonally close-packed polystyrene (PS) nanoparticle monolayer sample self-assembled on both a BD substrate and a metal one (glass slide deposited with the same multilayer coatings) can be discerned by BTG microsphere-assisted microscopy, and the contrast of the virtual images of the sample on the BD substrate is enhanced compared to that of the metal substrate. When the size of BTG microspheres is 20–30 μm, the contrast of the observed virtual images is the best. Moreover, the Talbot images of 300 nm PS nanoparticle arrays assembled on the BD substrate can also be experimentally observed. We can also distinguish 200 nm PS nanoparticles assembled into a one-dimensional stripe structure on the BD substrate. Numerical simulations reveal that the electric field among the gaps of PS nanoparticles and the contacting areas between the nanoparticles and a metal/BD substrate is enhanced, and the enhanced electric field of the sample placed on the BD substrate is stronger than that of the sample placed on the metal substrate. In addition, the enhanced electric field is the strongest when the BTG microsphere size is 20–30 μm in optoplamonic structures. The simulated results fit well with the experimental ones, indicating that the enhanced electric field improves the resolution of microsphere-assisted microscopy in imaging low-contrast PS nanoparticle samples.

Direct observation Brownian motion of individual nanoparticles in water using microsphere-assisted microscopy.

Observing Brownian motion of nanoscale objects through a traditional optical microscope is still a challenge. Here, we present a method to overcome this challenge by using a traditional optical microscope assisted with a removable microsphere-embedded thin film. The diffusion coefficient of individual unconstrained polystyrene (PS) nanoparticles with a diameter of 300 nm in water is calculated from their respective mean-square displacement versus time curves, and the measured diffusion coefficient shows good agreement with the theoretical Stokes-Einstein one, proving the feasibility of our method. In addition, the experimental results show that the movement of the PS nanoparticles is slowed down near a plane wall, and the diffusion coefficient is consistent with the theoretical constrained diffusion coefficient, which shows that our method can also study the constrained Brownian motion of nanoparticles constrained near a plane wall. Our research results are helpful for the application of microsphere-assisted microscopy in new fields and also provide a new method for nanoparticle tracking.

Near- to Far-Field Coupling of Evanescent Waves by Glass Microspheres

Through rigorous electromagnetic simulations, the natural coupling of high-spatial-frequency evanescent waves from the near field to the far field by dielectric microspheres is studied in air. The generation of whispering gallery modes inside the microspheres is shown independently of any resonance. In addition, the conversion mechanism of these evanescent waves into propagating waves is analysed. This latter point leads to key information that allows a better physical understanding of the super-resolution phenomenon in microsphere-assisted microscopy where sub-diffraction-limit revolving power is achieved.

Super-resolution imaging of low-contrast periodic nanoparticle arrays by microsphere-assisted microscopy* *Project supported by the National Natural Science Foundation of China (Grant No. 61673287).

We use the label-free microsphere-assisted microscopy to image low-contrast hexagonally close-packed polystyrene nanoparticle arrays with diameters of 300 and 250 nm. When a nanoparticle array is directly placed on a glass slide, it cannot be distinguished. If a 30-nm-thick Ag film is deposited on the surface of a nanoparticle array, the nanoparticle array with nanoparticle diameters of 300 and 250 nm can be distinguished. In addition, the Talbot effect of the 300-nm-diameter nanoparticle array is also observed. If a nanoparticle sample is assembled on a glass slide deposited with a 30-nm-thick Ag film, an array of 300-nm-diameter nanoparticles can be discerned. We propose that in microsphere-assisted microscopy imaging, the resolution can be improved by the excitation of surface plasmon polaritons (SPPs) on the sample surface or at the sample/substrate interface, and a higher near-field intensity due to the excited SPPs would benefit the resolution improvement. Our study of label-free super-resolution imaging of low-contrast objects will promote the applications of microsphere-assisted microscopy in life sciences.

Converting Evanescent Waves into Propagating Waves: The Super-Resolution Mechanism in Microsphere-Assisted Microscopy

The lateral resolution beyond the classical diffraction limit can be achieved by the combination of a dielectric microsphere and an optical microscope, and this paper utilizes microsphere-assisted ...

Coated High-Refractive-Index Barium Titanate Glass Microspheres for Optically Trapped Microsphere Super-Resolution Microscopy: A Simulation Study

As water is normally used as the immersion medium in optically trapped microsphere microscopy, the high-refractive-index barium titanate glass (BTG) microsphere shows a better imaging performance than the low-index polystyrene (PS) or melamine formaldehyde (MF) microsphere, but it is difficult to be trapped by single-beam optical trapping due to its overly high refractive index. In this study, coated BTG microspheres with a PS coating have been computationally explored for the combination of optical trapping with microsphere-assisted microscopy. The PS coating thickness affects both the optical trapping efficiency and photonic nanojet (PNJ) property of the coated BTG sphere. Compared to the uncoated BTG sphere, the coated BTG sphere with a proper PS coating thickness has a highly improved trapping efficiency which enables single-beam optical trapping, and a better PNJ with a higher optical intensity Imax and a narrower full width at half maximum (FWHM) corresponding to better imaging performance. These coated BTG spheres also have an advantage in trapping efficiency and imaging performance over conventional PS and MF spheres. The coated BTG microsphere is highly desirable for optically trapped microsphere super-resolution microscopy and potentially beneficial to other research areas, such as nanoparticle detection.

Surface plasmon enhancement for microsphere-assisted super-resolution imaging of metallodielectric nanostructures

Using a two-dimensional hexagonally close-packed SiO2/Ag nanoshell array coated with or without a dielectric layer as an imaging object, we find that the imaging quality of microsphere-assisted microscopy depends strongly on the optical properties of the metallodielectric sample. When a thin dielectric film is deposited on the top surface of a SiO2/Ag nanoshell array, a periodic enhanced near-field electric field is formed at the interface between the Ag layer and the deposited dielectric layer due to the excitation of surface plasmon polaritons. By increasing the dielectric constant of the coating layer, the enhanced near-field electric field region becomes larger and extends into the region which is closer to the imaging system. When the SiO2/Ag nanoshell array is coated with a SiO layer, the best resolution, the highest magnification, and the longest working distance are obtained. And, a SiO coated nanoshell array with a core size of 180 nm (λ/3, where λ = 540 nm is the central wavelength of illumination) can be resolved.

Selecting a Proper Microsphere to Combine Optical Trapping with Microsphere-Assisted Microscopy

Microsphere-assisted microscopy serves as an effective super-resolution technique in biological observations and nanostructure detections, and optical trapping is widely used for the manipulation of small particles like microspheres. In this study, we focus on the selection of microsphere types for the combination of the optical trapping and the super-resolution microsphere-assisted microscopy, by considering the optical trapping performances and the super-resolution imaging ability of index-different microspheres in water simultaneously. Finally, the polystyrene (PS) sphere and the melamine formaldehyde (MF) sphere have been selected from four typical index-different microspheres normally used in microsphere-assisted microscopy. In experiments, the optically trapped PS/MF microsphere in water has been used to achieve super-resolution imaging of a 139 nm line-width silicon nanostructure grating under white light illumination. The image quality and the magnification factor are affected by the refractive index contrast between the microspheres and the immersion medium, and the difference of image quality is partly explained by the photonic nanojet. This work guides us in selecting proper microspheres, and also provides a label-free super-resolution imaging technique in many research fields.

From 2D to 3D super-resolution imaging through glass microspheres -INVITED

Microsphere-assisted microscopy is a new imaging technique which allows the diffraction limit to be overcome using transparent microspheres. It makes it possible to reach a resolution of up to 100 nm in air while being label-free and full-field. An overview of the imaging technique is presented showing the influence of the photonic jet on the image nature and the unconventional behaviour of the magnification factor. Moreover, interferometry through microspheres is demonstrated for the 3D reconstruction of nanoelements.