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.

Self-Sensing Scanning Superlens for Three-Dimensional Noninvasive Visible-Light Nanoscale Imaging on Complex Surfaces.

Microsphere-assisted super-resolution imaging technology offers label-free, real-time dynamic imaging via white light, which has potential applications in living systems and the nanoscale detection of semiconductor chips. Scanning can aid in overcoming the limitations of the imaging area of a single microsphere superlens. However, the current scanning imaging method based on the microsphere superlens cannot achieve super-resolution optical imaging of complex curved surfaces. Unfortunately, most natural surfaces are composed of complex curved surfaces at the microscale. In this study, we developed a method to overcome this limitation through a microsphere superlens with a feedback capability. By maintaining a constant force between the microspheres and the sample, noninvasive super-resolution optical imaging of complex abiotic and biological surfaces was achieved, and the three-dimensional information on the sample was simultaneously obtained. The proposed method significantly expands the universality of scanning microsphere superlenses for samples and promotes their widespread use.

Novel Optofluidic Imaging System Integrated with Tunable Microlens Arrays.

Optofluidic tunable microlens arrays (MLAs) can manipulate and control light propagation using fluids. Lately, their applicability to miniature lab-on-a-chip systems is being extensively researched. However, it is difficult to incorporate 3D MLAs directly in a narrow microfluidic channel using common techniques. This has resulted in limited research on variable focal length imaging with optofluidic 3D MLAs. In this paper, we propose a method for fabricating MLAs in polydimethylsiloxane (PDMS)-based microchannels via electrohydrodynamic jet (E-jet) printing to achieve optofluidic tunable MLAs. Using this method, MLAs of diameters 15 to 80 µm can be fabricated in microfluidic channels with widths of 200 and 300 µm. By alternately using solutions with different refractive indices in the microchannel, the optofluidic microlenses exhibit reversible modulation properties while retaining the morphologies and refractive indices of the microlenses. The focal length of the resulting optofluidic chip can have threefold tunability, thereby achieving an imaging depth of approximately 450 µm. This outstanding advantage is useful in observing microspheres and cells flowing in the microfluidic system. Thus, the proposed optofluidic chip exhibits great potential for cell counting and imaging applications.

Characterization of interconnectivity of gelatin methacrylate hydrogels using photoacoustic imaging.

Hydrogels can provide a three-dimensional microenvironment for cells and thus serve as an extracellular matrix in a biofabrication process. The properties of hydrogels, such as their porosity and mechanical properties, significantly influence the cell growth. However, there is still a lack of effective methods for characterizing the hydrogel structure noninvasively. Herein, a photoacoustic (PA) imaging-based method is proposed for the characterization of gelatin methacrylate (GelMA) hydrogels. Owing to their high PA contrast, red blood cells (RBCs) are included as mediators in the GelMA hydrogel to analyze its pore distribution. The interconnectivity of the pores is further analyzed through the lysis of RBCs. The diffusion of the RBC lysis buffer in the GelMA is consistent with the trend observed in simulations. The analyzed vitality of HEK293 cells in different GelMA hydrogels reveals that understanding the diffusion of solutes (i.e., nutrients) is a potential strategy to optimize the hydrogel parameters during biofabrication.

In Situ Electrohydrodynamic Jet Printing-Based Fabrication of Tunable Microlens Arrays.

Tunable microlens arrays (MLAs) with controllable focal lengths have been extensively used in optical sensors, biochips, and electronic devices. The commonly used method is electrowetting on dielectric (EWOD) that controls the contact angle of the microlens to adjust the focal length. However, the fabrication of tunable MLAs at the microscale remains a challenge because the size of MLAs is limited by the external electrodes of EWOD. In this study, a highly integrated planar annular microelectrode array was proposed to achieve an electrowetting tunable MLA. The planar microelectrode was fabricated by electrohydrodynamic jet (E-jet) printing and the liquid microlens was then deposited in situ on the microelectrode. This method could realize 36 tunable liquid microlenses with an average diameter of 24 µm in a 320 × 320 µm2 plane. The fabricated tunable MLAs with higher integration levels and smaller sizes can be beneficial for cell imaging, optofluidic systems, and microfluidic chips.

Real-time red blood cell counting and osmolarity analysis using a photoacoustic-based microfluidic system.

Counting the number of red blood cells (RBCs) in blood samples is a common clinical diagnostic procedure, but conventional methods are unable to provide the size and other physical properties of RBCs at the same time. In this work, we explore photoacoustic (PA) detection as a rapid label-free and noninvasive analysis technique that can potentially be used for single RBC characterization based on their photoabsorption properties. We have demonstrated an on-chip PA flow cytometry system using a simple microfluidic chip combined with a PA imaging system to count and characterize up to ∼60 RBCs per second. Compared with existing microfluidic-based RBC analysis methods, which typically use camera-captured image sequences to characterize cell morphology and deformation, the PA method discussed here requires only the processing of one-dimensional time-series data instead of two- or three-dimensional time-series data acquired by computer vision methods. Therefore, the PA method will have significantly lower computational requirements when large numbers of RBCs are to be analyzed. Moreover, we have demonstrated that the PA signals of RBCs flowing in a microfluidic device could be directly used to acquire the osmolarity conditions (in the range of 124 to 497 mOsm L-1) of the medium surrounding the RBCs. This finding suggests a potential extension of applicability to blood tests via PA-based biomedical detection.

Self-assembled microcage fabrication for manipulating and selectively capturing microparticles and cells.

Single-cell-scale selective manipulation and targeted capture play a vital role in cell behavior analysis. However, selective microcapture has primarily been performed in specific circumstances to maintain the trapping state, making the subsequent in situ characterization and analysis of specific particles or cells difficult and imprecise. Herein, we propose a novel method that combines femtosecond laser two-photon polymerization (TPP) micromachining technology with the operation of optical tweezers (OTs) to achieve selective and targeted capture of single particles and cells. Diverse ordered microcages with different shapes and dimensions were self-assembled by micropillars fabricated via TPP. The micropillars with high aspect ratios were processed by single exposure, and the parameters of the micropillar arrays were investigated to optimize the capillary-force-driven self-assembly process of the anisotropic microcages. Finally, single microparticles and cells were selectively transported to the desired microcages by manipulating the flexibly of the OTs in a few minutes. The captured microparticles and cells were kept trapped without additional forces.

Scanning Super-Resolution Imaging in Enclosed Environment by Laser Tweezer Controlled Superlens.

Super-resolution imaging using microspheres has attracted tremendous scientific attention recently because it has managed to overcome the diffraction limit and allowed direct optical imaging of structures below 100 nm without the aid of fluorescent microscopy. To allow imaging of specific areas on the surface of samples, the migration of the microspheres to specific locations on two-dimensional planes should be controlled to be as precise as possible. The common approach involves the attachment of microspheres on the tip of a probe. However, this technology requires additional space for the probe and could not work in an enclosed environment, e.g., in a microfluidic enclosure, thereby reducing the range of potential applications for microlens-based super-resolution imaging. Herein, we explore the use of laser trapping to manipulate microspheres to achieve super-resolution imaging in an enclosed microfluidic environment. We have demonstrated that polystyrene microsphere lenses could be manipulated to move along designated routes to image features that are smaller than the optical diffraction limit. For example, a silver nanowire with a diameter of 90 nm could be identified and imaged. In addition, a mosaic image could be constructed by fusing a sequence of images of a sample in an enclosed environment. Moreover, we have shown that it is possible to image Escherichia coli bacteria attached on the surface of an enclosed microfluidic device with this method. This technology is expected to provide additional super-resolution imaging opportunities in enclosed environments, including microfluidic, lab-on-a-chip, and organ-on-a-chip devices.

Enhanced high-quality super-resolution imaging in air using microsphere lens groups.

Most microsphere-assisted super-resolution imaging experiments require a high-refractive-index microsphere to be immersed in a liquid to improve the super-resolution. However, samples are inevitably polluted by residuals in the liquid. This Letter presents a novel (to the best of our knowledge) method employing a microsphere lens group (MLG) that can easily achieve high-quality super-resolution imaging in air. The performance of this method is at par or better than that of the high-refractive-index microspheres immersed in liquid. In addition, the MLG generates a real image that is closely related to the photonic nanojet position of the microsphere super-lens. This imaging method is beneficial in microsphere imaging applications where liquids are impractical.

Thermometry of photosensitive and optically induced electrokinetics chips.

Optically induced electrokinetics (OEK)-based technologies, which integrate the high-resolution dynamic addressability of optical tweezers and the high-throughput capability of electrokinetic forces, have been widely used to manipulate, assemble, and separate biological and non-biological entities in parallel on scales ranging from micrometers to nanometers. However, simultaneously introducing optical and electrical energy into an OEK chip may induce a problematic temperature increase, which poses the potential risk of exceeding physiological conditions and thus inducing variations in cell behavior or activity or even irreversible cell damage during bio-manipulation. Here, we systematically measure the temperature distribution and changes in an OEK chip arising from the projected images and applied alternating current (AC) voltage using an infrared camera. We have found that the average temperature of a projected area is influenced by the light color, total illumination area, ratio of lighted regions to the total controlled areas, and amplitude of the AC voltage. As an example, optically induced thermocapillary flow is triggered by the light image-induced temperature gradient on a photosensitive substrate to realize fluidic hydrogel patterning. Our studies show that the projected light pattern needs to be properly designed to satisfy specific application requirements, especially for applications related to cell manipulation and assembly.

Spatial Manipulation and Assembly of Nanoparticles by Atomic Force Microscopy Tip-Induced Dielectrophoresis.

In this article, we present a novel method of spatial manipulation and assembly of nanoparticles via atomic force microscopy tip-induced dielectrophoresis (AFM-DEP). This method combines the high-accuracy positioning of AFM with the parallel manipulation of DEP. A spatially nonuniform electric field is induced by applying an alternating current (AC) voltage between the conductive AFM probe and an indium tin oxide glass substrate. The AFM probe acted as a movable DEP tweezer for nanomanipulation and assembly of nanoparticles. The mechanism of AFM-DEP was analyzed by numerical simulation. The effects of solution depth, gap distance, AC voltage, solution concentration, and duration time were experimentally studied and optimized. Arrays of 200 nm polystyrene nanoparticles were assembled into various nanostructures, including lines, ellipsoids, and arrays of dots. The sizes and shapes of the assembled structures were controllable. It was thus demonstrated that AFM-DEP is a flexible and powerful tool for nanomanipulation.

Scanning superlens microscopy for non-invasive large field-of-view visible light nanoscale imaging.

Nanoscale correlation of structural information acquisition with specific-molecule identification provides new insight for studying rare subcellular events. To achieve this correlation, scanning electron microscopy has been combined with super-resolution fluorescent microscopy, despite its destructivity when acquiring biological structure information. Here we propose time-efficient non-invasive microsphere-based scanning superlens microscopy that enables the large-area observation of live-cell morphology or sub-membrane structures with sub-diffraction-limited resolution and is demonstrated by observing biological and non-biological objects. This microscopy operates in both non-invasive and contact modes with ∼200 times the acquisition efficiency of atomic force microscopy, which is achieved by replacing the point of an atomic force microscope tip with an imaging area of microspheres and stitching the areas recorded during scanning, enabling sub-diffraction-limited resolution. Our method marks a possible path to non-invasive cell imaging and simultaneous tracking of specific molecules with nanoscale resolution, facilitating the study of subcellular events over a total cell period.