Acoustofluidic scanning fluorescence nanoscopy with a large field of view.

Large-field nanoscale fluorescence imaging is invaluable for many applications, such as imaging subcellular structures, visualizing protein interactions, and high-resolution tissue imaging. Unfortunately, conventional fluorescence microscopy requires a trade-off between resolution and field of view due to the nature of the optics used to form the image. To overcome this barrier, we developed an acoustofluidic scanning fluorescence nanoscope that simultaneously achieves superior resolution, a large field of view, and strong fluorescent signals. The acoustofluidic scanning fluorescence nanoscope utilizes the superresolution capabilities of microspheres that are controlled by a programmable acoustofluidic device for rapid fluorescence enhancement and imaging. The acoustofluidic scanning fluorescence nanoscope resolves structures that cannot be resolved with conventional fluorescence microscopes with the same objective lens and enhances the fluorescent signal by a factor of ~5 without altering the field of view of the image. The improved resolution realized with enhanced fluorescent signals and the large field of view achieved via acoustofluidic scanning fluorescence nanoscopy provides a powerful tool for versatile nanoscale fluorescence imaging for researchers in the fields of medicine, biology, biophysics, and biomedical engineering.

Advances in Microsphere-based Super-resolution Imaging.

Techniques to resolve images beyond the diffraction limit of light with a large field of view (FOV) are necessary to foster progress in various fields such as cell and molecular biology, biophysics, and nanotechnology, where nanoscale resolution is crucial for understanding the intricate details of large-scale molecular interactions. Although several means of achieving super-resolutions exist, they are often hindered by factors such as high costs, significant complexity, lengthy processing times, and the classical tradeoff between image resolution and FOV. Microsphere-based super-resolution imaging has emerged as a promising approach to address these limitations. In this review, we delve into the theoretical underpinnings of microsphere-based imaging and the associated photonic nanojet. This is followed by a comprehensive exploration of various microsphere-based imaging techniques, encompassing static imaging, mechanical scanning, optical scanning, and acoustofluidic scanning methodologies. This review concludes with a forward-looking perspective on the potential applications and future scientific directions of this innovative technology.

Acoustofluidic scanning fluorescence nanoscopy with large field of view.

Nanoscale fluorescence imaging with a large-field view is invaluable for many applications such as imaging of subcellular structures, visualizing protein interaction, and high-resolution tissue imaging. Unfortunately, conventional fluorescence microscopy has to make a trade-off between resolution and field of view due to the nature of the optics used to form an image. To overcome this barrier, we have developed an acoustofluidic scanning fluorescence nanoscope that can simultaneously achieve superior resolution, a large field of view, and enhanced fluorescent signal. The acoustofluidic scanning fluorescence nanoscope utilizes the super-resolution capability of microspheres that are controlled by a programable acoustofluidic device for rapid fluorescent enhancement and imaging. The acoustofluidic scanning fluorescence nanoscope can resolve structures that cannot be achieved with a conventional fluorescent microscope with the same objective lens and enhances the fluorescent signal by a factor of ~5 without altering the field of view of the image. The improved resolution with enhanced fluorescent signal and large field of view via the acoustofluidic scanning fluorescence nanoscope provides a powerful tool for versatile nanoscale fluorescence imaging for researchers in the fields of medicine, biology, biophysics, and biomedical engineering.

Acousto-dielectric tweezers for size-insensitive manipulation and biophysical characterization of single cells.

The intrinsic biophysical properties of cells, such as mechanical, acoustic, and electrical properties, are valuable indicators of a cell's function and state. However, traditional single-cell biophysical characterization methods are hindered by limited measurable properties, time-consuming procedures, and complex system setups. This study presents acousto-dielectric tweezers that leverage the balance between controllable acoustophoretic and dielectrophoretic forces applied on cells through surface acoustic waves and alternating current electric fields, respectively. Particularly, the balanced acoustophoretic and dielectrophoretic forces can trap cells at equilibrium positions independent of the cell size to differentiate between various cell-intrinsic mechanical, acoustic, and electrical properties. Experimental results show our mechanism has the potential for applications in single-cell analysis, size-insensitive cell separation, and cell phenotyping, which are all primarily based on cells' intrinsic biophysical properties. Our results also show the measured equilibrium position of a cell can inversely determine multiple biophysical properties, including membrane capacitance, cytoplasm conductivity, and acoustic contrast factor. With these features, our acousto-dielectric tweezing mechanism is a valuable addition to the resources available for biophysical property-based biological and medical research.

An acoustofluidic scanning nanoscope using enhanced image stacking and processing.

Nanoscale optical resolution with a large field of view is a critical feature for many research and industry areas, such as semiconductor fabrication, biomedical imaging, and nanoscale material identification. Several scanning microscopes have been developed to resolve the inverse relationship between the resolution and field of view; however, those scanning microscopes still rely upon fluorescence labeling and complex optical systems. To overcome these limitations, we developed a dual-camera acoustofluidic nanoscope with a seamless image merging algorithm (alpha-blending process). This design allows us to precisely image both the sample and the microspheres simultaneously and accurately track the particle path and location. Therefore, the number of images required to capture the entire field of view (200 × 200 µm) by using our acoustofluidic scanning nanoscope is reduced by 55-fold compared with previous designs. Moreover, the image quality is also greatly improved by applying an alpha-blending imaging technique, which is critical for accurately depicting and identifying nanoscale objects or processes. This dual-camera acoustofluidic nanoscope paves the way for enhanced nanoimaging with high resolution and a large field of view.

Intelligent nanoscope for rapid nanomaterial identification and classification.

Machine learning image recognition and classification of particles and materials is a rapidly expanding field. However, nanomaterial identification and classification are dependent on the image resolution, the image field of view, and the processing time. Optical microscopes are one of the most widely utilized technologies in laboratories across the world, due to their nondestructive abilities to identify and classify critical micro-sized objects and processes, but identifying and classifying critical nano-sized objects and processes with a conventional microscope are outside of its capabilities, due to the diffraction limit of the optics and small field of view. To overcome these challenges of nanomaterial identification and classification, we developed an intelligent nanoscope that combines machine learning and microsphere array-based imaging to: (1) surpass the diffraction limit of the microscope objective with microsphere imaging to provide high-resolution images; (2) provide large field-of-view imaging without the sacrifice of resolution by utilizing a microsphere array; and (3) rapidly classify nanomaterials using a deep convolution neural network. The intelligent nanoscope delivers more than 46 magnified images from a single image frame so that we collected more than 1000 images within 2 seconds. Moreover, the intelligent nanoscope achieves a 95% nanomaterial classification accuracy using 1000 images of training sets, which is 45% more accurate than without the microsphere array. The intelligent nanoscope also achieves a 92% bacteria classification accuracy using 50 000 images of training sets, which is 35% more accurate than without the microsphere array. This platform accomplished rapid, accurate detection and classification of nanomaterials with miniscule size differences. The capabilities of this device wield the potential to further detect and classify smaller biological nanomaterial, such as viruses or extracellular vesicles.

Acoustofluidic Scanning Nanoscope with High Resolution and Large Field of View.

Optical imaging with nanoscale resolution and a large field of view is highly desirable in many research areas. Unfortunately, it is challenging to achieve these two features simultaneously while using a conventional microscope. An objective lens with a low numerical aperture (NA) has a large field of view but poor resolution. In contrast, a high NA objective lens will have a higher resolution but reduced field of view. In an effort to close the gap between these trade-offs, we introduce an acoustofluidic scanning nanoscope (AS-nanoscope) that can simultaneously achieve high resolution with a large field of view. The AS-nanoscope relies on acoustofluidic-assisted scanning of multiple microsized particles. A scanned 2D image is then compiled by processing the microparticle images using an automated big-data image algorithm. The AS-nanoscope has the potential to be integrated into a conventional microscope or could serve as a stand-alone instrument for a wide range of applications where both high resolution and large field of view are required.

Lens-free shadow image based high-throughput continuous cell monitoring technique.

A high-throughput continuous cell monitoring technique which does not require any labeling reagents or destruction of the specimen is demonstrated. More than 6000 human alveolar epithelial A549 cells are monitored for up to 72 h simultaneously and continuously with a single digital image within a cost and space effective lens-free shadow imaging platform. In an experiment performed within a custom built incubator integrated with the lens-free shadow imaging platform, the cell nucleus division process could be successfully characterized by calculating the signal-to-noise ratios (SNRs) and the shadow diameters (SDs) of the cell shadow patterns. The versatile nature of this platform also enabled a single cell viability test followed by live cell counting. This study firstly shows that the lens-free shadow imaging technique can provide a continuous cell monitoring without any staining/labeling reagent and destruction of the specimen. This high-throughput continuous cell monitoring technique based on lens-free shadow imaging may be widely utilized as a compact, low-cost, and high-throughput cell monitoring tool in the fields of drug and food screening or cell proliferation and viability testing.