PROJECT SUMMARY Over the past two decades, a number of “super-resolution” 3D imaging technologies have been developed, enabling researchers to observe nanoscale biological structures that were previously invisible to traditional, diffraction-limited imaging techniques. The ability to visualize cellular and subcellular structures at the nanoscale has revealed key insights into a variety of biological processes. Although impressive progress has been made in the development of 3D super-resolution imaging techniques, researchers are often forced to accept a tradeoff in terms of the resolution, field-of-view, speed, and ease of use of their 3D imaging technique. Recently, we have developed an acoustofluidic scanning nanoscope that can simultaneously achieve both super-resolution and large field-of-view imaging in 2D. In this R01 project, we will develop and validate a 3D acoustofluidic scanning nanoscope with the following features: (1) Super-resolution imaging with lateral and axial resolutions of ~50 nm and ~120 nm, respectively: The proposed 3D imaging method will achieve a resolution that is four times better than that from a confocal microscope, which makes the optical imaging of more detailed inner architecture of many subcellular structures possible; (2) Large field-of-view (~1,100×1,100 µm2): Conventional optical imaging methods achieve high-throughput imaging at the cost of reduced resolution and vice versa. By utilizing acoustics to simultaneously manipulate multiple microsphere lenses, the proposed imaging method will solve this long-standing technical barrier for large field-of-view imaging while maintaining superior lateral and axial resolution; (3) Imaging speed 10 times faster than that from a confocal microscope: Rapid z-stacking at a speed 10 times faster than that of a confocal microscope can be achieved by using surface acoustic waves to scan an array of microspheres across the sample volume in a precise, controllable manner; (4) Seamless connection to a conventional optical microscope for ease of use: Our device can be seamlessly connected to a conventional optical microscope without modification of the optical setup, which can significantly reduce the cost and the complexity of operation. With the aforementioned advantages, the proposed 3D acoustofluidic scanning nanoscope technology has the potential to significantly exceed current standards in the field and address many unmet needs. We will validate its performance by imaging 3D nanorod samples and the organelles of live HeLa cells. In this regard, we aim to demonstrate the far-reaching potential of our 3D acoustofluidic scanning nanoscope technology to enable improved research in areas ranging from subcellular imaging to the visualization of 3D neural activity.

Project Narrative 3D imaging is an indispensable tool for understanding biological processes. Although impressive progress has been made in the development of 3D imaging, existing techniques suffer from tradeoffs in terms of their resolution, field-of-view, and speed. The objective of this project is to develop an acoustic-based 3D nano- imaging technology that attaches seamlessly to a conventional optical microscope and achieves high resolution, large field-of-view, and high speed, simultaneously.