DBlink: dynamic localization microscopy in super spatiotemporal resolution via deep learning.

Single-molecule localization microscopy (SMLM) has revolutionized biological imaging, improving the spatial resolution of traditional microscopes by an order of magnitude. However, SMLM techniques require long acquisition times, typically a few minutes, to yield a single super-resolved image, because they depend on accumulation of many localizations over thousands of recorded frames. Hence, the capability of SMLM to observe dynamics at high temporal resolution has always been limited. In this work, we present DBlink, a deep-learning-based method for super spatiotemporal resolution reconstruction from SMLM data. The input to DBlink is a recorded video of SMLM data and the output is a super spatiotemporal resolution video reconstruction. We use a convolutional neural network combined with a bidirectional long short-term memory network architecture, designed for capturing long-term dependencies between different input frames. We demonstrate DBlink performance on simulated filaments and mitochondria-like structures, on experimental SMLM data under controlled motion conditions and on live-cell dynamic SMLM. DBlink's spatiotemporal interpolation constitutes an important advance in super-resolution imaging of dynamic processes in live cells.

Monocular kilometer-scale passive ranging by point-spread function engineering.

Standard imaging systems are designed for 2D representation of objects, while information about the third dimension remains implicit, as imaging-based distance estimation is a difficult challenge. Existing long-range distance estimation technologies mostly rely on active emission of signal, which as a subsystem, constitutes a significant portion of the complexity, size and cost of the active-ranging apparatus. Despite the appeal of alleviating the requirement for signal-emission, passive distance estimation methods are essentially nonexistent for ranges greater than a few hundreds of meters. Here, we present monocular long-range, telescope-based passive ranging, realized by integration of point-spread-function engineering into a telescope, extending the scale of point-spread-function engineering-based ranging to distances where it has never been tested before. We provide experimental demonstrations of the optical system in a variety of challenging imaging scenarios, including adversarial weather conditions, dynamic targets and scenes of diversified textures, at distances extending beyond 1.7 km. We conclude with brief quantification of the effect of atmospheric turbulence on estimation precision, which becomes a significant error source in long-range optical imaging.

Multiplexed PSF Engineering for Three-Dimensional Multicolor Particle Tracking.

Three-dimensional spatiotemporal tracking of microscopic particles in multiple colors is a challenging optical imaging task. Existing approaches require a trade-off between photon efficiency, field of view, mechanical complexity, spectral specificity, and speed. Here, we introduce multiplexed point-spread-function engineering that achieves photon-efficient, 3D multicolor particle tracking over a large field of view. This is accomplished by first chromatically splitting the emission path of a microscope to different channels, engineering the point-spread function of each, and then recombining them onto the same region of the camera. We demonstrate our technique for simultaneously tracking five types of emitters in vitro as well as colocalization of DNA loci in live yeast cells.

Large-FOV 3D localization microscopy by spatially variant point spread function generation.

Accurate characterization of the microscopic point spread function (PSF) is crucial for achieving high-performance localization microscopy (LM). Traditionally, LM assumes a spatially invariant PSF to simplify the modeling of the imaging system. However, for large fields of view (FOV) imaging, it becomes important to account for the spatially variant nature of the PSF. Here, we propose an accurate and fast principal components analysis-based field-dependent 3D PSF generator (PPG3D) and localizer for LM. Through simulations and experimental three-dimensional (3D) single-molecule localization microscopy (SMLM), we demonstrate the effectiveness of PPG3D, enabling super-resolution imaging of mitochondria and microtubules with high fidelity over a large FOV. A comparison of PPG3D with a shift-variant PSF generator for 3D LM reveals a threefold improvement in accuracy. Moreover, PPG3D is approximately 100 times faster than existing PSF generators, when used in image plane-based interpolation mode. Given its user-friendliness, we believe that PPG3D holds great potential for widespread application in SMLM and other imaging modalities.

Depth-enhanced high-throughput microscopy by compact PSF engineering.

High-throughput microscopy is vital for screening applications, where three-dimensional (3D) cellular models play a key role. However, due to defocus susceptibility, current 3D high-throughput microscopes require axial scanning, which lowers throughput and increases photobleaching and photodamage. Point spread function (PSF) engineering is an optical method that enables various 3D imaging capabilities, yet it has not been implemented in high-throughput microscopy due to the cumbersome optical extension it typically requires. Here we demonstrate compact PSF engineering in the objective lens, which allows us to enhance the imaging depth of field and, combined with deep learning, recover 3D information using single snapshots. Beyond the applications shown here, this work showcases the usefulness of high-throughput microscopy in obtaining training data for deep learning-based algorithms, applicable to a variety of microscopy modalities.

Near index matching enables solid diffractive optical element fabrication via additive manufacturing.

Diffractive optical elements (DOEs) have a wide range of applications in optics and photonics, thanks to their capability to perform complex wavefront shaping in a compact form. However, widespread applicability of DOEs is still limited, because existing fabrication methods are cumbersome and expensive. Here, we present a simple and cost-effective fabrication approach for solid, high-performance DOEs. The method is based on conjugating two nearly refractive index-matched solidifiable transparent materials. The index matching allows for extreme scaling up of the elements in the axial dimension, which enables simple fabrication of a template using commercially available 3D printing at tens-of-micrometer resolution. We demonstrated the approach by fabricating and using DOEs serving as microlens arrays, vortex plates, including for highly sensitive applications such as vector beam generation and super-resolution microscopy using MINSTED, and phase-masks for three-dimensional single-molecule localization microscopy. Beyond the advantage of making DOEs widely accessible by drastically simplifying their production, the method also overcomes difficulties faced by existing methods in fabricating highly complex elements, such as high-order vortex plates, and spectrum-encoding phase masks for microscopy.

3D printable diffractive optical elements by liquid immersion.

Diffractive optical elements (DOEs) are used to shape the wavefront of incident light. This can be used to generate practically any pattern of interest, albeit with varying efficiency. A fundamental challenge associated with DOEs comes from the nanoscale-precision requirements for their fabrication. Here we demonstrate a method to controllably scale up the relevant feature dimensions of a device from tens-of-nanometers to tens-of-microns by immersing the DOEs in a near-index-matched solution. This makes it possible to utilize modern 3D-printing technologies for fabrication, thereby significantly simplifying the production of DOEs and decreasing costs by orders of magnitude, without hindering performance. We demonstrate the tunability of our design for varying experimental conditions, and the suitability of this approach to ultrasensitive applications by localizing the 3D positions of single molecules in cells using our microscale fabricated optical element to modify the point-spread-function (PSF) of a microscope.