Deep learning-driven adaptive optics for single-molecule localization microscopy.

The inhomogeneous refractive indices of biological tissues blur and distort single-molecule emission patterns generating image artifacts and decreasing the achievable resolution of single-molecule localization microscopy (SMLM). Conventional sensorless adaptive optics methods rely on iterative mirror changes and image-quality metrics. However, these metrics result in inconsistent metric responses and thus fundamentally limit their efficacy for aberration correction in tissues. To bypass iterative trial-then-evaluate processes, we developed deep learning-driven adaptive optics for SMLM to allow direct inference of wavefront distortion and near real-time compensation. Our trained deep neural network monitors the individual emission patterns from single-molecule experiments, infers their shared wavefront distortion, feeds the estimates through a dynamic filter and drives a deformable mirror to compensate sample-induced aberrations. We demonstrated that our method simultaneously estimates and compensates 28 wavefront deformation shapes and improves the resolution and fidelity of three-dimensional SMLM through >130-µm-thick brain tissue specimens.

Acute Transcriptomic and Epigenetic Alterations at T12 After Rat T10 Spinal Cord Contusive Injury.

Spinal cord injury is a severely debilitating condition affecting a significant population in the USA. Spinal cord injury patients often have increased risk of developing persistent neuropathic pain and other neurodegenerative conditions beyond the primary lesion center later in their life. The molecular mechanism conferring to the "latent" damages at distal tissues, however, remains elusive. Here, we studied molecular changes conferring abnormal functionality at distal spinal cord (T12) beyond the lesion center (T10) by combining next-generation sequencing (RNA- and bisulfite sequencing), super-resolution microscopy, and immunofluorescence staining at 7 days post injury. We observed significant transcriptomic changes primarily enriched in neuroinflammation and synaptogenesis associated pathways. Transcription factors (TFs) that regulate neurogenesis and neuron plasticity, including Egr1, Klf4, and Myc, are significantly upregulated. Along with global changes in chromatin arrangements and DNA methylation, including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), bisulfite sequencing further reveals the involvement of DNA methylation changes in regulating cytokine, growth factor, and ion channel expression. Collectively, our results pave the way towards understanding transcriptomic and epigenomic mechanism in conferring long-term disease risks at distal tissues away from the primary lesion center and shed light on potential molecular targets that govern the regulatory mechanism at distal spinal cord tissues.

Sub-diffraction error mapping for localisation microscopy images.

Assessing the quality of localisation microscopy images is highly challenging due to the difficulty in reliably detecting errors in experimental data. The most common failure modes are the biases and errors produced by the localisation algorithm when there is emitter overlap. Also known as the high density or crowded field condition, significant emitter overlap is normally unavoidable in live cell imaging. Here we use Haar wavelet kernel analysis (HAWK), a localisation microscopy data analysis method which is known to produce results without bias, to generate a reference image. This enables mapping and quantification of reconstruction bias and artefacts common in all but low emitter density data. By avoiding comparisons involving intensity information, we can map structural artefacts in a way that is not adversely influenced by nonlinearity in the localisation algorithm. The HAWK Method for the Assessment of Nanoscopy (HAWKMAN) is a general approach which allows for the reliability of localisation information to be assessed.

Live-Cell Probe for In Situ Single-Cell Monitoring of Mitochondrial DNA Methylation.

Mitochondria, as the center of energy production, play an important role in cell homeostasis by regulating the cellular metabolism and mediating the cellular response to stress. Epigenetic changes such as DNA and histone methylation have been increasingly recognized to play a significant role in homeostasis and stress response. The cross-talking between the metabolome and the epigenome has attracted significant attention in recent years but with a major focus on how metabolism contributes to epigenomic changes. Few studies have focused on how epigenetic modifications may alter the mitochondrial composition and activity. In this work, we designed a novel probe targeting methylated CpGs of mitochondrial DNA (mtDNA). We demonstrated the capability of our probe to reveal the spatial distribution of methylated mtDNA and capture the mtDNA methylation changes at a single-cell level. We were also able to track single-cell mtDNA and nDNA methylation simultaneously and discovered the unsynchronized dynamics of the nucleus and mitochondria. Our tool offers a unique opportunity to understand the epigenetic regulation of mtDNA and its dynamic response to the microenvironment and cellular changes.

Three-dimensional nanoscopy of whole cells and tissues with in situ point spread function retrieval.

Single-molecule localization microscopy is a powerful tool for visualizing subcellular structures, interactions and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly degrades resolution with increasing depth. We propose a method that enables the construction of an in situ 3D response of single emitters directly from single-molecule blinking datasets, and therefore allows their locations to be pinpointed with precision that achieves the Cramér-Rao lower bound and uncompromised fidelity. We demonstrate this method, named in situ PSF retrieval (INSPR), across a range of cellular and tissue architectures, from mitochondrial networks and nuclear pores in mammalian cells to amyloid-ß plaques and dendrites in brain tissues and elastic fibers in developing cartilage of mice. This advancement expands the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra- and extracellular targets deep inside tissues.

Dynein-mediated microtubule translocation powering neurite outgrowth in chick and Aplysia neurons requires microtubule assembly.

Previously, we have shown that bulk microtubule (MT) movement correlates with neurite elongation, and blocking either dynein activity or MT assembly inhibits both processes. However, whether the contributions of MT dynamics and dynein activity to neurite elongation are separate or interdependent is unclear. Here, we investigated the underlying mechanism by testing the roles of dynein and MT assembly in neurite elongation of Aplysia and chick neurites using time-lapse imaging, fluorescent speckle microscopy, super-resolution imaging and biophysical analysis. Pharmacologically inhibiting either dynein activity or MT assembly reduced neurite elongation rates as well as bulk and individual MT anterograde translocation. Simultaneously suppressing both processes did not have additive effects, suggesting a shared mechanism of action. Single-molecule switching nanoscopy revealed that inhibition of MT assembly decreased the association of dynein with MTs. Finally, inhibiting MT assembly prevented the rise in tension induced by dynein inhibition. Taken together, our results suggest that MT assembly is required for dynein-driven MT translocation and neurite outgrowth.

A single tyrosine phosphorylation site in cortactin is important for filopodia formation in neuronal growth cones.

Cortactin is a Src tyrosine phosphorylation substrate that regulates multiple actin-related cellular processes. While frequently studied in nonneuronal cells, the functions of cortactin in neuronal growth cones are not well understood. We recently reported that cortactin mediates the effects of Src tyrosine kinase in regulating actin organization and dynamics in both lamellipodia and filopodia of Aplysia growth cones. Here, we identified a single cortactin tyrosine phosphorylation site (Y499) to be important for the formation of filopodia. Overexpression of a 499F phospho-deficient cortactin mutant decreased filopodia length and density, whereas overexpression of a 499E phospho-mimetic mutant increased filopodia length. Using an antibody against cortactin pY499, we showed that tyrosine-phosphorylated cortactin is enriched along the leading edge. The leading edge localization of phosphorylated cortactin is Src2-dependent, F-actin-independent, and important for filopodia formation. In vitro kinase assays revealed that Src2 phosphorylates cortactin at Y499, although Y505 is the preferred site in vitro. Finally, we provide evidence that Arp2/3 complex acts downstream of phosphorylated cortactin to regulate density but not length of filopodia. In conclusion, we have characterized a tyrosine phosphorylation site in Aplysia cortactin that plays a major role in the Src/cortactin/Arp2/3 signaling pathway controlling filopodia formation.

Analyzing complex single-molecule emission patterns with deep learning.

A fluorescent emitter simultaneously transmits its identity, location, and cellular context through its emission pattern. We developed smNet, a deep neural network for multiplexed single-molecule analysis to retrieve such information with high accuracy. We demonstrate that smNet can extract three-dimensional molecule location, orientation, and wavefront distortion with precision approaching the theoretical limit, and therefore will allow multiplexed measurements through the emission pattern of a single molecule.

Holographic fabrication of an arrayed one-axis scale grating for a two-probe optical linear encoder.

We present a method for the fabrication of an arrayed one-axis scale grating for a two-probe optical linear encoder using a dual-beam interference lithography (IL) system with a compact diode laser source. We employ a multiple-exposure stitching method to form an arrayed scale grating. This allows a scale grating with small line spacing to be achieved over a large width. This stitched scale grating integrates well with a newly developed two-probe optical encoder, allowing the measurement results to be numerically connected. Since neither the gap width nor the grating phase of the two adjacent gratings must be controlled, the fabrication process is both simplified and made more robust. This flexible and cost-effective fabrication technique can benefit many precision measurement applications. Experiments are carried out to demonstrate the feasibility of this technology.

Achieving unlimited recording length in interference lithography via broad-beam scanning exposure with self-referencing alignment.

Large-area holographic gratings are of great importance in diverse fields including long-range interference metrology, high-resolution astronomical telescopes, and chirped-pulse-amplification systems. However, in conventional interference lithography, the recording length is limited by the aperture of the collimating lenses. Here we propose broad-beam scanning exposure which employs the latent grating generated continuously during scanning for real-time dynamic fringe locking and thus achieves unlimited recording length. This method is experimentally proved to make high-quality gratings, and is expected to be a new type of interference lithography.

Fabrication of low-stray-light gratings by broad-beam scanning exposure in the direction perpendicular to the grating grooves.

Previous research on making low-stray-light gratings is mainly focused on process steps after the photoresist mask has been made. We propose to improve the quality of the photoresist mask directly in exposure. We present a broad-beam scanning exposure method along the grating vector (i.e., in the direction perpendicular to the grating grooves), utilizing a reference grating clamped below the substrate on the translation stage for phase and attitude locking. Scanning-exposed gratings with a size of 40 mm×40 mm are successfully made, which have straighter grooves and smoother surfaces, and their stray light levels around the first and second diffraction orders are decreased significantly.