Self-inspired learning for denoising live-cell super-resolution microscopy.

Every collected photon is precious in live-cell super-resolution (SR) microscopy. Here, we describe a data-efficient, deep learning-based denoising solution to improve diverse SR imaging modalities. The method, SN2N, is a Self-inspired Noise2Noise module with self-supervised data generation and self-constrained learning process. SN2N is fully competitive with supervised learning methods and circumvents the need for large training set and clean ground truth, requiring only a single noisy frame for training. We show that SN2N improves photon efficiency by one-to-two orders of magnitude and is compatible with multiple imaging modalities for volumetric, multicolor, time-lapse SR microscopy. We further integrated SN2N into different SR reconstruction algorithms to effectively mitigate image artifacts. We anticipate SN2N will enable improved live-SR imaging and inspire further advances.

Bright and stable monomeric green fluorescent protein derived from StayGold.

The high brightness and photostability of the green fluorescent protein StayGold make it a particularly attractive probe for long-term live-cell imaging; however, its dimeric nature precludes its application as a fluorescent tag for some proteins. Here, we report the development and crystal structures of a monomeric variant of StayGold, named mBaoJin, which preserves the beneficial properties of its precursor, while serving as a tag for structural proteins and membranes. Systematic benchmarking of mBaoJin against popular green fluorescent proteins and other recently introduced monomeric and pseudomonomeric derivatives of StayGold established mBaoJin as a bright and photostable fluorescent protein, exhibiting rapid maturation and high pH/chemical stability. mBaoJin was also demonstrated for super-resolution, long-term live-cell imaging and expansion microscopy. We further showed the applicability of mBaoJin for neuronal labeling in model organisms, including Caenorhabditis elegans and mice.

Miniature three-photon microscopy maximized for scattered fluorescence collection.

In deep-tissue multiphoton microscopy, diffusion and scattering of fluorescent photons, rather than ballistic emanation from the focal point, have been a confounding factor. Here we report on a 2.17-g miniature three-photon microscope (m3PM) with a configuration that maximizes fluorescence collection when imaging in highly scattering regimes. We demonstrate its capability by imaging calcium activity throughout the entire cortex and dorsal hippocampal CA1, up to 1.2 mm depth, at a safe laser power. It also enables the detection of sensorimotor behavior-correlated activities of layer 6 neurons in the posterior parietal cortex in freely moving mice during single-pellet reaching tasks. Thus, m3PM-empowered imaging allows the study of neural mechanisms in deep cortex and subcortical structures, like the dorsal hippocampus and dorsal striatum, in freely behaving animals.

Tracking endogenous proteins based on RNA editing-mediated genetic code expansion.

Protein labeling approaches are important to study proteins in living cells, and genome editing tools make it possible to tag endogenous proteins to address the concerns associated with overexpression. Here we established RNA editing-mediated noncanonical amino acids (ncAAs) protein tagging (RENAPT) to site-specifically label endogenous proteins with ncAAs in living cells. RENAPT labels protein in a temporary and nonheritable manner and is not restricted by protospacer adjacent motif sequence. Using a fluorescent ncAA or ncAA with a bio-orthogonal reaction handle for subsequent dye labeling, we demonstrated that a variety of endogenous proteins can be imaged at their specific subcellular locations. In addition, two proteins can be tagged individually and simultaneously using two different ncAAs. Furthermore, endogenous ion channels and neuron-specific proteins can be real-time labeled in primary neurons. Thus, RENAPT presents a promising platform with broad applicability for tagging endogenous proteins in living cells to study their localization and functions.

Physical Contacts Between Mitochondria and WPBs Participate in WPB Maturation.

BACKGROUND: Weibel-Palade bodies (WPBs) are endothelial cell-specific cigar-shaped secretory organelles containing various biologically active molecules. WPBs play crucial roles in thrombosis, hemostasis, angiogenesis, and inflammation. The main content of WPBs is the procoagulant protein vWF (von Willebrand factor). Physical contacts and functional cross talk between mitochondria and other organelles have been demonstrated. Whether an interorganellar connection exists between mitochondria and WPBs is unknown. METHODS: We observed physical contacts between mitochondria and WPBs in human umbilical vein endothelial cells by electron microscopy and living cell confocal microscopy. We developed an artificial intelligence-assisted method to quantify the duration and length of organelle contact sites in live cells. RESULTS: We found there existed physical contacts between mitochondria and WPBs. Disruption of mitochondrial function affected the morphology of WPBs. Furthermore, we found that Rab3b, a small GTPase on the WPBs, was enriched at the mitochondrion-WPB contact sites. Rab3b deficiency reduced interaction between the two organelles and impaired the maturation of WPBs and vWF multimer secretion. CONCLUSIONS: Our results reveal that Rab3b plays a crucial role in mediating the mitochondrion-WPB contacts, and that mitochondrion-WPB coupling is critical for the maturation of WPBs in vascular endothelial cells.

Functional network topography of the medial entorhinal cortex.

The medial entorhinal cortex (MEC) creates a map of local space, based on the firing patterns of grid, head-direction (HD), border, and object-vector (OV) cells. How these cell types are organized anatomically is debated. In-depth analysis of this question requires collection of precise anatomical and activity data across large populations of neurons during unrestrained behavior, which neither electrophysiological nor previous imaging methods fully afford. Here, we examined the topographic arrangement of spatially modulated neurons in the superficial layers of MEC and adjacent parasubiculum using miniaturized, portable two-photon microscopes, which allow mice to roam freely in open fields. Grid cells exhibited low levels of co-occurrence with OV cells and clustered anatomically, while border, HD, and OV cells tended to intermingle. These data suggest that grid cell networks might be largely distinct from those of border, HD, and OV cells and that grid cells exhibit strong coupling among themselves but weaker links to other cell types.

Optimal sidestepping of intraflagellar transport kinesins regulates structure and function of sensory cilia.

Cytoskeletal-based molecular motors produce force perpendicular to their direction of movement. However, it remains unknown whether and why motor proteins generate sidesteps movement along their filamentous tracks in vivo. Using Hessian structured illumination microscopy, we located green fluorescent protein (GFP)-labeled intraflagellar transport (IFT) particles inside sensory cilia of live Caenorhabditis elegans with 3-6-nanometer accuracy and 3.4-ms resolution. We found that IFT particles took sidesteps along axoneme microtubules, demonstrating that IFT motors generate torque in a living animal. Kinesin-II and OSM-3-kinesin collaboratively drive anterograde IFT. We showed that the deletion of kinesin-II, a torque-generating motor protein, reduced sidesteps, whereas the increase of neck flexibility of OSM-3-kinesin upregulated sidesteps. Either increase or decrease of sidesteps of IFT kinesins allowed ciliogenesis to the regular length, but changed IFT speeds, disrupted axonemal ninefold symmetry, and inhibited sensory cilia-dependent animal behaviors. Thus, an optimum level of IFT kinesin sidestepping is associated with the structural and functional fidelity of cilia.

Miniature two-photon microscopy for enlarged field-of-view, multi-plane and long-term brain imaging.

We have developed a miniature two-photon microscope equipped with an axial scanning mechanism and a long-working-distance miniature objective to enable multi-plane imaging over a volume of 420 × 420 × 180 µm3 at a lateral resolution of ~1 µm. Together with the detachable design that permits long-term recurring imaging, our miniature two-photon microscope can help decipher neuronal mechanisms in freely behaving animals.

RNA-binding protein-regulated fibronectin is essential for EGFR-activated metastasis of head and neck squamous cell carcinoma.

There is a higher expression level of epidermal growth factor receptor (EGFR) in up to 90% of advanced head and neck squamous cell carcinoma (HNSCC) tissue than in normal surrounding tissues. However, the role of RNA-binding proteins (RBPs) in EGFR-associated metastasis of HNSCC remains unclear. In this study, we reveal that RBPs, specifically nucleolin (NCL) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), correlated with the mesenchymal phenotype of HNSCC. The depletion of RBPs significantly attenuated EGF-induced HNSCC metastasis. Intriguingly, the EGF-induced EMT markers, such as fibronectin, were regulated by RBPs through the ERK and NF-κB pathway, followed by the enhancement of mRNA stability of fibronectin through the 5' untranslated region (5'-UTR) of the gene. The upregulation of fibronectin triggered the integrin signaling activation to enhance tumor cells' attachment to endothelial cells and increase endothelial permeability. In addition, the concurrence of EGFR and RBPs or EGFR and fibronectin was associated with overall survival and disease-free survival of HNSCC. The in vivo study showed that depletion of NCL, hnRNPA2B1, and fibronectin significantly inhibited EGF-promoted extravasation of tumor cells into lung tissues. The depletion of fibronectin or treatment with integrin inhibitors dramatically attenuated EGF-induced HNSCC metastatic nodules in the lung. Our data suggest that the RBPs/fibronectin axis is essential for EGF-induced tumor-endothelial cell interactions to enhance HNSCC cell metastasis.

A practical multicellular sample preparation pipeline broadens the application of in situ cryo-electron tomography.

The structural studies of macromolecules in their physiological context, particularly in tissue, is constrained by the bottleneck of sample preparation. In this study, we present a practical pipeline for preparing multicellular samples for cryo-electron tomography. The pipeline comprises sample isolation, vitrification, and lift-out-based lamella preparation using commercially available instruments. We demonstrate the efficacy of our pipeline by visualizing pancreatic ß cells from mouse islets at the molecular level. This pipeline enables the determination of the properties of insulin crystals in situ for the first time, using unperturbed samples.

New imaging instrument in animal models: Two-photon miniature microscope and large field of view miniature microscope for freely behaving animals.

Over the past decade, novel optical imaging tools have been developed for imaging neuronal activities along with the evolution of fluorescence indicators with brighter expression and higher sensitivity. Miniature microscopes, as revolutionary approaches, enable the imaging of large populations of neuron ensembles in freely behaving rodents and mammals, which allows exploring the neural basis of behaviors. Recent progress in two-photon miniature microscopes and mesoscale single-photon miniature microscopes further expand those affordable methods to navigate neural activities during naturalistic behaviors. In this review article, two-photon miniature microscopy techniques are summarized historically from the first documented attempt to the latest ones, and comparisons are made. The driving force behind and their potential for neuroscientific inquiries are also discussed. Current progress in terms of the mesoscale, i.e., the large field-of-view miniature microscopy technique, is addressed as well. Then, pipelines for registering single cells from the data of two-photon and large field-of-view miniature microscopes are discussed. Finally, we present the potential evolution of the techniques.

An optimized acetylcholine sensor for monitoring in vivo cholinergic activity.

The ability to directly measure acetylcholine (ACh) release is an essential step toward understanding its physiological function. Here we optimized the GRABACh (GPCR-activation-based ACh) sensor to achieve substantially improved sensitivity in ACh detection, as well as reduced downstream coupling to intracellular pathways. The improved version of the ACh sensor retains the subsecond response kinetics, physiologically relevant affinity and precise molecular specificity for ACh of its predecessor. Using this sensor, we revealed compartmental ACh signals in the olfactory center of transgenic flies in response to external stimuli including odor and body shock. Using fiber photometry recording and two-photon imaging, our ACh sensor also enabled sensitive detection of single-trial ACh dynamics in multiple brain regions in mice performing a variety of behaviors.

Unraveling the Vibrational Spectral Signatures of a Dislocated H Atom in Model Proton-Coupled Electron Transfer Dyad Systems.

Phenol-benzimidazole and phenol-pyridine proton-coupled electron transfer (PCET) dyad systems are computationally investigated to resolve the origins of the asymmetrically broadened H-bonded OH stretch transitions that have been previously reported using cryogenic ion vibrational spectroscopy in the ground electronic state. Two-dimensional (2D) potentials describing the strongly shared H atom are predicted to be very shallow along the H atom transfer coordinate, enabling dislocation of the H atom between the donor and acceptor groups upon excitation of the OH vibrational modes. These soft H atom potentials result in strong coupling between the OH modes, which exhibit significant bend-stretch mixing, and a large number of normal mode coordinates. Vibrational spectra are calculated using a Hamiltonian that linearly and quadratically couples the H atom potentials to over two dozen of the most strongly coupled normal modes treated at the harmonic level. The calculated vibrational spectra qualitatively reproduce the asymmetric shape and breadth of the experimentally observed bands in the 2300-3000 cm-1 range. Interestingly, these transitions fall well above the predicted OH stretch fundamentals, which are computed to be surprisingly red-shifted (<2000 cm-1). Time-dependent calculations predict rapid (<100 fs) relaxation of the excited OH modes and instant response from the lower-frequency normal modes, corroborating the strong coupling predicted by the model Hamiltonian. The results highlight a unique broadening mechanism and complicated anharmonic effects present within these biologically relevant PCET model systems.

Ultrafast transient vibrational action spectroscopy of cryogenically cooled ions.

Ultrafast transient vibrational action spectra of cryogenically cooled Re(CO)3(CH3CN)3+ ions are presented. Nonlinear spectra were collected in the time domain by monitoring the photodissociation of a weakly bound N2 messenger tag as a function of delay times and phases between a set of three infrared pulses. Frequency-resolved spectra in the carbonyl stretch region show relatively strong bleaching signals that oscillate at the difference frequency between the two observed vibrational features as a function of the pump-probe waiting time. This observation is consistent with the presence of nonlinear pathways resulting from underlying cross-peak signals between the coupled symmetric-asymmetric C≡O stretch pair. The successful demonstration of frequency-resolved ultrafast transient vibrational action spectroscopy of dilute molecular ion ensembles provides an exciting, new framework for the study of molecular dynamics in isolated, complex molecular ion systems.

Outcomes of surgery and subsequent therapy for central nervous system oligoprogression in EGFR-mutated NSCLC patients.

BACKGROUND: Oligoprogression is an emerging issue in patients with epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC). However, the surgical treatment for central nervous system (CNS) oligoprogression is not widely discussed. We investigated the outcomes of craniotomy with adjuvant whole-brain radiotherapy (WBRT) and subsequent therapies for CNS oligoprogression in patients with EGFR-mutated NSCLC. METHODS: NSCLC patients with CNS oligoprogression were identified from a tertiary medical center. The outcomes of surgery with adjuvant WBRT or WBRT alone were analyzed, along with other variables. Overall survival and progression-free survival were analyzed using the log-rank test as the primary and secondary endpoints. A COX regression model was used to identify the possible prognostic factors. RESULTS: Thirty-seven patients with CNS oligoprogression who underwent surgery or WBRT were included in the study after reviewing 728 patients. Twenty-one patients underwent surgery with adjuvant WBRT, and 16 received WBRT alone. The median overall survival for surgery and WBRT alone groups was 43 (95% CI 17-69) and 22 (95% CI 15-29) months, respectively. Female sex was a positive prognostic factor for overall survival (OR 0.19, 95% CI 0.06-0.57). Patients who continued previous tyrosine kinase inhibitors (OR 3.48, 95% CI 1.06-11.4) and induced oligoprogression (OR 3.35, 95% CI 1.18-9.52) were associated with worse overall survival. Smoking history (OR 4.27, 95% CI 1.54-11.8) and induced oligoprogression (OR 5.53, 95% CI 2.1-14.7) were associated with worse progression-free survival. CONCLUSIONS: Surgery combined with adjuvant WBRT is a feasible treatment modality for CNS oligoprogression in patients with EGFR-mutated NSCLC. Changing the systemic-targeted therapy after local treatments may be associated with improved overall survival.

Evaluation of the safety, radiographic and resident training results of thoracic pedicle screws placement using resection of the transverse process.

OBJECTIVE: This study aimed at the evaluation and assessment of a simple method, the transverse process resection (TPR) technique, for freehand thoracic pedicle screw placement and the learning curve for trainee surgeons. METHODS: In the TPR technique, the tip of the thoracic transverse process (TP) is removed to create an entry point in the cancellous bone of the TP, and the thoracic pedicle is cannulated from the TP. We retrospectively evaluated the safety and radiographic results of the TPR technique and compared with that of conventional pedicle screws. The training performance of seven neurosurgical residents with TPR techniques were evaluated. RESULTS: Among 46 patients, a total of 322 thoracic screws were analyzed, including 178 screws placed using the TPR technique and 144 screws using the conventional straight-forward (SF) technique. TPR screws had greater medial angulations in all levels from T2 to T12 compared to SF screws (p < 0.001). The incidence of pedicle breach was lower in the TPR screws compared to SF screws (6.2% vs. 21.5%, p < 0.001), especially for screws placed by residents (6.7% vs. 29.6%, p < 0.001). Residents had improved performance following a cadaveric training course on the TPR technique (p = 0.001). CONCLUSION: This study demonstrated the safety of the TPR technique for thoracic pedicle screw placement and its short learning curve for trainee surgeons.

Spectrin-based membrane skeleton supports ciliogenesis.

Cilia are remarkable cellular devices that power cell motility and transduce extracellular signals. To assemble a cilium, a cylindrical array of 9 doublet microtubules push out an extension of the plasma membrane. Membrane tension regulates cilium formation; however, molecular pathways that link mechanical stimuli to ciliogenesis are unclear. Using genome editing, we introduced hereditary elliptocytosis (HE)- and spinocerebellar ataxia (SCA)-associated mutations into the Caenorhabditis elegans membrane skeletal protein spectrin. We show that these mutations impair mechanical support for the plasma membrane and change cell shape. RNA sequencing (RNA-seq) analyses of spectrin-mutant animals uncovered a global down-regulation of ciliary gene expression, prompting us to investigate whether spectrin participates in ciliogenesis. Spectrin mutations affect intraflagellar transport (IFT), disrupt axonemal microtubules, and inhibit cilium formation, and the endogenous spectrin periodically distributes along cilia. Mammalian spectrin also localizes in cilia and regulates ciliogenesis. These results define a previously unrecognized yet conserved role of spectrin-based mechanical support for cilium biogenesis.

Origins of the diffuse shared proton vibrational signatures in proton-coupled electron transfer model dyad complexes.

Phenol-benzimidazole and phenol-pyridine dyad complexes have served as popular model systems for the study of proton-coupled electron transfer (PCET) kinetics in solution-phase experiments. Interpretation of measured PCET rates in terms of key structural parameters, such as the H-bond donor-acceptor distance, however, remains challenging. Herein, we report vibrational spectra in the electronic ground state for a series of phenol-benzimidazole and phenol-pyridine complexes isolated and cryogenically cooled in an ion trap. The four models studied each display highly red-shifted and broadened OH stretching transitions that arise from strong H-bonding interactions between the phenol OH group and the basic N site on benzimidazole/pyridine rings. The OH stretch transition in each model displays relatively strong absorption onsets near 2500 cm-1 with broad shoulders that extend asymmetrically to higher frequencies over hundreds of wavenumbers. In contrast, the deuterated isotopologues yield much weaker OD stretch transitions that appear symmetrically broadened. The spectral breadth and shape of the OD stretch transitions are ascribed to variations in OD stretch frequencies that arise from zero-point distributions in the proton donor-acceptor low-frequency soft mode vibration. The asymmetric structure of the OH stretch transitions is attributed to a set of combination bands between the OH stretch and a series of low-frequency H-bond soft modes. The spectra and modeling highlight the importance of OH stretch-soft mode couplings, which are thought to play important roles in PCET and proton transfer dynamics.

Space-variant Shack-Hartmann wavefront sensing based on affine transformation estimation.

The space-variant wavefront reconstruction problem inherently exists in deep tissue imaging. In this paper, we propose a framework of Shack-Hartmann wavefront space-variant sensing with extended source illumination. The space-variant wavefront is modeled as a four-dimensional function where two dimensions are in the spatial domain and two are in the Fourier domain with priors that both gently vary. Here, the affine transformation is used to characterize the wavefront space-variant function. Correspondingly, the zonal and modal methods are both escalated to adapt to four-dimensional representation and reconstruction. Experiments and simulations show double to quadruple improvements in space-variant wavefront reconstruction accuracy compared to the conventional space-invariant correlation method.

Loss of the voltage-gated proton channel Hv1 decreases insulin secretion and leads to hyperglycemia and glucose intolerance in mice.

Insulin secretion by pancreatic islet ß-cells is regulated by glucose levels and is accompanied by proton generation. The voltage-gated proton channel Hv1 is present in pancreatic ß-cells and extremely selective for protons. However, whether Hv1 is involved in insulin secretion is unclear. Here we demonstrate that Hv1 promotes insulin secretion of pancreatic ß-cells and glucose homeostasis. Hv1-deficient mice displayed hyperglycemia and glucose intolerance because of reduced insulin secretion but retained normal peripheral insulin sensitivity. Moreover, Hv1 loss contributed much more to severe glucose intolerance as the mice got older. Islets of Hv1-deficient and heterozygous mice were markedly deficient in glucose- and K+-induced insulin secretion. In perifusion assays, Hv1 deletion dramatically reduced the first and second phase of glucose-stimulated insulin secretion. Islet insulin and proinsulin content was reduced, and histological analysis of pancreas slices revealed an accompanying modest reduction of ß-cell mass in Hv1 knockout mice. EM observations also indicated a reduction in insulin granule size, but not granule number or granule docking, in Hv1-deficient mice. Mechanistically, Hv1 loss limited the capacity for glucose-induced membrane depolarization, accompanied by a reduced ability of glucose to raise Ca2+ levels in islets, as evidenced by decreased durations of individual calcium oscillations. Moreover, Hv1 expression was significantly reduced in pancreatic ß-cells from streptozotocin-induced diabetic mice, indicating that Hv1 deficiency is associated with ß-cell dysfunction and diabetes. We conclude that Hv1 regulates insulin secretion and glucose homeostasis through a mechanism that depends on intracellular Ca2+ levels and membrane depolarization.