The Cell Tracking Challenge: 10 years of objective benchmarking.

The Cell Tracking Challenge is an ongoing benchmarking initiative that has become a reference in cell segmentation and tracking algorithm development. Here, we present a significant number of improvements introduced in the challenge since our 2017 report. These include the creation of a new segmentation-only benchmark, the enrichment of the dataset repository with new datasets that increase its diversity and complexity, and the creation of a silver standard reference corpus based on the most competitive results, which will be of particular interest for data-hungry deep learning-based strategies. Furthermore, we present the up-to-date cell segmentation and tracking leaderboards, an in-depth analysis of the relationship between the performance of the state-of-the-art methods and the properties of the datasets and annotations, and two novel, insightful studies about the generalizability and the reusability of top-performing methods. These studies provide critical practical conclusions for both developers and users of traditional and machine learning-based cell segmentation and tracking algorithms.

Morphological and Morphometrical Analyses of Fracture-Healing Sites of an Atypical Femoral Fracture in Patients with and without Long-Term Bisphosphonate Treatment for Osteoporosis: A Report of Two Cases.

Bisphosphonates have been the first drug of choice for osteoporosis in the recent years because of their known ability to suppress osteoclast activity. The adverse effect of long-term bisphosphonate administration in the fracture-healing process is controversial. The aim of our study was to observe not only morphology but also morphometry of the fracture site of atypical femoral fracture with and without long-term bisphosphonate administration, in a case study of two difficult-to-obtain human samples. The patients with insufficient healing of atypical femoral fracture were treated with valgus wedge osteotomy. Histomorphometrical analysis was performed in bone samples of fracture sites harvested during osteotomy. The thickness of the femoral cortex was measured in the fracture site and the adjacent, non-fracture site. A comparative analysis of the content of hypertrophic osteoclasts in fracture sites, shape and size of osteons, mass, and ratio of the woven bone to the total bone mass was performed, comparing bisphosphonate-treated and untreated samples. In bisphosphonate-treated samples, we observed femoral cortex thickening at the fracture site; the appearance of hypertrophic osteoclasts; decreased bone resorption surface, decreased osteoclast numbers on the bone resorption surface, and increased ratio of multinuclear osteoclasts; osteons were misshapen and thin; and the mass and ratio of the woven bone to the total bone mass were higher. This study demonstrated that long-term bisphosphonate administration can alter the morphological features of the fracture site compared to its physiological state.

[A Case of Granulocyte-Colony Stimulating Factor-Producing Esophageal Squamous Cell Carcinoma Treated with Chemoradiation Therapy].

The patient was a 64-year-old man who presented with a hoarse voice, pharyngalgia, and high fever.Despite receiving therapy, he presented with dysphagia, and endoscopy revealed a tumor in the thoracic esophagus.A biopsy indicated squamous cell carcinoma.Despite no evidence of infection, laboratory findings revealed leukocytosis and high serum levels of granulocyte-colony stimulating factor(G-CSF).An immunohistochemical study showed positive staining for G-CSF in the tumor cells.Chemoradiation therapy(CRT)with 5-fluorouracil and cisplatin was administered, but his response to treatment was evaluated as progressive disease.Bone, brain, and liver metastases were detected consecutively, and he died 7 months after diagnosis.There are few reports of G-CSF-producing esophageal tumors, and the prognosis is very poor.

Direct observation of cytoskeleton-dependent trafficking of miRNA visualized by the introduction of pre-miRNA.

MicroRNA (miRNA) plays physiologically and pathologically important roles in post-transcriptional regulation. Although miRNA has been suggested to dynamically interact with cellular organelles, the dynamicity of intracellular miRNA behavior has remained unclear. Here, by introducing fluorescently labeled pre-miRNA into living cells, we improved the miRNA visualization method using exogenous miRNA precursors. Through the combination of our miRNA visualization method and single-molecule sensitive fluorescence microscopy, we quantitatively analyzed the process of miRNA maturation. Furthermore, single-particle tracking of fluorescent miRNA in cells revealed the directed movements of miRNA on cytoskeletal components (i.e., microtubules and actin filaments). Our results also suggest that cytoskeleton-dependent miRNA trafficking is associated with the interaction of miRNAs with the nucleus and the endoplasmic reticulum/Golgi apparatus. Our method should facilitate the elucidation of the mechanism and physiological significance of the subcellular localization and organelle interaction of miRNA.

Tracking cell lineages in 3D by incremental deep learning.

Deep learning is emerging as a powerful approach for bioimage analysis. Its use in cell tracking is limited by the scarcity of annotated data for the training of deep-learning models. Moreover, annotation, training, prediction, and proofreading currently lack a unified user interface. We present ELEPHANT, an interactive platform for 3D cell tracking that addresses these challenges by taking an incremental approach to deep learning. ELEPHANT provides an interface that seamlessly integrates cell track annotation, deep learning, prediction, and proofreading. This enables users to implement cycles of incremental learning starting from a few annotated nuclei. Successive prediction-validation cycles enrich the training data, leading to rapid improvements in tracking performance. We test the software's performance against state-of-the-art methods and track lineages spanning the entire course of leg regeneration in a crustacean over 1 week (504 timepoints). ELEPHANT yields accurate, fully-validated cell lineages with a modest investment in time and effort.

IMACEL: A cloud-based bioimage analysis platform for morphological analysis and image classification.

Automated quantitative image analysis is essential for all fields of life science research. Although several software programs and algorithms have been developed for bioimage processing, an advanced knowledge of image processing techniques and high-performance computing resources are required to use them. Hence, we developed a cloud-based image analysis platform called IMACEL, which comprises morphological analysis and machine learning-based image classification. The unique click-based user interface of IMACEL's morphological analysis platform enables researchers with limited resources to evaluate particles rapidly and quantitatively without prior knowledge of image processing. Because all the image processing and machine learning algorithms are performed on high-performance virtual machines, users can access the same analytical environment from anywhere. A validation study of the morphological analysis and image classification of IMACEL was performed. The results indicate that this platform is an accessible and potentially powerful tool for the quantitative evaluation of bioimages that will lower the barriers to life science research.

Heterogeneous expression of DNA-dependent protein kinase in esophageal cancer and normal epithelium.

Esophageal cancer tissues and adjacent normal mucosae in 13 patients with primary esophageal cancer were examined for quantitative differences in DNA-dependent protein kinase (DNA-PK) activity and for expressions of Ku70, Ku80 and DNA-PKcs proteins by Western blotting and immunohistochemistry. The tumor tissues showed higher DNA-PK activity than the normal mucosae. Protein levels of Ku70, Ku80 and DNA-PKcs correlated with DNA-PK activities in the tumor tissues. Immunohistochemical analysis revealed that Ku70, Ku80 and DNA-PKcs located predominantly in the nuclei in both the tumor tissues and normal mucosae. In the normal epithelium, Ku70, Ku80 and DNA-PKcs were expressed only in the nuclei of the basal cell layers and not in those of the lumenal cell layers. In the tumor tissues, the expressions of DNA-PK proteins showed intratumoral heterogeneity. The different portions in the same tumor showed different expression levels of DNA-PK proteins, and even each tumor cell showed different expression levels. These results suggest that cell differentiation and tumor progression affect cellular DNA-PK protein levels and its activity. Furthermore, the intratumoral heterogeneity of DNA-PK protein expression in esophageal cancer cells/ tissues also suggests the difficulty in prediction of radio- or chemo-sensitivity of the tumor through estimation of DNA-PK activity/protein levels in tumor specimens.

Role of stress in the self-limiting oxidation of copper nanoparticles.

The oxidation process of Cu nanoparticles has been investigated by means of an in-situ X-ray diffraction method. A self-limiting oxidation process involving an unusually drastic decrease (about 4 orders in magnitude) in the oxidation rate was observed at 298 K, whereas a non-self-limiting oxidation emerged at 323 K with a rate of at least 4 orders in magnitude faster than 298 K. The drastic slowing at 298 K and the big differences between the two close temperatures in the oxidation kinetics were found to be directly correlated to whether the compressive stress in the Cu(2)O(111) layers that commensurately formed on the Cu(111) surface is relaxed or not.

A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging.

Single-molecule localization microscopy is used to construct super-resolution images, but generally requires prior intense laser irradiation and in some cases additives, such as thiols, to induce on-off switching of fluorophores. These requirements limit the potential applications of this methodology. Here, we report a first-in-class spontaneously blinking fluorophore based on an intramolecular spirocyclization reaction. Optimization of the intramolecular nucleophile and rhodamine-based fluorophore (electrophile) provide a suitable lifetime for the fluorescent open form, and equilibrium between the open form and the non-fluorescent closed form. We show that this spontaneously blinking fluorophore is suitable for single-molecule localization microscopy imaging deep inside cells and for tracking the motion of structures in living cells. We further demonstrate the advantages of this fluorophore over existing methodologies by applying it to nuclear pore structures located far above the coverslip with a spinning-disk confocal microscope and for repetitive time-lapse super-resolution imaging of microtubules in live cells for up to 1 h.

ZEKE spectroscopy and theoretical calculations of copper-methylamine complexes.

The copper-monomethylamine and -dimethylamine complexes were produced in a supersonic jet and examined using single-photon zero kinetic energy (ZEKE) photoelectron spectroscopy and theoretical calculations. The adiabatic ionization potentials (I.P.) of the complexes and vibrational frequencies of the corresponding ions were measured from their ZEKE spectra. The equilibrium geometries, binding energies, and vibrational frequencies of the neutral and ionized complexes were obtained from MP2 and B3LYP calculations. The observed vibrational frequencies of the ionic complexes were well-reproduced by both calculations, whereas the Franck-Condon intensity patterns of the spectra were simulated better by MP2 than B3LYP. The observed I.P. and vibrational frequencies of the Cu-NH(n)(CH3)(3-n) (n = 0-3) complexes were compared, and methyl substitution effects on their ZEKE spectra were discussed.

Pulsed-field ionization electron spectroscopy and conformation of copper-diammonia.

Copper-diammonia, Cu(NH3)2, and its deuterated species, Cu(ND3)2, are produced in supersonic molecular beams and studied by pulsed-field ionization zero electron kinetic energy photoelectron spectroscopy and ab initio calculations. Structural isomers with a copper atom binding to an ammonia dimer or two ammonia molecules are obtained by the calculations. By comparing the experimental measurements to the theoretical calculations, the neutral and ionic forms of copper-diammonia are determined to be in a doubly bound linear conformation in their ground electronic states. The adiabatic ionization potentials of Cu(NH3)2 and Cu(ND3)2 are measured as 29,532 (5) and 29,313 (5) cm(-1), respectively. The metal-ligand symmetric stretching frequencies are measured to be 436 cm(-1) for Cu+-(NH3)2 and 398 cm(-1) for Cu+-(ND3)2, and the metal-ligand bending frequencies 75,139 cm(-1) for CuCu+-(NH3)2 and 70125 cm(-1) for CuCu+-(ND3)2. Moreover, the dissociation energy of Cu(NH3)2-->CuNH3+NH3 is determined to be 11(3) kcal mol(-1) through a thermodynamic relationship.

Size- and temperature-dependent structural transitions in gold nanoparticles.

Size- and temperature-dependent structural transitions in gold nanoparticles were revealed with morphology statistics obtained by high-resolution electron microscopic observations for thousands of particles annealed in a helium heat bath. We found that gold nanoparticles over a wide size range, 3-14 nm, undergo a structural transformation from icosahedral to decahedral morphology just below the melting points. It was also clarified that the formation of bulk crystalline structures from the decahedral morphology requires the melt-freeze process due to an insurmountable high free-energy barrier.