Deep learning for Alzheimer's disease: Mapping large-scale histological tau protein for neuroimaging biomarker validation.

Abnormal tau inclusions are hallmarks of Alzheimer's disease and predictors of clinical decline. Several tau PET tracers are available for neurodegenerative disease research, opening avenues for molecular diagnosis in vivo. However, few have been approved for clinical use. Understanding the neurobiological basis of PET signal validation remains problematic because it requires a large-scale, voxel-to-voxel correlation between PET and (immuno) histological signals. Large dimensionality of whole human brains, tissue deformation impacting co-registration, and computing requirements to process terabytes of information preclude proper validation. We developed a computational pipeline to identify and segment particles of interest in billion-pixel digital pathology images to generate quantitative, 3D density maps. The proposed convolutional neural network for immunohistochemistry samples, IHCNet, is at the pipeline's core. We have successfully processed and immunostained over 500 slides from two whole human brains with three phospho-tau antibodies (AT100, AT8, and MC1), spanning several terabytes of images. Our artificial neural network estimated tau inclusion from brain images, which performs with ROC AUC of 0.87, 0.85, and 0.91 for AT100, AT8, and MC1, respectively. Introspection studies further assessed the ability of our trained model to learn tau-related features. We present an end-to-end pipeline to create terabytes-large 3D tau inclusion density maps co-registered to MRI as a means to facilitate validation of PET tracers.

Smartphone adapter time trial analysis: A low-cost, time-efficient method to disseminate quality photomicrographs at the microscope.

BACKGROUND: Within the field of pathology there is a need for a uniform low-cost option for securing high-quality photomicrographs. Advances in smartphone photography and 3D-printing technology allow for custom adapters to be designed for the purpose of photomicrograph capture. METHODS: Photomicrograph acquisition was performed using four core modalities: a novel 3D-printed smartphone-to-microscope adapter, freehand smartphone-to-microscope technique, a commercial adaptor (LabCam Pro), and a microscope-mounted digital camera. Eight skin diagnoses were photographed using each of the modalities and time to image capture was measured. The photomicrographs were blindly reviewed by two academic dermatopathologists and one pathologist using a side-by-side comparison technique to determine the image quality. Cost assessments were evaluated by obtaining free pricing information on manufacturer websites. RESULTS: The 3D-printed adapter was the most efficient method of capturing a high-quality photomicrograph in addition to being budget neutral. The microscope-mounted camera produced the highest quality photomicrographs followed by the 3D-printed adapter. CONCLUSIONS: The 3D-printed smartphone-to-microscope adapter offers a low-cost, time-efficient method of capturing high-quality photomicrographs.

PALM and STORM: Into large fields and high-throughput microscopy with sCMOS detectors.

Single Molecule Localization Microscopy (SMLM) techniques such as Photo-Activation Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) enable fluorescence microscopy super-resolution: the overcoming of the resolution barrier imposed by the diffraction of light. These techniques are based on acquiring hundreds or thousands of images of single molecules, locating them and reconstructing a higher-resolution image from the high-precision localizations. These methods generally imply a considerable trade-off between imaging speed and resolution, limiting their applicability to high-throughput workflows. Recent advancements in scientific Complementary Metal-Oxide Semiconductor (sCMOS) camera sensors and localization algorithms reduce the temporal requirements for SMLM, pushing it toward high-throughput microscopy. Here we outline the decisions researchers face when considering how to adapt hardware on a new system for sCMOS sensors with high-throughput in mind.

Phenotyping transgenic embryos: a rapid 3-D screening method based on episcopic fluorescence image capturing.

We describe a technique suitable for routine three-dimensional (3-D) analysis of mouse embryos that is based on episcopic fluorescence images captured during serial sectioning of wax-embedded specimens. We have used this procedure to describe the cardiac phenotype and associated blood vessels of trisomic 16 (Ts16) and Cited2-null mutant mice, as well as the expression pattern of an Myf5 enhancer/beta-galactosidase transgene. The consistency of the images and their precise alignment are ideally suited for 3-D analysis using video animations, virtual resectioning or commercial 3-D reconstruction software packages. Episcopic fluorescence image capturing (EFIC) provides a simple and powerful tool for analyzing embryo and organ morphology in normal and transgenic embryos.

Combined optical and acoustical detection of single microbubble dynamics.

A detailed understanding of the response of single microbubbles subjected to ultrasound is fundamental to a full understanding of the contrast-enhancing abilities of microbubbles in medical ultrasound imaging, in targeted molecular imaging with ultrasound, and in ultrasound-mediated drug delivery with microbubbles. Here, single microbubbles are isolated and their ultrasound-induced radial dynamics recorded with an ultra-high-speed camera at up to 25 million frames per second. The sound emission is recorded simultaneously with a calibrated single element transducer. It is shown that the sound emission can be predicted directly from the optically recorded radial dynamics, and vice versa, that the nanometer-scale radial dynamics can be predicted from the acoustic response recorded in the far field.

[Imaging of single molecules in live cells]. / Microscopies cellulaires à l'échelle de la molécule individuelle.

Progress in optical microscopy, combined to the emergence of new fluorescent probes and advanced instrumentation, now permits the imaging of single molecules in fixed and live cells. This extreme detection sensitivity has opened new modalities in cellular imaging. On the one hand, optical images with an unprecedented resolution in the 10-50 nm range, well below the diffraction limit of light, can be recorded. These super-resolution images give new insights into the properties of cellular structures. On the other hand, proteins, either in the membrane or intracellular, can be tracked in live cells and in physiological conditions. Their individual trajectories provide invaluable information on the molecular interactions that control their dynamics and their spatial organization. Single molecule imaging is rapidly becoming a unique tool to understand the biochemical and biophysical processes that determine the properties of molecular assemblies in a cellular context.

[Development and application of a fundus transverse microscopic imaging system].

A human fundus transverse microscopic imaging system based on a MEMS deformable membrane mirror was developed. A 37 element small MEMS deformable membrane mirror was used as wave front corrector in this system. Wavefront errors were measured by a Hartman-Shack wave front sensor which contains 127 micro lens lets. After the wavefront error of human eye had been corrected by the deformable membrane mirror under the control of a computer, the imaging illumination light was triggered by a electronic shutter to illuminate the retina, the images were captured by a CCD camera. It has been showed in model eye's test that the system could measure and correct the eye's wavefront aberration efficiently. The fundus image achieved the diffraction limit after aberration correction. It was showed in clinic that except a few patients with turbid eye, most patients could finish the process of measuring and correcting wavefront aberration and then taking fundus image. The examination process could be finished safely, quickly and reliably.

Evaluation of the safety of time-lapse observations for human embryos.

PURPOSE: To assess the effects of light from an integrated optical microscope and evaluate the safety of time-lapse observations using a built-in microscope incubator. METHODS: We prospectively compared the fertilization rate and embryonic morphology after intracytoplasmic sperm injection between embryos cultured with time-lapse observations every 15 min in an incubator with an integrated optical microscope and embryos with intermittent observations (once a day) in conventional incubators. RESULTS: No significant differences were observed in the fertilization rate (57.5% vs. 57.5%) or the rate of excellent-good cleavage embryos (36.0% vs. 36.0%). CONCLUSIONS: These results suggest that time-lapse observations using an incubator with an integrated optical microscope may therefore be safely utilized in clinical practice.

A Script-Assisted Microscopy (SAM) package to improve data acquisition rates on FEI Tecnai electron microscopes equipped with Gatan CCD cameras.

High throughput methods of data acquisition are advantageous for cryoelectron microscopy and single particle reconstruction as high-resolution structure determination requires thousands of particle images. We have developed a semi-automated data collection method that utilizes the scripting languages provided by FEI for their Tecnai User Interface (TUI) and by Gatan for their Digital Micrograph package. Our Script-Assisted Microscopy (SAM) method allows for the selection of multiple locations within a low magnification, search mode, micrograph and for subsequent automated imaging of these locations at a higher exposure magnification. The SAM approach permits the user to retain control over the microscope, while streamlining the most repetitive steps of collecting and evaluating micrographs. With SAM, we have found an average of 1000 micrographs can be collected per day on any grid type, either irregular homemade grids or prefabricated grids with regularly spaced holes. This rate of data collection represents a fivefold improvement over our manual collection rates. SAM provides an example of an individually tailored approach to data acquisition utilizing the scripting interfaces provided by the equipment manufacturers. The SAM method has proven valuable for determination of a subnanometer resolution cryoEM structure of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a 469kDa protein.

The structure of Lippmann heliochromes: Cajal and the 1908 Nobel Prize in Physics.

The 1908 Nobel Prize in Physics was awarded to Luxembourgeois Gabriel Lippmann (1848-1921), Professor of Mathematical and Experimental Physics at la Sorbonne, for his method of reproducing colors photographically based on the theory of wave interference. In the preceding several years, the eminent neurohistologist - and avid photographer - Santiago Ramón y Cajal (1852-1934) had been experimenting with Lippmann heliochromes, studying under the microscope the structure of the laminae of Zenker that produce mixed colors, and especially white. Those studies led to a series of technical papers by Cajal, the culmination being an article published 100 years ago in the Annual Report of the Board of Regents of the Smithsonian Institution in Washington, DC. A few years later, Cajal published Photography in Colors, his classic monograph on the physicochemical principles of the 'art of Daguerre,' bearing further testimony to his exuberant productivity, far-reaching interests, and scientific genius. The present article reflects on the workings of the mind of Cajal and his fundamental knowledge that was a precondition for his success in neurohistology. It highlights the links between the early photographic studies of Cajal and Lippmann, masters of the biological and physical sciences, respectively. Special emphasis is placed on Lippmann's discovery of heliochromes and the microscopic analyses performed on them by Cajal, including elements from relevant contemporary studies and discoveries.

Imaging of molecular dynamics regulated by electrical activities in neural circuits and in synapses.

One of the major challenges in brain research is to unravel a network of molecules, neurons, circuits and systems that are responsible for dynamic and hierarchical brain functions. To understand molecular events that occur in synapses could be an important key to exploring the mechanism of information processing. A spatiotemporal recording method is required to observe neuronal activities in a particular local circuit and to resolve single synaptic potential with high resolution. As alternative methods, real-time imaging using fluorescent probes and optical recording methods are also a powerful approach for investigating the molecular dynamics of biological events in neurons in vitro and in vivo. Recently, optical imaging techniques have become of great importance to visualize the molecular dynamics in a micron-sized compartment of a single neuron such as neuronal synapse. In general, the presynaptic axon forms synapses at the postsynaptic site on the dendritic spines in the mammalian central nervous system. Subsets of the synapses undergo a series of enduring changes in spine shape and density as well as alterations in electrophysiological functions. Here we describe recent optical imaging studies conducted by elaborate methods and techniques that provide evidence for the link between neural activity and molecular dynamics.

Rapid and sensitive detection of viable bacteria in contaminated platelet concentrates using a newly developed bioimaging system.

BACKGROUND: Rapid and sensitive methods for the detection of bacteria in platelet concentrates (PCs) are required as well as inactivation techniques to decrease the transfusion-associated risk of infection from bacterially contaminated PCs. In this study, a rapid microbiologic method for the sensitive counting of viable bacteria in PCs was developed by combining a fluorescent staining technique and a bioimaging system. STUDY DESIGN AND METHODS: An esterase indicator, carboxyfluorescein diacetate, was used to detect physiologically active bacteria. Treatment was optimized to selectively remove platelets (PLTs). Bacterial cells trapped on a filter were automatically discriminated from other particles or PLT debris and counted by a bioimaging system. The sensitivity, rapidity, and recovery rates were evaluated using PCs spiked with 14 reference bacterial strains and clinical isolates. RESULTS: Lysis treatment with enzyme and detergent was effective to remove PLTs and white blood cells. Two buffers for fluorescent vital staining were needed for highly sensitive detection of pathogenic bacteria. Fewer than 100 cells spiked in 5-mL PCs were detected by the bioimaging system after treatment and fluorescent staining, and this result shows that PLTs are selectively digested by the treatment. Bacterial cells spiked in 25-mL PCs were detected within 45 minutes (treatment, 15 min; filtration and fluorescent staining, 15 min; automated counting and precise image analysis, 10-15 min). CONCLUSION: The microbiologic method described here is rapid and sensitive, and this method has potential for the screening of PCs contaminated with bacterial cells. Furthermore, this method could contribute to further evaluation of inactivation techniques.

Lymphoma and leukemia cells possess fractal dimensions that correlate with their biological features.

BACKGROUND: Living cells can be viewed as complex adaptive systems that exhibit non-linear dynamics and fractal features. We investigated the fractal qualities of normal and malignant hematological cells and their potential as a tool for characterizing cell phenotype and clinical behavior. METHODS: A mathematical algorithm and an optic tool for fractal analysis of nuclei were developed. A total of 4,713 lymphoid cells derived from 66 patients of five distinct diagnostic groups (normal and reactive lymphocytes, low-grade lymphomas and an aggressive lymphoma) were assessed for their fractal dimension. In addition, in 19 patients fractal analysis of leukemia cells was compared to clinical endpoints. RESULTS: After validating our method, hematological cells possessed fractal dimensions corresponding to their clinical entity. There was a highly significant overall difference in fractal dimensions between various types of hematological malignancies. A preliminary correlation was found between the fractal dimension and the clinical outcome of leukemia patients. CONCLUSIONS: Hematological cells possess fractal dimensions that correlate with their biological properties. Measurement of fractal dimension seems to be a sensitive method to assess the hematological cell phenotype and to define a clinical group. This tool may be potentially useful for the evaluation of clinical behavior of hematological diseases.

A circulating myocardial depressant substance in humans with septic shock. Septic shock patients with a reduced ejection fraction have a circulating factor that depresses in vitro myocardial cell performance.

We have previously described a subpopulation of patients with septic shock who had a reversible depression of radionuclide-determined left ventricular ejection fraction (EF). To investigate the mechanism of this myocardial depression, an in vitro model of mammalian myocardial cell performance was established employing primary spontaneously beating rat myocardial cells. The contraction of a single cardiac cell was quantitated by recording the changes in area occupied by the cell during contraction and relaxation. In 20 septic shock patients during the acute phase, the mean left ventricular EF was decreased (mean = 0.33, normal mean = 0.50), and serum obtained during this acute phase induced a mean (+/- standard error of the mean) 33 +/- 4% decrease in extent and 25 +/- 4% decrease in velocity of myocardial cell shortening during contraction (P less than 0.001). In contrast, serum obtained from 11 of these same patients before shock (n = 2) or after recovery (n = 9) of the left ventricular EF (mean = 0.50) showed a return toward normal in extent and velocity of shortening (P less than 0.001). Sera from 17 critically ill nonseptic patients, from 10 patients with structural heart disease as a cause for a depressed EF, and from 12 healthy laboratory personnel, induced no significant changes in in vitro myocardial cell performance. In 20 patients during the acute phase of septic shock, the decreased EF in vivo demonstrated a significant correlation (r = +0.52, P less than 0.01) with a decrease in the extent of myocardial cell shortening in vitro. The quantitative and temporal correlation between the decreased left ventricular EF and this serum myocardial depressant substance argues for a pathophysiologic role for this depressant substance in producing the reversible cardiomyopathy seen during septic shock in humans.

[A phototube connecting digital cameras with light optic microscopes].

We present here a phototube model making possible connection of a digital camera with light optic microscopes in order to obtain images of microobjects and for their further computer treatment. The advantage of this model is simplicity of its manufacturing and small required expenses as well as an increase in information density for microobject studies. This phototube has been covered by a patent for a useful model N2 48228 registered in the Public Register of RF on September 27, 2005.