Reconstruction of axial tomographic high resolution data from confocal fluorescence microscopy: a method for improving 3D FISH images.

Fluorescent confocal laser scanning microscopy allows an improved imaging of microscopic objects in three dimensions. However, the resolution along the axial direction is three times worse than the resolution in lateral directions. A method to overcome this axial limitation is tilting the object under the microscope, in a way that the direction of the optical axis points into different directions relative to the sample. A new technique for a simultaneous reconstruction from a number of such axial tomographic confocal data sets was developed and used for high resolution reconstruction of 3D-data both from experimental and virtual microscopic data sets. The reconstructed images have a highly improved 3D resolution, which is comparable to the lateral resolution of a single deconvolved data set. Axial tomographic imaging in combination with simultaneous data reconstruction also opens the possibility for a more precise quantification of 3D data. The color images of this publication can be accessed from http://www.esacp.org/acp/2000/20-1/heintzmann.++ +htm. At this web address an interactive 3D viewer is additionally provided for browsing the 3D data. This java applet displays three orthogonal slices of the data set which are dynamically updated by user mouse clicks or keystrokes.     

Neural Signals Evoked by Stimuli of Increasing Social Scene Complexity Are Detectable at the Single-Trial Level and Right Lateralized

Classification of neural signals at the single-trial level and the study of their relevance in affective and cognitive neuroscience are still in their infancy. Here we investigated the neurophysiological correlates of conditions of increasing social scene complexity using 3D human models as targets of attention, which may also be important in autism research. Challenging single-trial statistical classification of EEG neural signals was attempted for detection of oddball stimuli with increasing social scene complexity. Stimuli had an oddball structure and were as follows: 1) flashed schematic eyes, 2) simple 3D faces flashed between averted and non-averted gaze (only eye position changing), 3) simple 3D faces flashed between averted and non-averted gaze (head and eye position changing), 4) animated avatar alternated its gaze direction to the left and to the right (head and eye position), 5) environment with 4 animated avatars all of which change gaze and one of which is the target of attention. We found a late (> 300 ms) neurophysiological oddball correlate for all conditions irrespective of their complexity as assessed by repeated measures ANOVA. We attempted single-trial detection of this signal with automatic classifiers and obtained a significant balanced accuracy classification of around 79%, which is noteworthy given the amount of scene complexity. Lateralization analysis showed a specific right lateralization only for more complex realistic social scenes. In sum, complex ecological animations with social content elicit neurophysiological events which can be characterized even at the single-trial level. These signals are right lateralized. These finding paves the way for neuroscientific studies in affective neuroscience based on complex social scenes, and given the detectability at the single trial level this suggests the feasibility of brain computer interfaces that can be applied to social cognition disorders such as autism.     

UCSF tomography: an integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction

A real-time alignment and reconstruction scheme for electron microscopic tomography (EMT) has been developed and integrated within our UCSF tomography data collection software. This newly integrated software suite provides full automation from data collection to real-time reconstruction by which the three-dimensional (3D) reconstructed volume is immediately made available at the end of each data collection. Real-time reconstruction is achieved by calculating a weighted back-projection on a small Linux cluster (five dual-processor compute nodes) concurrently with the UCSF tomography data collection running on the microscope's computer, and using the fiducial-marker free alignment data generated during the data collection process. The real-time reconstructed 3D volume provides users with immediate feedback to fully asses all aspects of the experiment ranging from sample choice, ice thickness, experimental parameters to the quality of specimen preparation. This information can be used to guide subsequent data collections. Access to the reconstruction is especially useful in low-dose cryo EMT where such information is very difficult to obtain due to extraordinary low signal to noise ratio in each 2D image. In our environment, we generally collect 2048 x 2048 pixel images which are subsequently computationally binned four-fold for the on-line reconstruction. Based upon experiments performed with thick and cryo specimens at various CCD magnifications (50000x-80000x), alignment accuracy is sufficient to support this reduced resolution but should be refined before calculating a full resolution reconstruction. The reduced resolution has proven to be quite adequate to assess sample quality, or to screen for the best data set for full-resolution reconstruction, significantly improving both productivity and efficiency of system resources. The total time from start of data collection to a final reconstructed volume (512 x 512 x 256 pixels) is about 50 min for a +/-70 degrees 2k x 2k pixel tilt series acquired at every 1 degrees.     

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs^1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.     

One- and Two-Dimensional Electron Paramagnetic Resonance Imaging in Skin

EPR imaging with modulated field gradients provides the possibility for obtaining an EPR spectrum in a selected volume We demonstrate the feasibility of X-band (9.5GHz) electron paramagnetic resonance (EPR) imaging in skin biopsies of hairless mice. One- (ID) and two-dimensional (2D) EPR images of the persistent free radical di-tertiary-butyl-nitroxide are measured. At a microwave frequency of 9.5 GHz (X-band), 2D images are obtained in skin biopsies with an actual point distinction resolution of 25 μm. In a biological model system. 2D images are measured at L-band frequency (2.0 GHz) with a pixel resolution of 61 μm. and a theoretical spatial resolution of 12.5 μm. In combination with the spin labeling and spin trapping technique. EPR imaging is the most direct approach to analyzing spatial distribution of physico-chemical properties in skin, such as membrane fluidity and polarity. as well as detection of free radicals.     

Giga-Pixel Lensfree Holographic Microscopy and Tomography Using Color Image Sensors

We report Giga-pixel lensfree holographic microscopy and tomography using color sensor-arrays such as CMOS imagers that exhibit Bayer color filter patterns. Without physically removing these color filters coated on the sensor chip, we synthesize pixel super-resolved lensfree holograms, which are then reconstructed to achieve ∼350 nm lateral resolution, corresponding to a numerical aperture of ∼0.8, across a field-of-view of ∼20.5 mm². This constitutes a digital image with ∼0.7 Billion effective pixels in both amplitude and phase channels (i.e., ∼1.4 Giga-pixels total). Furthermore, by changing the illumination angle (e.g., ±50°) and scanning a partially-coherent light source across two orthogonal axes, super-resolved images of the same specimen from different viewing angles are created, which are then digitally combined to synthesize tomographic images of the object. Using this dual-axis lensfree tomographic imager running on a color sensor-chip, we achieve a 3D spatial resolution of ∼0.35 µm×0.35 µm×∼2 µm, in x, y and z, respectively, creating an effective voxel size of ∼0.03 µm³ across a sample volume of ∼5 mm³, which is equivalent to >150 Billion voxels. We demonstrate the proof-of-concept of this lensfree optical tomographic microscopy platform on a color CMOS image sensor by creating tomograms of micro-particles as well as a wild-type C. elegans nematode.     

Three‐dimensional multiphoton/optical coherence tomography for diagnostic applications in dermatology

A preliminary clinical trial using state‐of‐the‐art multiphoton tomography (MPT) and optical coherence tomography (OCT) for three‐dimensional (3D) multimodal in vivo imaging of normal skin, nevi, scars and pathologic skin lesions has been conducted. MPT enabled visualization of sub‐cellular details with axial and transverse resolutions of <2 μm and <0.5 μm, respectively, from a volume of 0.35 × 0.35 × 0.2 mm³ at a frame rate of 0.14 Hz (512 × 512 pixels). State‐of‐the‐art OCT, operating at a center wavelength of 1300 nm, was capable of acquiring 3D images depicting the layered architecture of skin with axial and transverse resolutions ～8 μm and ～20 μm, respectively, from a volume of 7 × 3.5 × 1.5 mm³ at a frame rate of 46 Hz (1024 × 1024 pixels). This study demonstrates the clinical diagnostic potential of MPT/OCT for pre‐screening relatively large areas of skin using 3D OCT to identify suspicious regions at microscopic level and subsequently using high resolution MPT to obtain zoomed in, sub‐cellular level information of the respective regions (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Serial section scanning electron microscopy (S3EM) on silicon wafers for ultra-structural volume imaging of cells and tissues

High resolution, three-dimensional (3D) representations of cellular ultrastructure are essential for structure function studies in all areas of cell biology. While limited subcellular volumes have been routinely examined using serial section transmission electron microscopy (ssTEM), complete ultrastructural reconstructions of large volumes, entire cells or even tissue are difficult to achieve using ssTEM. Here, we introduce a novel approach combining serial sectioning of tissue with scanning electron microscopy (SEM) using a conductive silicon wafer as a support. Ribbons containing hundreds of 35 nm thick sections can be generated and imaged on the wafer at a lateral pixel resolution of 3.7 nm by recording the backscattered electrons with the in-lens detector of the SEM. The resulting electron micrographs are qualitatively comparable to those obtained by conventional TEM. S(3)EM images of the same region of interest in consecutive sections can be used for 3D reconstructions of large structures. We demonstrate the potential of this approach by reconstructing a 31.7 μm(3) volume of a calyx of Held presynaptic terminal. The approach introduced here, Serial Section SEM (S(3)EM), for the first time provides the possibility to obtain 3D ultrastructure of large volumes with high resolution and to selectively and repetitively home in on structures of interest. S(3)EM accelerates process duration, is amenable to full automation and can be implemented with standard instrumentation.     

Creating physical 3D stereolithograph models of brain and skull

The human brain and skull are three dimensional (3D) anatomical structures with complex surfaces. However, medical images are often two dimensional (2D) and provide incomplete visualization of structural morphology. To overcome this loss in dimension, we developed and validated a freely available, semi-automated pathway to build 3D virtual reality (VR) and hand-held, stereolithograph models. To evaluate whether surface visualization in 3D was more informative than in 2D, undergraduate students (n = 50) used the Gillespie scale to rate 3D VR and physical models of both a living patient-volunteer's brain and the skull of Phineas Gage, a historically famous railroad worker whose misfortune with a projectile tamping iron provided the first evidence of a structure-function relationship in brain. Using our processing pathway, we successfully fabricated human brain and skull replicas and validated that the stereolithograph model preserved the scale of the VR model. Based on the Gillespie ratings, students indicated that the biological utility and quality of visual information at the surface of VR and stereolithograph models were greater than the 2D images from which they were derived. The method we developed is useful to create VR and stereolithograph 3D models from medical images and can be used to model hard or soft tissue in living or preserved specimens. Compared to 2D images, VR and stereolithograph models provide an extra dimension that enhances both the quality of visual information and utility of surface visualization in neuroscience and medicine.     

An iterative deconvolution algorithm for image recovery in clinical CT: A phantom study

PurposeTo study the feasibility of using an iterative reconstruction algorithm to improve previously reconstructed CT images which are judged to be non-diagnostic on clinical review. A novel rapidly converging, iterative algorithm (RSEMD) to reduce noise as compared with standard filtered back-projection algorithm has been developed.Materials and methodsThe RSEMD method was tested on in-silico, Catphan^®500, and anthropomorphic 4D XCAT phantoms. The method was applied to noisy CT images previously reconstructed with FBP to determine improvements in SNR and CNR. To test the potential improvement in clinically relevant CT images, 4D XCAT phantom images were used to simulate a small, low contrast lesion placed in the liver.ResultsIn all of the phantom studies the images proved to have higher resolution and lower noise as compared with images reconstructed by conventional FBP. In general, the values of SNR and CNR reached a plateau at around 20 iterations with an improvement factor of about 1.5 for in noisy CT images. Improvements in lesion conspicuity after the application of RSEMD have also been demonstrated. The results obtained with the RSEMD method are in agreement with other iterative algorithms employed either in image space or with hybrid reconstruction algorithms.ConclusionsIn this proof of concept work, a rapidly converging, iterative deconvolution algorithm with a novel resolution subsets-based approach that operates on DICOM CT images has been demonstrated. The RSEMD method can be applied to sub-optimal routine-dose clinical CT images to improve image quality to potentially diagnostically acceptable levels.     

CT based conformal brachytherapy treatment planning

PURPOSE: To introduce the CT based three dimensional (3D) conformal brachytherapy treatment planning for interstitial implants, to compare the conventional X-ray film based planning with the 3D planning from the point of view of reconstruction and dosimetry, to discuss the differences and highlight the advantages. MATERIAL AND METHODS: On 10 patients with breast and 5 with head and neck tumor treated with HDR interstitial implants, following the catheter implantations, CT scans were taken at 5 mm spacing. The images were loaded into the PLATO BPS v14.0 3D planning system for brachytherapy. The contours of the target volume and critical structures were outlined on each slice, the catheter describing points were identified and the anatomical structures and catheter positions were reconstructed in 3D. Having taken into account the target volume, the active lengths were determined in each catheter, and dose optimization on dose points on target was performed. RESULTS: The 3D treatment planning was applied at interstitial breast treatments and head and neck implants. We investigated the dose distribution on axial, reconstructed coronal / sagittal planes and in 3D view with respect to anatomical structures. Dose volume histograms related to the target volume and critical structures were used for quantitative assessment of the plans. We found that the conformal dose distribution might result in increase of dose inhomogeneity within the target volume. CONCLUSIONS: The three dimensional brachytherapy treatment planning can be introduced into the clinical practice under proper technical conditions. A tradeoff between conformality and dose homogeneity results in an acceptable dose plan. The dose inhomogeneity can be decreased with the use of CT scans taken before the implantation. The guidelines and quantitative parameters describing the dose distribution, which can be used for determining the optimal dose distribution in clinical point of view, are still waiting to be established.     

A compact high‐speed full‐field optical coherence microscope for high‐resolution in vivo skin imaging

A compact high‐speed full‐field optical coherence microscope has been developed for high‐resolution in vivo imaging of biological tissues. The interferometer, in the Linnik configuration, has a size of 11 × 11 × 5 cm³ and a weight of 210 g. Full‐field illumination with low‐coherence light is achieved with a high‐brightness broadband light‐emitting diode. High‐speed full‐field detection is achieved by using part of the image sensor of a high‐dynamic range CMOS camera. En face tomographic images are acquired at a rate of 50 Hz, with an integration time of 0.9 ms. The image spatial resolution is 0.9 μm × 1.2 μm (axial × transverse), over a field of view of 245 × 245 μm². Images of human skin, revealing in‐depth cellular‐level structures, were obtained in vivo and in real‐time without the need for stabilization of the subject. The system can image larger fields, up to 1 × 1 mm², but at a reduced depth.      

SwarmPS: rapid, semi-automated single particle selection software

Single particle analysis (SPA) coupled with high-resolution electron cryo-microscopy is emerging as a powerful technique for the structure determination of membrane protein complexes and soluble macromolecular assemblies. Current estimates suggest that approximately 10(4)-10(5) particle projections are required to attain a 3A resolution 3D reconstruction (symmetry dependent). Selecting this number of molecular projections differing in size, shape and symmetry is a rate-limiting step for the automation of 3D image reconstruction. Here, we present Swarm(PS), a feature rich GUI based software package to manage large scale, semi-automated particle picking projects. The software provides cross-correlation and edge-detection algorithms. Algorithm-specific parameters are transparently and automatically determined through user interaction with the image, rather than by trial and error. Other features include multiple image handling (approximately 10(2)), local and global particle selection options, interactive image freezing, automatic particle centering, and full manual override to correct false positives and negatives. Swarm(PS) is user friendly, flexible, extensible, fast, and capable of exporting boxed out projection images, or particle coordinates, compatible with downstream image processing suites.     

31 Effects of light and ammonium on growth,ammonium uptake and pigmentation of four porphyra (bangiales,rhodophyta) species native to the northwestern atlantic

“Phytopia: Discovery of the Marine Ecosystem” is an educational CD‐ROM that features a wealth of new images, animations, microscope‐ and satellite‐derived data sets, and multimedia discovery tools. It has received support from NASA and the National Science Foundation and can ordered on‐line through http://www.bigelow.org/phytopia . A pre‐release version of the “Phytopia” was tested by students, educators, and scientists at 34 institutions in Britain, Germany, Russia, and throughout the U.S. This product is designed for use in undergraduate classes; however, our testing has shown that it provides an exciting and interactive learning experience appropriate for Grades 7 and higher. The virtual microscope tool allows the user to view prepared assemblages of plankton at various magnifications, under different epifluorescence conditions, or by scanning electron microscopy. For several species, the user can view movies showing the organism's motility. Some species are featured as three‐dimensional models that can be viewed from any perspective. “Phytopia” also helps users connect ocean primary productivity patterns with environmental factors in several geographic areas. Innovative tools allow investigation of co‐registered temperature, wind, current, nutrient, and ocean color data. This tool is aligned with research efforts to better understand plankton ecology using remotely sensed data.     

Structural analysis of theridiid spider's testicular cyst using 3D reconstruction rendering

A three dimensional reconstruction technique was used for the analysis of a theridiid spider's (Achaearanea tepidariorum) testicular cyst. Although microscopic techniques have greatly improved, most of the information gathered is still based on two‐dimensional images. Particularly in spiders, it is very difficult to count the exact number of sperm in a single cyst, since their spermatogenetic processes takes place within the spherical cysts through the flagellar coiling process. Since morphological features of spider sperm provide detailed information on the whole spermatogenetic processes, we analyzed the exact number of germ cells per cyst in A. tepidariorum through a three‐dimensional image reconstruction technique. For image processing, serially sectioned histological images were scanned using a light microscope and 3D rendering images were reconstructed from these sections. Based on the three dimensional image analysis of the testicular cyst, the number of secondary spermatocytes per cyst was calculated to be 32 (2⁵). Therefore the total number of sperm produced from a single cyst can be calculated as 64 (2⁶), which indicates that a single spermatogonium undergoes four mitotic divisions and an additional two meiotic divisions to produce mature spermatozoa.     

CMEIAS Color Segmentation: An Improved Computing Technology to Process Color Images for Quantitative Microbial Ecology Studies at Single-Cell Resolution

Quantitative microscopy and digital image analysis are underutilized in microbial ecology largely because of the laborious task to segment foreground object pixels from background, especially in complex color micrographs of environmental samples. In this paper, we describe an improved computing technology developed to alleviate this limitation. The system’s uniqueness is its ability to edit digital images accurately when presented with the difficult yet commonplace challenge of removing background pixels whose three-dimensional color space overlaps the range that defines foreground objects. Image segmentation is accomplished by utilizing algorithms that address color and spatial relationships of user-selected foreground object pixels. Performance of the color segmentation algorithm evaluated on 26 complex micrographs at single pixel resolution had an overall pixel classification accuracy of 99+%. Several applications illustrate how this improved computing technology can successfully resolve numerous challenges of complex color segmentation in order to produce images from which quantitative information can be accurately extracted, thereby gain new perspectives on the in situ ecology of microorganisms. Examples include improvements in the quantitative analysis of (1) microbial abundance and phylotype diversity of single cells classified by their discriminating color within heterogeneous communities, (2) cell viability, (3) spatial relationships and intensity of bacterial gene expression involved in cellular communication between individual cells within rhizoplane biofilms, and (4) biofilm ecophysiology based on ribotype-differentiated radioactive substrate utilization. The stand-alone executable file plus user manual and tutorial images for this color segmentation computing application are freely available at . This improved computing technology opens new opportunities of imaging applications where discriminating colors really matter most, thereby strengthening quantitative microscopy-based approaches to advance microbial ecology in situ at individual single-cell resolution.     

New head models extracted from thermal infrared (IR) images for dosimetry computations

In electromagnetic dosimetry, anatomical human models are commonly obtained by segmentation of magnetic resonance imaging or computed tomography scans. In this paper, a human head model extracted from thermal infrared images is examined in terms of its applicability to specific absorption rate (SAR) calculations. Since thermal scans are two-dimensional (2D) representation of surface temperature, this allows researchers to overcome the extensive computational demand associated with 3D simulation. The numerical calculations are performed using the finite-difference time-domain method with mesh sizes of 2 mm at 900 MHz plane wave irradiation. The power density of the incident plane wave is assumed to be 10 W/m². Computations were compared with a realistic anatomical head model. The results show that although there were marked differences in the local SAR distribution in the various tissues in the two models, the 1 g peak SAR values are approximately similar in the two models.     

Spectral signal-to-noise ratio and resolution assessment of 3D reconstructions

Measuring the quality of three-dimensional (3D) reconstructed biological macromolecules by transmission electron microscopy is still an open problem. In this article, we extend the applicability of the spectral signal-to-noise ratio (SSNR) to the evaluation of 3D volumes reconstructed with any reconstruction algorithm. The basis of the method is to measure the consistency between the data and a corresponding set of reprojections computed for the reconstructed 3D map. The idiosyncrasies of the reconstruction algorithm are taken explicitly into account by performing a noise-only reconstruction. This results in the definition of a 3D SSNR which provides an objective indicator of the quality of the 3D reconstruction. Furthermore, the information to build the SSNR can be used to produce a volumetric SSNR (VSSNR). Our method overcomes the need to divide the data set in two. It also provides a direct measure of the performance of the reconstruction algorithm itself; this latter information is typically not available with the standard resolution methods which are primarily focused on reproducibility alone.