Optical Slices Research Articles

光学薄片点胶的数值分析及优化

光学薄片点胶的数值分析及优化

Light microscopy of whole plant organs.

Plants are ideal organisms for light microscopical studies of cellular mechanisms controlling cell organisation and cell functioning. However, most plant organs are not transparent to light which prevents high resolution imaging deep within plant tissues. Classically, access into plant organs is achieved by sectioning or whole-mount tissue clearing. Until recently, the protocols for clearing destroyed the signal from fluorescent markers which prevented the imaging of the distribution of fluorescent proteins and the three-dimensional reconstruction from optical slices of whole plant organs. From 2011, a number of protocols have been developed for whole brain and whole organism imaging for animal studies. Now, these protocols have been adapted for in-depth imaging of whole plant organs. Here, I present an overview of clearing techniques of plant organs and highlight the latest developments of plant tissue clearing in combination with high resolution fluorescence microscopy.

Application-Centric, Energy-Efficient Network Architecture ACTION, Based on Virtual Optical Slice Core and Deterministic Optical Access Network

The Internet traffic is forecasted to grow at a compound annual rate of 21 % from 2013 to 2018, according to surveys carried out by Cisco [1]. Network resources are significantly over-provisioned in today’s networks, and it is quite common to see link utilization in the 30-40% range [2]. Additionally, the multi-media services have widely divergent bandwidth and Quality of Experience (QoE) requirements. Unfortunately, the huge transmission capacity will increase the power consumption of network equipment [3]. Applications Coordinated with Transport, Internet Protocol and Optical Networks (ACTION) [4] has been proposed to realize a multi-QoE, application-centric, and highly energy-efficient network that leverages flexible elastic optical network technologies [5-7]. This paper provides key network technologies for realizing the ACTION, which are a virtual optical slice core network and a Time Division Multiplexing (TDM)-based deterministic active optical access network.

Modulation of TRIM72 Alters the Repair Capacity of Lung Epithelial Cells

The molecular mechanisms for alveolar epithelial cell plasma membrane wounding and repair are largely unknown. Previous studies identified TRIM72 (also known as mitsugumin 53) from striated muscle tissues and linked its physiological functions in these tissues to membrane repair, cell proliferation, and glucose metabolism. Here, using biochemical and immunochemical assays, we have characterized TRIM72 expression in lung tissue and cells, and investigated the physiological function of TRIM72 in the repair of alveolar epithelial cells. In vivo injury of alveolus resident cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled by postinjury administration of propidium iodide and quantified in optical slices of subpleural alveoli. Primary alveolar epithelial cells were isolated to study plasma membrane wounding and repair in vitro, using a dual-dye incorporation assay following glass bead–induced damage to separately quantify injured versus repaired cells. Our results show that TRIM72 protein was expressed in alveolar epithelia of mouse, rat, and human lungs. High tidal volume ventilation of trim72 knockout mice caused significantly more fatal damage of alveolus cells as compared with the wild-type mice. Strikingly, injury estimates in trim72 overexpressor lungs were significantly lower than in the wild-type. The in vitro cell wounding assay further revealed that absence of TRIM72 led to significant repair defects in alveolar epithelial cells, whereas the total number of wounded cells (repaired plus nonrepaired) was similar in the wild-type and trim72 knockout groups. The cellular function of TRIM72 in the lung may be further linked to caveolin 1, as physical and physiological interactions were identified between these two proteins. These data suggest an important role for TRIM72 in the repair of alveolar epithelial cells under plasma membrane stress failure and potential reversal of cell repair defects by TRIM72 overexpression.

Quantitative Analysis of Nanoscale Lipid Bilayer Modifications via Second Harmonic Generating Probes

Second Harmonic Generation (SHG) microscopy is an effective tool for studying the order of symmetry-breaking interfaces, such as lipid bilayers. Recently, we have shown that disruptions in the interfacial nature of the membrane can be studied with the addition of lipophilic SHG probes, specifically Di-4-ANEPPDHQ (Di-4). In addition to the conformational information provided by the SHG signal, this particular probe can be used to determine lipid phase and transmembrane voltage through the dye's fluorescence behavior. We now strengthen our technique by changing the polarity of the incident laser beam from linear to circular polarization. The modification provides SHG signal around the circumference of the cell in every optical slice, while maintaining a sufficient signal-to-noise ratio. Utilizing nanosecond pulsed electric fields (nsPEFs) of varying pulse widths and intensities as a tool for provoking spatially and temporally minute perturbations in the cell membrane, we observe membrane poration on the nano-scale. To verify our results, we adapted a previously-published electrical membrane model to map the theoretical angular area affected by these nsPEFs. The tests further establish the effectiveness of SHG probes, in our case Di-4, as a tool for observing subtle membrane alterations and document the extent to which electric fields of varying pulse parameters affect the cellular membrane.

Research on Preparation of Ni/SiO2 Optical Attenuation Slice by Magnetron Sputtering

In order to find new methods for preparing and improving the performance of optical attenuation slice. Vacuum magnetron sputtering method was used to prepare Ni/SiO2 composite film optical attenuation slices, with vacuum magnetron sputtering apparatus, at 5,10,15,20,25min sputtering time, 0.2,0.4,0.6,0.8,1.0Pa sputtering pressure and sputtering power 300~1200W. The XRD, SEM, EDS, AFM and 722 spectrophotometer also were used to study the effects of different sputtering time, sputtering pressure and sputtering power on the film structure, surface morphology, composition, three-dimensional structure, surface roughness and light attenuation rate of optical attenuation slice samples. The results indicated that: Ni/SiO2 composite films were formed, instead of simple physical adsorption between Ni film and SiO2 substrates with magnetron sputtering by XRD analysis and calculation, with uniform grain size of 25.96, 32.38, 32.29,26.95, 25.92nm, respectively. The main component element was Ni, but there were few impurities deposited on the substrates. Impurities were reduced gradually with the increase of sputtering time, mainly resulted from two sputtering of Ni atoms by EDS; The film surface was smooth and dense, flatness and organizational structure of Ni film were better, surface roughness was 1.267nm at 25min sputtering time, 0.4Pa pressure and 400W power with SEM and AFM. Light attenuation rate of optical attenuation slices was different in different process parameters, and the maximum reached 0.52, film flatness, impurities and defects were the main reason by the light attenuation performance analysis.

A Fractal Nature for Polymerized Laminin

Polylaminin (polyLM) is a non-covalent acid-induced nano- and micro-structured polymer of the protein laminin displaying distinguished biological properties. Polylaminin stimulates neuritogenesis beyond the levels achieved by ordinary laminin and has been shown to promote axonal regeneration in animal models of spinal cord injury. Here we used confocal fluorescence microscopy (CFM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize its three-dimensional structure. Renderization of confocal optical slices of immunostained polyLM revealed the aspect of a loose flocculated meshwork, which was homogeneously stained by the antibody. On the other hand, an ordinary matrix obtained upon adsorption of laminin in neutral pH (LM) was constituted of bulky protein aggregates whose interior was not accessible to the same anti-laminin antibody. SEM and AFM analyses revealed that the seed unit of polyLM was a flat polygon formed in solution whereas the seed structure of LM was highly heterogeneous, intercalating rod-like, spherical and thin spread lamellar deposits. As polyLM was visualized at progressively increasing magnifications, we observed that the morphology of the polymer was alike independently of the magnification used for the observation. A search for the Hausdorff dimension in images of the two matrices showed that polyLM, but not LM, presented fractal dimensions of 1.55, 1.62 and 1.70 after 1, 8 and 12 hours of adsorption, respectively. Data in the present work suggest that the intrinsic fractal nature of polymerized laminin can be the structural basis for the fractal-like organization of basement membranes in the neurogenic niches of the central nervous system.

Synucleins regulate the kinetics of synaptic vesicle endocytosis.

Genetic and pathological studies link α-synuclein to the etiology of Parkinson's disease (PD), but the normal function of this presynaptic protein remains unknown. α-Synuclein, an acidic lipid binding protein, shares high sequence identity with β- and γ-synuclein. Previous studies have implicated synucleins in synaptic vesicle (SV) trafficking, although the precise site of synuclein action continues to be unclear. Here we show, using optical imaging, electron microscopy, and slice electrophysiology, that synucleins are required for the fast kinetics of SV endocytosis. Slowed endocytosis observed in synuclein null cultures can be rescued by individually expressing mouse α-, β-, or γ-synuclein, indicating they are functionally redundant. Through comparisons to dynamin knock-out synapses and biochemical experiments, we suggest that synucleins act at early steps of SV endocytosis. Our results categorize α-synuclein with other familial PD genes known to regulate SV endocytosis, implicating this pathway in PD.

Evaluation of New Morphometric Parameters of Neoangiogenesis in Human Colorectal Cancer Using Confocal Laser Endomicroscopy (CLE) and Targeted Panendothelial Markers

The tumor microcirculation is characterized by an abnormal vascular network with dilated, tortuous and saccular vessels. Therefore, imaging the tumor vasculature and determining its morphometric characteristics represent a critical goal for optimizing the cancer treatment that targets the blood vessels (i.e. antiangiogenesis therapy). The aim of this study was to evaluate new vascular morphometric parameters in colorectal cancer, difficult to achieve through conventional immunohistochemistry, by using the confocal laser endomicroscopy method. Fresh biopsies from tumor and normal tissue were collected during colonoscopy from five patients with T3 colorectal carcinoma without metastasis and were marked with fluorescently labeled anti-CD31 antibodies. A series of optical slices spanning 250 µm inside the tissue were immediately collected for each sample using a confocal laser endomicroscope. All measurements were expressed as the mean ± standard error. The mean diameter of tumor vessels was significantly larger than the normal vessels (9.46±0.4 µm vs. 7.60±0.3 µm, p = 0.0166). The vessel density was also significantly higher in the cancer vs. normal tissue samples (5541.05±262.81 vs. 3755.79±194.96 vessels/mm3, p = 0.0006). These results were confirmed by immunohistochemistry. In addition, the tortuosity index and vessel lengths were not significantly different (1.05±0.016 and 28.30±3.27 µm in normal tissue, vs. 1.07±0.008 and 26.49±3.18 µm in tumor tissue respectively, p = 0.5357 and p = 0.7033). The daughter/mother ratio (ratio of the sum of the squares of daughter vessel radii over the square of the mother vessel radius) was 1.15±0.09 in normal tissue, and 1.21±0.08 in tumor tissue (p = 0.6531). The confocal laser endomicroscopy is feasible for measuring more vascular parameters from fresh tumor biopsies than conventional immunohistochemistry alone. Provided new contrast agents will be clinically available, future in vivo use of CLE could lead to identification of novel biomarkers based on the morphometric characteristics of tumor vasculature.

NIR to NIR upconversion in KYb2F7: RE3+ (RE = Tm, Er) nanoparticles for biological imaging.

Until recently, many contrast agents widely used in biological imaging have absorbed and emitted in the visible region, limiting their usefulness for deeper tissue imaging. In order to push the boundaries of deep tissue imaging with non-ionizing radiation, contrast agents in the near infrared (NIR) regime, which is not strongly absorbed or scattered by most tissues, are being sought after. Upconverting nanoparticles (UCNPs) are attractive candidates since their upconversion emission is tunable with a very narrow bandwidth and they do not photobleach or blink. The upconversion produced by the nanoparticles can be tailored for NIR to NIR by carefully choosing the lanthanide dopants and dopant ratios such as KYb2F7: RE3+ (RE = Tm, Er). Spectroscopic characterization was done by analyzing absorption, fluorescence, and quantum yield data. In order to study the toxicity of the nanoparticles Monkey Retinal Endothelial Cells (MREC) were cultivated in 24 well plates and then treated with nanoparticles at different concentrations in triplicate to obtain the optimal concentration for in vivo experiments. It will be shown that these UCNPs do not elicit a strong toxic response such as quantum dots and some noble metal nanoparticles. 3-D optical slices of nanoparticle treated fibroblast cells were imaged using a confocal microscope where the nucleus and cytoplasm were stained with DAPI and Alexa Fluor respectively. These results presented support the initial assumption, which suggests that KYb2F7: RE3+ would be excellent candidates for NIR contrast agents.

Visualization and quantitation of abundant macroautophagy in virus-infected cells by confocal three-dimensional fluorescence imaging

Varicella-zoster virus (VZV) is a human herpesvirus. Primary infection causes varicella (chickenpox), a viremic illness typified by an exanthem consisting of several hundred vesicles. When VZV reactivates from latency in the spinal ganglia during late adulthood, the emerging virus causes a vesicular dermatomal rash (herpes zoster or shingles). To expand investigations of autophagy during varicella and zoster, newer 3D imaging technology was combined with laser scanning confocal microscopy to provide animations of autophagosomes in the vesicular rash. First, the cells were immunolabeled with antibodies against VZV proteins and the LC3 protein, an integral autophagosomal protein. Antibody reagents lacking activity against the human blood group A1 antigen were selected. After laser excitation of the samples, optimized emission detection bandwidths were configured by Zeiss Zen control software. Confocal Z-stacks comprising up to 40 optical slices were reconstructed into 3D animations with the aid of Imaris software. With this imaging technology, individual autophagosomes were clearly detectable as spheres within each vesicular cell. To enumerate the number of autophagosomes, data sets from 50 cells were reconstructed as 3D fluorescence images and analyzed with MeasurementPro software. The mean number of autophagosomes per infected vesicular cell was >100, although over 200 autophagosomes were seen in a few cells. In summary, macroautophagy was easily quantitated within VZV-infected cells after immunolabeling and imaging by 3D confocal animation technology. These same 3D imaging techniques will be applicable for investigations of autophagy in other virus-infected cells.

Neurogliaform cells of amygdala: a source of slow phasic inhibition in the basolateral complex

Synaptic inhibition in the amygdala actively participates in processing emotional information. To improve the understanding of interneurons in amygdala networks it is necessary to characterize the GABAergic cell types, their connectivity and physiological roles. We used a mouse line expressing a green fluorescent protein (GFP) under the neuropeptide Y (NPY) promoter. Paired recordings between presynaptic NPY-GFP-expressing (+) cells and postsynaptic principal neurons (PNs) of the basolateral amygdala (BLA) were performed. The NPY-GFP+ neurons displayed small somata and short dendrites embedded in a cloud of highly arborized axon, suggesting a neurogliaform cell (NGFC) type. We discovered that a NPY-GFP+ cell evoked a GABA(A) receptor-mediated slow inhibitory postsynaptic current (IPSC) in a PN and an autaptic IPSC. The slow kinetics of these IPSCs was likely caused by the low concentration and spillover of extracellular GABA. We also report that NGFCs of the BLA fired action potentials phase-locked to hippocampal theta oscillations in anaesthetized rats. When this firing was re-played in NPY+-NGFCs in vitro, it evoked a transient depression of the IPSCs. Presynaptic GABA(B) receptors and functional depletion of synaptic vesicles determined this short-term plasticity. Synaptic contacts made by recorded NGFCs showed close appositions, and rarely identifiable classical synaptic structures. Thus, we report here a novel interneuron type of the amygdala that generates volume transmission of GABA. The peculiar functional mode of NGFCs makes them unique amongst all GABAergic cell types of the amygdala identified so far.

Extended Depth of Focus 알고리듬 파라메타 초기설정에 관한 연구

카메라로부터 획득한 여러 장의 영상에서 3차원 정보를 얻어내기 위한 Extended Depth of Focus(EDF) 알고리듬은 최근 많은 연구가 이루어지고 있다. 피사물체의 깊이정보에 따른 제한된 초점으로 인해 초점이 일부분 맞는 여러 장의 이미지를 가지고 EDF알고리듬은 각 영상들의 focus 영역에서 하나의 focused 영상과 depth영상을 취득한다. 대부분의 영상처리 알고리듬이 그렇듯, EDF 알고리듬에 사용되는 파라메타들의 초기설정에 따라 결과에 큰 영향을 준다. 본 논문에서는 EDF알고리듬을 적용하기 전 입력영상의 기반으로 pyramid, wavelet transform, complex wavelet transform을 사용하였으며 EDF알고리즘에서 사용되는 파라메타들의 설정에 따른 각 알고리즘의 성능을 분석하였다. 본 논문에서 제시한 파라메타들은 입력영상의 크기에 따른 down sampling의 단계, 영상의 기반 알고리듬의 영상 복원에 사용되는 최하위 레벨의 이미지에 대한 취득 형태, 연산에 쓰이는 window size의 크기이다. 우리는 실험을 통해 제시한 입력영상에 따라 각 파라메타들이 미치는 영향에 대해 분석하였고, 기존에 사용되었던 일반적인 파라메타 선정방식보다 최적화된 파라메타 선정방식을 통해 얻어진 결과영상이 3dB ~ 19dB정도 개선된 것을 확인하였다. Extended Depth of Focus (EDF) algorithms for extracting three-dimensional (3D) information from a set of optical image slices are studied by many researches recently. Due to the limited depth of focus of the microscope, only a small portion of the image slices are in focus. Most of the EDF algorithms try to find the in-focus area to generate a single focused image and a 3D depth image. Inherent to most image processing algorithms, the EDF algorithms need parameters to be properly initialized to perform successfully. In this paper, we select three popular transform-based EDF algorithms which are each based on pyramid, wavelet transform, and complex wavelet transform, and study the performance of the algorithms according to the initialization of its parameters. The parameters we considered consist of the number of levels used in the transform, the selection of the lowest level image, the window size used in high frequency filter, the noise reduction method, etc. Through extended simulation, we find a good relationship between the initialization of the parameters and the properties of both the texture and 3D ground truth images. Typically, we find that a proper initialization of the parameters improve the algorithm performance 3dB ~ 19dB over a default initialization in recovering the 3D information.

Correcting the axial shrinkage of skeletal muscle thick sections visualized by confocal microscopy

Confocal microscopy is a suitable method for measurements and visualization of skeletal muscle fibres and the neighbouring capillaries. When using 3D images of thick sections the tissue deformation effects should be avoided. We studied the deformation in thick sections of the rat skeletal muscle from complete stacks of images captured with confocal microscope. We measured the apparent thickness of the stacks and compared it to the slice thickness deduced from calibrated microtome settings. The ratio of both values yielded the axial scaling factor for every image stack. Careful sample preparation and treatment of the tissue cryosections with cold Ringer solution minimize the tissue deformation. We conclude that rescaling by the inverse of the axial scaling factor of the stack of optical slices in the direction of the microscope optical axis satisfactorily corrects the axial deformation of skeletal muscle samples.

Stereological Quantification of Cerebellar Purkinje Cells: Literature Review and Description of a Variation of the Physical Disector Method Adapted to Confocal Laser Microscopy

Estimation of cerebellar Purkinje cell number is an important evaluation parameter for the study of the development of the cerebellum as well as for investigating the changes occurring in various experimental and physiological conditions. The introduction of stereological techniques in biological and biomedical research has made quantification of Purkinje cells not only more reliable, because independent from morphology-related sources of bias, but also easier and less time consuming. In this paper, we will briefly overview the stereological techniques that have been used so far for estimation of Purkinje cell number in different animal species and we will describe a simple method (the confocal physical disector) for Purkinje cell quantification on confocal microscope optical slices, based on a variation of the physical disector technique that can be adopted without the need of any dedicated stereological workstation. NeuroQuantology | March 2012 | Volume 10 | Issue 1 | Page 125-131

Thermal gradients and residual stresses in veneered Y-TZP frameworks

Objectives The occurrence of “chipping” of all-ceramic restorations with Y-TZP frameworks has resulted in various designs and cooling procedures recommended for reducing such behavior. In this paper the temperature gradients during fast and slow cooling for conventional and anatomical designs are compared as well as an optical procedure to directly compare the influence of cooling rate on residual stress. Methods This investigation quantifies the temperature gradients between the inner and outer surfaces of crowns measured with thermocouples during two different cooling methods with uniform and anatomical frameworks. In the first method the crown was removed from the furnace after commencement of cooling whereas for the second method the crown was cooled to the glass transition temperature (600 °C) before removal. Direct observation of the residual stresses was made with an optical polarimeter and thin slices of veneered copings. Results This study observed that slow cooling decreases the temperature differences but still differences of up to 88 °C were observed. For the fast cooled crown, temperature differences of more than 100 °C for the uniform and 140 °C for the anatomical framework at temperatures above the glass transition temperature were recorded. Optical polarimeter observations indicated much lower stresses within the porcelain layer upon cooling by removing the crown below the glass transition temperature. Conclusion Slow cooling during the final veneering of dental restorations with zirconia frameworks reduces the temperature gradients and residual stresses within the porcelain layer, which represent one possible cause for chipping. An anatomical designed framework did not show the same reduction extent.

Reconstructing micrometer-scale fiber pathways in the brain: Multi-contrast optical coherence tomography based tractography

Comprehensive understanding of connective neural pathways in the brain has put great challenges on the current imaging techniques, for which three-dimensional (3D) visualization of fiber tracts with high spatiotemporal resolution is desirable. Here we present optical imaging and tractography of rat brain ex-vivo using multi-contrast optical coherence tomography (MC-OCT), which is capable of simultaneously generating depth-resolved images of reflectivity, phase retardance, optic axis orientation and, for in-vivo studies, blood flow images. Using the birefringence property of myelin sheath, nerve fiber tracts as small as a few tens of micrometers can be resolved and neighboring fiber tracts with different orientations can be distinguished in cross-sectional optical slices, 2D en-face images and 3D volumetric images. Combinational contrast of MC-OCT images enables visualization of the spatial architecture and nerve fiber orientations in the brain with unprecedented detail. The results suggest that optical tractography, by virtue of its direct accessibility to nerve fibers, has the potential to validate diffusion magnetic resonance images and investigate structural connections in normal brain and neurological disorders. In addition, an endoscopic MC-OCT may be useful in neurosurgical interventions to aid in placement of deep brain stimulating electrodes.

Ultra Smooth Polishing Research Based on the Cluster Magnetorheological Effect

Based on the principle of MR polishing processing, cluster MR-effect polishing method which formed by cluster distributed magnetic body has been put forward in this article. The author analyzed the principle of the cluster MR-effect plane polishing, developed experimental device and conducted processing experiment to K9 optical glass and silicon slice. Results show that the method is feasible and can realize high precision polishing. The surface roughness of K9 glass and silicon slice can achieve respectively Ra0.005µm and Ra0.016µm finally after processed. It has high efficiency at the same time, the surface roughness can decrease 1 order of magnitude after processed 10 minutes, the surface roughness of K9 glass and silicon slice can realize respectively decrease 3 orders of magnitude and 1 order of magnitude after processed 50 minutes.

Three dimensional microvascular measurements in human endometrium using optical slices from laser scanning confocal microscopy (LSCM)

There is increasing interest in the structure of the microvascular environment in human endometrium because of the recognition of the complexity and functional importance of this tissue. Endometrial microcirculatory networks and their relationships have rarely been studied in three-dimensions. Longitudinal uterine slices containing endometrial tissue were carefully selected from women undergoing a hysterectomy. Formalin-fixed endometrial sections (≤50 μm) representing the fundal and isthmic regions were immunofluorescently labeled with monoclonal antibody (CD34) to target the endothelium of microvessel and fluorescein isothiocyanate (FITC) labeled goat anti-mouse. Digital images were acquired using a Nikon Eclipse E800 microscope equipped with a Radiance 2000 confocal scanning laser attachment. ImarisBasic 4.1 visualization suite was utilized for qualitative interpretation. NeuronTracer 1.0 software was utilized to derive the length and numerical densities. There were significant changes across the phases of the menstrual cycle in functional and basal endometrial layers in vessel length density (LD v) and branch point density (ND v) within both fundal and isthmic regions of the uterus ( P < 0.001). There was also a significant effect of menstrual cycle phase on mean vessel segment length (SL v) within each region and within each of the layers ( P < 0.001). The capillary radial diffusion distance r(diff) was negatively correlated with LD v. In general, within each of the menstrual cycle phases, LD v, ND v were greater in the fundal than the isthmic regions while, in contrast, SL v was found to be greatest in the isthmic region. Utilization of immunofluorescence and laser scanning confocal microscopy has enabled us to demonstrate significant vascular changes in human endometrial layers illustrating that in general, within each of the menstrual cycle phases, vessel length and branch point densities were greater in the fundal than the isthmic regions, while vessel segment lengths were found to be greatest in the isthmic region.

3-D Image Pre-processing Algorithms for Improved Automated Tracing of Neuronal Arbors

The accuracy and reliability of automated neurite tracing systems is ultimately limited by image quality as reflected in the signal-to-noise ratio, contrast, and image variability. This paper describes a novel combination of image processing methods that operate on images of neurites captured by confocal and widefield microscopy, and produce synthetic images that are better suited to automated tracing. The algorithms are based on the curvelet transform (for denoising curvilinear structures and local orientation estimation), perceptual grouping by scalar voting (for elimination of non-tubular structures and improvement of neurite continuity while preserving branch points), adaptive focus detection, and depth estimation (for handling widefield images without deconvolution). The proposed methods are fast, and capable of handling large images. Their ability to handle images of unlimited size derives from automated tiling of large images along the lateral dimension, and processing of 3-D images one optical slice at a time. Their speed derives in part from the fact that the core computations are formulated in terms of the Fast Fourier Transform (FFT), and in part from parallel computation on multi-core computers. The methods are simple to apply to new images since they require very few adjustable parameters, all of which are intuitive. Examples of pre-processing DIADEM Challenge images are used to illustrate improved automated tracing resulting from our pre-processing methods.