Data and image transfer using mobile phones to strengthen microscopy-based diagnostic services in low and middle income country laboratories

Background

The emerging market of mobile phone technology and its use in the health sector is rapidly expanding and connecting even the most remote areas of world. Distributing diagnostic images over the mobile network for knowledge sharing, feedback or quality control is a logical innovation.

Objective

To determine the feasibility of using mobile phones for capturing microscopy images and transferring these to a central database for assessment, feedback and educational purposes.

Methods

A feasibility study was carried out in Uganda. Images of microscopy samples were taken using a prototype connector that could fix a variety of mobile phones to a microscope. An Information Technology (IT) platform was set up for data transfer from a mobile phone to a website, including feedback by text messaging to the end user.

Results

Clear images were captured using mobile phone cameras of 2 megapixels (MP) up to 5MP. Images were sent by mobile Internet to a website where they were visualized and feedback could be provided to the sender by means of text message.

Conclusion

The process of capturing microscopy images on mobile phones, relaying them to a central review website and feeding back to the sender is feasible and of potential benefit in resource poor settings. Even though the system needs further optimization, it became evident from discussions with stakeholders that there is a demand for this type of technology.     

Development,Alteration and Real Time Dynamics of Conjunctiva-Associated Lymphoid Tissue

Purpose

Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model.

Methods

Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy.

Results

Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 µm/min.

Conclusions

CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration.     

Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo

Background

The generation of transgenic mice expressing combinations of fluorescent proteins has greatly aided the reporting of activity and identification of specific neuronal populations. Methods capable of separating multiple overlapping fluorescence emission spectra, deep in the living brain, with high sensitivity and temporal resolution are therefore required. Here, we investigate to what extent spectral unmixing addresses these issues.

Methodology/Principal Findings

Using fluorescence resonance energy transfer (FRET)-based reporters, and two-photon laser scanning microscopy with synchronous multichannel detection, we report that spectral unmixing consistently improved FRET signal amplitude, both in vitro and in vivo. Our approach allows us to detect odor-evoked FRET transients 180–250 µm deep in the brain, the first demonstration of in vivo spectral imaging and unmixing of FRET signals at depths greater than a few tens of micrometer. Furthermore, we determine the reporter efficiency threshold for which FRET detection is improved by spectral unmixing.

Conclusions/Significance

Our method allows the detection of small spectral variations in depth in the living brain, which is essential for imaging efficiently transgenic animals expressing combination of multiple fluorescent proteins.     

How Accurate Are the Fusion of Cone-Beam CT and 3-D Stereophotographic Images?

Background

Cone-beam Computed Tomography (CBCT) and stereophotography are two of the latest imaging modalities available for three-dimensional (3-D) visualization of craniofacial structures. However, CBCT provides only limited information on surface texture. This can be overcome by combining the bone images derived from CBCT with 3-D photographs. The objectives of this study were 1) to evaluate the feasibility of integrating 3-D Photos and CBCT images 2) to assess degree of error that may occur during the above processes and 3) to identify facial regions that would be most appropriate for 3-D image registration.

Methodology

CBCT scans and stereophotographic images from 29 patients were used for this study. Two 3-D images corresponding to the skin and bone were extracted from the CBCT data. The 3-D photo was superimposed on the CBCT skin image using relatively immobile areas of the face as a reference. 3-D colour maps were used to assess the accuracy of superimposition were distance differences between the CBCT and 3-D photo were recorded as the signed average and the Root Mean Square (RMS) error.

Principal Findings:

The signed average and RMS of the distance differences between the registered surfaces were −0.018 (±0.129) mm and 0.739 (±0.239) mm respectively. The most errors were found in areas surrounding the lips and the eyes, while minimal errors were noted in the forehead, root of the nose and zygoma.

Conclusions

CBCT and 3-D photographic data can be successfully fused with minimal errors. When compared to RMS, the signed average was found to under-represent the registration error. The virtual 3-D composite craniofacial models permit concurrent assessment of bone and soft tissues during diagnosis and treatment planning.     

Development of Low-Cost Inverted Microscope to Detect Early Growth of Mycobacterium tuberculosis in MODS Culture

Background

The microscopic observation drug susceptibility (MODS) assay for rapid, low-cost detection of tuberculosis and multidrug resistant tuberculosis depends upon visualization of the characteristic cording colonies of Mycobacterium tuberculosis in liquid media. This has conventionally required an inverted light microscope in order to inspect the MODS culture plates from below. Few tuberculosis laboratories have this item and the capital cost of $5,000 for a high-end microscope could be a significant obstacle to MODS roll-out.

Methodology

We hypothesized that the precise definition provided by costly high-specification inverted light microscopes might not be necessary for pattern recognition.

Significance

In this work we describe the development of a low-cost artesenal inverted microscope that can operate in both a standard or digital mode to effectively replace the expensive commercial inverted light microscope, and an integrated system that could permit a local and remote diagnosis of tuberculosis.     

Integrative Advances for OCT-Guided Ophthalmic Surgery and Intraoperative OCT: Microscope Integration,Surgical Instrumentation,and Heads-Up Display Surgeon Feedback

Purpose

To demonstrate key integrative advances in microscope-integrated intraoperative optical coherence tomography (iOCT) technology that will facilitate adoption and utilization during ophthalmic surgery.

Methods

We developed a second-generation prototype microscope-integrated iOCT system that interfaces directly with a standard ophthalmic surgical microscope. Novel features for improved design and functionality included improved profile and ergonomics, as well as a tunable lens system for optimized image quality and heads-up display (HUD) system for surgeon feedback. Novel material testing was performed for potential suitability for OCT-compatible instrumentation based on light scattering and transmission characteristics. Prototype surgical instruments were developed based on material testing and tested using the microscope-integrated iOCT system. Several surgical maneuvers were performed and imaged, and surgical motion visualization was evaluated with a unique scanning and image processing protocol.

Results

High-resolution images were successfully obtained with the microscope-integrated iOCT system with HUD feedback. Six semi-transparent materials were characterized to determine their attenuation coefficients and scatter density with an 830 nm OCT light source. Based on these optical properties, polycarbonate was selected as a material substrate for prototype instrument construction. A surgical pick, retinal forceps, and corneal needle were constructed with semi-transparent materials. Excellent visualization of both the underlying tissues and surgical instrument were achieved on OCT cross-section. Using model eyes, various surgical maneuvers were visualized, including membrane peeling, vessel manipulation, cannulation of the subretinal space, subretinal intraocular foreign body removal, and corneal penetration.

Conclusions

Significant iterative improvements in integrative technology related to iOCT and ophthalmic surgery are demonstrated.     

Spectral characterization and unmixing of intrinsic contrast in intact normal and diseased gastric tissues using hyperspectral two-photon microscopy

Background

Living tissues contain a range of intrinsic fluorophores and sources of second harmonic generation which provide contrast that can be exploited for fresh tissue imaging. Microscopic imaging of fresh tissue samples can circumvent the cost and time associated with conventional histology. Further, intrinsic contrast can provide rich information about a tissue''s composition, structure and function, and opens the potential for in-vivo imaging without the need for contrast agents.

Methodology/Principal Findings

In this study, we used hyperspectral two-photon microscopy to explore the characteristics of both normal and diseased gastrointestinal (GI) tissues, relying only on their endogenous fluorescence and second harmonic generation to provide contrast. We obtained hyperspectral data at subcellular resolution by acquiring images over a range of two-photon excitation wavelengths, and found excitation spectral signatures of specific tissue types based on our ability to clearly visualize morphology. We present the two-photon excitation spectral properties of four major tissue types that are present throughout the GI tract: epithelium, lamina propria, collagen, and lymphatic tissue. Using these four excitation signatures as basis spectra, linear unmixing strategies were applied to hyperspectral data sets of both normal and neoplastic tissue acquired in the colon and small intestine. Our results show that hyperspectral unmixing with excitation spectra allows segmentation, showing promise for blind identification of tissue types within a field of view, analogous to specific staining in conventional histology. The intrinsic spectral signatures of these tissue types provide information relating to their biochemical composition.

Conclusions/Significance

These results suggest hyperspectral two-photon microscopy could provide an alternative to conventional histology either for in-situ imaging, or intraoperative ‘instant histology’ of fresh tissue biopsies.     

Complex Collagen Fiber and Membrane Morphologies of the Whole Porcine Aortic Valve

Objectives

Replacement aortic valves endeavor to mimic native valve function at the organ, tissue, and in the case of bioprosthetic valves, the cellular levels. There is a wealth of information about valve macro and micro structure; however, there presently is limited information on the morphology of the whole valve fiber architecture. The objective of this study was to provide qualitative and quantitative analyses of whole valve and leaflet fiber bundle branching patterns using a novel imaging system.

Methods

We developed a custom automated microscope system with motor and imaging control. Whole leaflets (n = 25) were imaged at high resolution (e.g. 30,000×20,000 pixels) using elliptically polarized light to enhance contrast between structures without the need for staining or other methods. Key morphologies such as fiber bundle size and branching were measured for analyses.

Results

The left coronary leaflet displayed large asymmetry in fiber bundle organization relative to the right coronary and non-coronary leaflets. We observed and analyzed three main patterns of fiber branching; tree-like, fan-like, and pinnate structures. High resolution images and quantitative metrics are presented such as fiber bundle sizes, positions, and branching morphological parameters.

Significance

To our knowledge there are currently no high resolution images of whole fresh leaflets available in the literature. The images of fiber/membrane structures and analyses presented here could be highly valuable for improving the design and development of more advanced bioprosthetic and/or bio-mimetic synthetic valve replacements.     

Non-invasive microstructure and morphology investigation of the mouse lung: qualitative description and quantitative measurement

Background

Early detection of lung cancer is known to improve the chances of successful treatment. However, lungs are soft tissues with complex three-dimensional configuration. Conventional X-ray imaging is based purely on absorption resulting in very low contrast when imaging soft tissues without contrast agents. It is difficult to obtain adequate information of lung lesions from conventional X-ray imaging.

Methods

In this study, a recently emerged imaging technique, in-line X-ray phase contrast imaging (IL-XPCI) was used. This powerful technique enabled high-resolution investigations of soft tissues without contrast agents. We applied IL-XPCI to observe the lungs in an intact mouse for the purpose of defining quantitatively the micro-structures in lung.

Findings

The three-dimensional model of the lung was successfully established, which provided an excellent view of lung airways. We highlighted the use of IL-XPCI in the visualization and assessment of alveoli which had rarely been studied in three dimensions (3D). The precise view of individual alveolus was achieved. The morphological parameters, such as diameter and alveolar surface area were measured. These parameters were of great importance in the diagnosis of diseases related to alveolus and alveolar scar.

Conclusion

Our results indicated that IL-XPCI had the ability to represent complex anatomical structures in lung. This offered a new perspective on the diagnosis of respiratory disease and may guide future work in the study of respiratory mechanism on the alveoli level.     

Helium: visualization of large scale plant pedigrees

Background

Plant breeders use an increasingly diverse range of data types to identify lines with desirable characteristics suitable to be taken forward in plant breeding programmes. There are a number of key morphological and physiological traits, such as disease resistance and yield that need to be maintained and improved upon if a commercial variety is to be successful. Computational tools that provide the ability to integrate and visualize this data with pedigree structure, will enable breeders to make better decisions on the lines that are used in crossings to meet both the demands for increased yield/production and adaptation to climate change.

Results

We have used a large and unique set of experimental barley (H. vulgare) data to develop a prototype pedigree visualization system. We then used this prototype to perform a subjective user evaluation with domain experts to guide and direct the development of an interactive pedigree visualization tool called Helium.

Conclusions

We show that Helium allows users to easily integrate a number of data types along with large plant pedigrees to offer an integrated environment in which they can explore pedigree data. We have also verified that users were happy with the abstract representation of pedigrees that we have used in our visualization tool.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-259) contains supplementary material, which is available to authorized users.     

ISS-An Electronic Syndromic Surveillance System for Infectious Disease in Rural China

Background

syndromic surveillance system has great advantages in promoting the early detection of epidemics and reducing the necessities of disease confirmation, and it is especially effective for surveillance in resource poor settings. However, most current syndromic surveillance systems are established in developed countries, and there are very few reports on the development of an electronic syndromic surveillance system in resource-constrained settings.

Objective

this study describes the design and pilot implementation of an electronic surveillance system (ISS) for the early detection of infectious disease epidemics in rural China, complementing the conventional case report surveillance system.

Methods

ISS was developed based on an existing platform ‘Crisis Information Sharing Platform’ (CRISP), combining with modern communication and GIS technology. ISS has four interconnected functions: 1) work group and communication group; 2) data source and collection; 3) data visualization; and 4) outbreak detection and alerting.

Results

As of Jan. 31^st 2012, ISS has been installed and pilot tested for six months in four counties in rural China. 95 health facilities, 14 pharmacies and 24 primary schools participated in the pilot study, entering respectively 74256, 79701, and 2330 daily records into the central database. More than 90% of surveillance units at the study sites are able to send daily information into the system. In the paper, we also presented the pilot data from health facilities in the two counties, which showed the ISS system had the potential to identify the change of disease patterns at the community level.

Conclusions

The ISS platform may facilitate the early detection of infectious disease epidemic as it provides near real-time syndromic data collection, interactive visualization, and automated aberration detection. However, several constraints and challenges were encountered during the pilot implementation of ISS in rural China.     

ITAM Signaling by Vav Family Rho Guanine Nucleotide Exchange Factors Regulates Interstitial Transit Rates of Neutrophils In Vivo

Background

In response to infection, neutrophils are quickly recruited from the blood into inflamed tissues. The interstitial migration of neutrophils is crucial for the efficient capture and control of rapidly proliferating microbes before microbial growth can overwhelm the host''s defenses. However, the molecular mechanisms that regulate interstitial migration are incompletely understood.

Methodology/Principal Findings

Here, we use two-photon microscopy (2PM) to study discrete steps of neutrophil responses during subcutaneous infection with bacteria. Our study demonstrates that signals emanating from ITAM-containing receptors mediated by Vav family Rho GEFs control the velocity, but not the directionality, of neutrophil migration towards sites of bacterial infection.

Conclusions/Significance

Here we show that during neutrophil migration towards sites of bacterial infection, signals emanating from ITAM-containing receptors specifically control interstitial neutrophil velocity.     

Comparative structural analysis of lipid binding START domains

Background

Steroidogenic acute regulatory (StAR) protein related lipid transfer (START) domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response to ligand binding. More than a dozen human genes encode START domains, and several of them are implicated in a disease.

Principal Findings

We report crystal structures of the human STARD1, STARD5, STARD13 and STARD14 lipid transfer domains. These represent four of the six functional classes of START domains.

Significance

Sequence alignments based on these and previously reported crystal structures define the structural determinants of human START domains, both those related to structural framework and those involved in ligand specificity.

Enhanced version

This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.     

Primary mucosal sinonasal melanoma—Case report and review of the literature. The role of complex treatment-surgery and adjuvant radiotherapy

Aim

The place of adjuvant radiotherapy in the treatment of sinonasal melanoma.

Background

Sinonasal mucosal melanoma is a rare disease with poor prognosis and requires a complex treatment. Elective neck dissection in patients with N0 and adjuvant radiotherapy has been a source of controversy. High late regional recurrence rates rise questions about elective irradiation of the neck nodes in patients with N0 stage disease.

Methods

We present our two years’ follow up in a case of locally advanced sinonasal melanoma and literature review of the treatment options for mucosal melanoma.

Results

In locally advanced sinonasal melanoma treated with surgical resection, postoperative radiotherapy and chemotherapy we had local tumor control. Two years later, a regional contralateral recurrence without distant metastasis occurred.

Conclusions

Literature data for frequent neck lymph nodes recurrences justify elective neck dissection. Postoperative elective neck radiotherapy for patients with locally advanced sinonasal melanoma and clinically N0 appears to decrease the rate of late regional recurrences.     

Immunolocalization of arabinogalactan proteins (AGPs) in reproductive structures of an early-divergent angiosperm,Trithuria (Hydatellaceae)

Background and Aims Trithuria

is the sole genus of Hydatellaceae, a family of the early-divergent angiosperm lineage Nymphaeales (water-lilies). In this study different arabinogalactan protein (AGP) epitopes in T. submersa were evaluated in order to understand the diversity of these proteins and their functions in flowering plants.

Methods

Immunolabelling of different AGPs and pectin epitopes in reproductive structures of T. submersa at the stage of early seed development was achieved by immunofluorescence of specific antibodies.

Key Results

AGPs in Trithuria pistil tissues could be important as structural proteins and also as possible signalling molecules. Intense labelling was obtained with anti-AGP antibodies both in the anthers and in the intine wall, the latter associated with pollen tube emergence.

Conclusions

AGPs could play a significant role in Trithuria reproduction, due to their specific presence in the pollen tube pathway. The results agree with labellings obtained for Arabidopsis and confirms the importance of AGPs in angiosperm reproductive structures as essential structural components and probably important signalling molecules.     

Experimental Differentiation of Intraocular Masses Using Ultrahigh-Field Magnetic Resonance Imaging – A Case Series

Purpose

The case reports presented here were compiled to demonstrate the potential for improved diagnosis and monitoring of disease progress of intraocular lesions using ultrahigh-field magnetic resonance microscopy (MRM) at 7.1 Tesla.

Methods

High-resolution ex vivo ocular magnetic resonance (MR) images were acquired on an ultrahigh-field MR system (7.1 Tesla, ClinScan, Bruker BioScan, Germany) using a 2-channel coil with 4 coil elements and T2-weighted turbo spin echo (TSE) sequences of human eyes enucleated because of different intraocular lesions. Imaging parameters were: 40×40 mm field of view, 512×512 matrix, and 700 µm slice thickness. The results were correlated with in vivo ultrasound and histology of the enucleated eyes.

Results

Imaging was performed in enucleated eyes with choroidal melanoma, malignant melanoma of iris and ciliary body with scleral perforation, ciliary body melanoma, intraocular metastasis of esophageal cancer, subretinal bleeding in the presence of perforated corneal ulcer, hemorrhagic choroidal detachment, and premature retinopathy with phthisis and ossification of bulbar structures. MR imaging allowed differentiation between solid and cystic tumor components. In case of hemorrhage, fluid-fluid levels were identified. Melanin and calcifications caused significant hypointensity. Microstructural features of eye lesions identified by MRM were confirmed by histology.

Conclusion

This study demonstrates the potential of MRM for the visualization and differential diagnosis of intraocular lesions. At present, the narrow bore of the magnet still limits the use of this technology in humans in vivo. Further advances in ultrahigh-field MR imaging will permit visualization of tumor extent and evaluation of nonclassified intraocular structures in the near future.     

SFMetrics: an analysis tool for scanning force microscopy images of biomolecules

Background

Scanning force microscopy (SFM) allows direct, rapid and high-resolution visualization of single molecular complexes; irregular shapes and differences in sizes are immediately revealed by the scanning tip in three-dimensional images. However, high-throughput analysis of SFM data is limited by the lack of versatile software tools accessible to SFM users. Most existing SFM software tools are aimed at broad general use: from material-surface analysis to visualization of biomolecules.

Results

We present SFMetrics as a metrology toolbox for SFM, specifically aimed at biomolecules like DNA and proteins, which features (a) semi-automatic high-throughput analysis of individual molecules; (b) ease of use working within MATLAB environment or as a stand-alone application; (c) compatibility with MultiMode (Bruker), NanoWizard (JPK instruments), Asylum (Asylum research), ASCII, and TIFF files, that can be adjusted with minor modifications to other formats.

Conclusion

Assembled in a single user interface, SFMetrics serves as a semi-automatic analysis tool capable of measuring several geometrical properties (length, volume and angles) from DNA and protein complexes, but is also applicable to other samples with irregular shapes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0457-8) contains supplementary material, which is available to authorized users.     

Cellular quantitative analysis of neuroblastoma tumor and splitting overlapping cells

Background

Neuroblastoma Tumor (NT) is one of the most aggressive types of infant cancer. Essential to accurate diagnosis and prognosis is cellular quantitative analysis of the tumor. Counting enormous numbers of cells under an optical microscope is error-prone. There is therefore an urgent demand from pathologists for robust and automated cell counting systems. However, the main challenge in developing these systems is the inability of them to distinguish between overlapping cells and single cells, and to split the overlapping cells. We address this challenge in two stages by: 1) distinguishing overlapping cells from single cells using the morphological differences between them such as area, uniformity of diameters and cell concavity; and 2) splitting overlapping cells into single cells. We propose a novel approach by using the dominant concave regions of cells as markers to identify the overlap region. We then find the initial splitting points at the critical points of the concave regions by decomposing the concave regions into their components such as arcs, chords and edges, and the distance between the components is analyzed using the developed seed growing technique. Lastly, a shortest path determination approach is developed to determine the optimum splitting route between two candidate initial splitting points.

Results

We compare the cell counting results of our system with those of a pathologist as the ground-truth. We also compare the system with three state-of-the-art methods, and the results of statistical tests show a significant improvement in the performance of our system compared to state-of-the-art methods. The F-measure obtained by our system is 88.70%. To evaluate the generalizability of our algorithm, we apply it to images of follicular lymphoma, which has similar histological regions to NT. Of the algorithms tested, our algorithm obtains the highest F-measure of 92.79%.

Conclusion

We develop a novel overlapping cell splitting algorithm to enhance the cellular quantitative analysis of infant neuroblastoma. The performance of the proposed algorithm promises a reliable automated cell counting system for pathology laboratories. Moreover, the high performance obtained by our algorithm for images of follicular lymphoma demonstrates the generalization of the proposed algorithm for cancers with similar histological regions and histological structures.