An Image Analysis Workstation Designed for Multiple Users: Application of Quantitative Digital Imaging Techniques to Electron Microscopy

The purpose of the present study is to describe the setup of an image analysis workstation designed for multiple users, and to show the application of digital imaging techniques to the analysis of electron microscopic images. The image analysis system consists of a conventional light microscope mounted on a table-top, vibration-free platform, a light box for viewing negatives, two separate video cameras, a switch box, a video monitor, a digitizing tablet, a computer, and mor-phometric software packages. The system can quantitate the amount that each of the 256 gray levels contributes to the image, perform morpho-metric analysis (eg, shape and size) on individual gray level-defined subimages, and perform statistical analysis. Each operator has access to his or her own data and program setups through the use of 21.4-Mb removable Bernoulli cartridges. This setup for multiple users prevents the cluttering of the hard drive of the computer and avoids the possibility of accidentally removing the stored data of another user. The quantitative capabilities of the digital imaging system is demonstrated using an image of a normal lymphocyte and an apoptotic cell (ie, a cell which has undergone programmed cell death), both captured on the same electron microscopic negative. A comparison of the histograms of nuclear densities determined for these two cells reveals subtleties in gray level distribution not appreciated by the naked eye.     

Thick-section histometry of porous hydroxyapatite implants using backscattered electron imaging

A histometry system has been developed to measure bone ingrowth into porous hydroxyapatite implants utilizing the backscattered electron image of thick sections. The system consists of a scanning electron microscope (SEM) with backscattered electron detector, digital beam controller, minicomputer based image digitization, and microcomputer based image processing, point counting, and lineal analysis. The SEM backscattered electron imaging mode yields high tissue contrast and sharp tissue boundaries, substantially reducing the lost cap and projection effect errors of thick sections. High-resolution digitization of the image substantially reduces the standard error of the estimates. By using the digitized image the tedious process of filtering artifacts and recording actual point counts, intersections, and intercept lengths is delegated to computer software. Performance of this system in a recent study demonstrated substantial ease of operator use and speed of measurement. In the absence of a digital beam controller an inexpensive video digitizer circuit board may be used to digitize photographic prints of the SEM images. The combination of increased accuracy, precision, operator ease, and speed suggests that this system can be useful for soft tissue-bone-implant histometry.     

Computerized classification of nuclear profiles in non-Hodgkin's lymphomas

The authors present experimental technics for the diagnosis of non-Hodgkin's lymphomas, based on instrumental classification of nuclear profiles using a video-based system for computerized interactive morphometry (CIM). In their system, the real time video image of a specimen is superimposed to a graphics overlay generated by a computer, consisting of a test area with four visual markers for random sampling of cells and a menu with several options to send direct commands to the system. Using a touch-sensitive screen mounted on a video monitor as an interactive peripheral, a trained observer traces 100 randomly selected lymphoid cells, counts mitoses in 25 microscopic fields, and categorizes the lesion as diffuse or nodular. Each cell is instrumentally classified into either small cell noncleaved, small cell cleaved, or large, based on the length of their nuclear profiles, their enclosed nuclear area, and a circularity factor. Thereafter the computer provides a "diagnosis," based on hierarchic analysis of the data. The morphometric data are also interpreted by alternate statistical methods of discriminatory classificatory analysis that provide a diagnosis and a probability statement derived from matching unknown cases with a data base. Forty-two lymphoid lesions have been categorized with the CIM system. Studies of interobserver and intraobserver variations in data collection are discussed. The potential advantages of CIM for the objective classification of non-Hodgkin's lymphomas are discussed.     

多路电视腹腔镜图像数字化处理系统的设计与实现

目的设计多路电视腹腔镜图像数字化处理系统。方法通过VC++建立模版,设计程序。软件设计对多床位视频信号实时进行采集、同时显示,同时对各路视频进行动态录制、保存。模块包括视频流截获模块、视频窗口的创建和显示、视频显示的切换、录像和拍照模块、视频回放模块、报告编辑保存打印模块、查询模块、图像处理模块。结果可以对多床位视频信号进行实时采集、能同时进行显示,能同时对各路视频进行动态录制、保存,能根据医生的需要,即时拍下对应床位的图片并能对图片进行处理。结论该系统较现行的腹腔镜数字图像处理系统先进,腹腔镜图像实现数字化。     

Immunofluorescence-digital image processing system for the quantitation of secreted immunoglobulin by single cells

To quantitate the amount of secreted immunoglobulin (Ig) by a single cell, the immunofluorescence digital image processing (IDIP) system was adapted to the modified enzyme-linked immunospot (ELI-SPOT) assay. In this assay, an immunofluorescence (tetramethylrhodamine isothiocyanate) conjugated antibody was used for the detection of spots instead of the usual method of enzyme coupling. We have named this the immunofluorescence-linked immunospot (ILISPOT) assay. In addition to the quantitation of secreted Ig by single cells, this method allowed us to objectively determine the exact number of Ig producing spot forming cells (SFC). 96 well culture plates were pre-coated with goat anti-mouse Ig. The mouse IgM producing hybridoma (E-3-4) was incubated in the plates for 4 h at 37 degrees C. Cells were removed prior to the addition of biotinylated goat anti-mouse mu antibody. After overnight incubation, immunofluorescence conjugated avidin was added for the visualization of spots by the IDIP system. The IDIP system consists of a fluorescent microscope equipped with a video camera and computer. The gray scale of secreted IgM was initially established as a standard by the known amount of purified IgM. By using digital image processing, the number of spots and the gray scale of individual spots were computed. The shape and pattern of gray scale data were used to distinguish between the real spots and pseudo spots. This IDIP system could detect as little as 0.19 pg of secreted IgM (1.2 x 10(5) molecules) and an average of approximately 1.33 pg (8.3 x 10(5) molecules) produced by a single cell. Adaptation of the digital image processing system to the ILISPOT assay allowed the measurement of both the amount of Ig produced at the single cell level and also the exact numbers of SFC present in a totally objective fashion.     

Comparison between Different Cost Devices for Digital Capture of X-ray Films: An Image Characteristics Detection Approach

A common teleradiology practice is digitizing films. The costs of specialized digitizers are very high, that is why there is a trend to use conventional scanners and digital cameras. Statistical clinical studies are required to determine the accuracy of these devices, which are very difficult to carry out. The purpose of this study was to compare three capture devices in terms of their capacity to detect several image characteristics. Spatial resolution, contrast, gray levels, and geometric deformation were compared for a specialized digitizer ICR (US$ 15,000), a conventional scanner UMAX (US$ 15,000), a conventional scanner UMAX (US 1,800), and a digital camera LUMIX (US$ 450, but require an additional support system and a light box for about US$ 450, but require an additional support system and a light box for about US 400). Test patterns printed in films were used. The results detected gray levels lower than real values for all three devices; acceptable contrast and low geometric deformation with three devices. All three devices are appropriate solutions, but a digital camera requires more operator training and more settings.     

Methods in laboratory investigation. Digital reversal and enhancement of negatives in video-based interactive morphometry

We describe a system for the digital reversal of negatives in interactive morphometry. Minimum requirements, in addition to a high-resolution black and white video camera and monitor, are a readily available digital video frame store and a computer system as simple as a microprocessor-based personal computer. In a video-based analysis system, the addition of a digital image memory device and computer obviates the time and expense of photographic processing of the large number of negatives to positive prints required for an appropriate sampling size in morphometry. Additionally, the enhancement of digital images to recover details in the negative is feasible. This study represents a part of a continuing effort to develop an efficient, interactive morphometry system that encourages the use of a large number of random samples by simplifying processing.     

Erratum to Decomposition cross-correlation for analysis of collagen matrix deformation by single smooth muscle cells

Microvascular remodeling is known to depend on cellular interactions with matrix tissue. However, it is difficult to study the role of specific cells or matrix elements in an in vivo setting. The aim of this study is to develop an automated technique that can be employed to obtain and analyze local collagen matrix remodeling by single smooth muscle cells. We combined a motorized microscopic setup and time-lapse video microscopy with a new cross-correlation based image analysis algorithm to enable automated recording of cell-induced matrix reorganization. This method rendered 60–90 single cell studies per experiment, for which collagen deformation over time could be automatically derived. Thus, the current setup offers a tool to systematically study different components active in matrix remodeling.     

Going virtual with quicktime VR: new methods and standardized tools for interactive dynamic visualization of anatomical structures

Continuing evolution of computer-based multimedia technologies has produced QuickTime, a multiplatform digital media standard that is supported by stand-alone commercial programs and World Wide Web browsers. While its core functions might be most commonly employed for production and delivery of conventional video programs (e.g., lecture videos), additional QuickTime VR "virtual reality" features can be used to produce photorealistic, interactive "non-linear movies" of anatomical structures ranging in size from microscopic through gross anatomic. But what is really included in QuickTime VR and how can it be easily used to produce novel and innovative visualizations for education and research? This tutorial introduces the QuickTime multimedia environment, its QuickTime VR extensions, basic linear and non-linear digital video technologies, image acquisition, and other specialized QuickTime VR production methods. Four separate practical applications are presented for light and electron microscopy, dissectable preserved specimens, and explorable functional anatomy in magnetic resonance cinegrams.     

Analysis of facial sebum distribution using a digital fluorescent imaging system

Current methods for analysis of sebum excretion have limitations, such as irreproducible results in repeatable measurements due to the point measurement method, user-dependent artifacts due to contact measurement or qualitative evaluation of the image, and long measurement time. A UV-induced fluorescent digital imaging system is developed to acquire facial images so that the distribution of sebum excretion on the face could be analyzed. The imaging system consists of a constant UV-A light source, digital color camera, and head-positioning device. The system for acquisition of a fluorescent facial image and the image analysis method is described. The imaging modality provides uniform light distribution and presents a discernible color fluorescent image. Valuable parameters of sebum excretion are obtained after image analysis. The imaging system, which provides a noncontact method, is proved to be a useful tool to evaluate the amount and pattern of sebum excretion. When compared to conventional "Wood's lamp" and "Sebutape" methods that provide similar parameters for sebum excretion, the described method is simpler and more reliable to evaluate the dynamics of sebum excretion in nearly real-time.     

Visualization of fast-moving cells in vivo using digital holographic video microscopy

Digital in-line holography offers some significant advantages over conventional optical holography and microscopy to image biological specimens. By combining holography with digital video microscopy, an in-line holographic video microscope is developed and is capable of recording spatial 3D holographic images of biological specimens, while preserving the time dimension. The system enables high-speed video recording of fast cell movement, such as the rapid movement of blood cells in the blood stream in vivo. This capability is demonstrated with observations of fast 3-D movement of live cells in suspension cultures in response to a gentle shake to the Petri dish. The experimental and numerical procedures are incorporated with a fast reconstruction algorithm for reconstruction of holographic video frames at various planes (z axis) from the hologram and along the time axis. The current system enables both lateral and longitudinal resolutions down to a few micrometers. Postreconstruction processing of background subtraction is utilized to eliminate noise caused by scattered light, thereby enabling visualization of, for example, blood streams of live Xenopos tadpoles. The combination of digital holography and microscopy offers unique advantages for imaging of fast moving cells and other biological particles in three dimensions in vivo with high spatial and temporal resolution.     

Electron Microscopic and Confocal Laser Scanning Microscopic Observation of Subcellular Organelles and Pituitary Hormone mRNA: Application of Ultrastructural In Situ Hybridization and Immunohistochemistry to the Pathophysiological Studies of Pituitary Cells

Nonradioisotopic electron microscopic (EM)in situ hybridization (ISH) (EM-ISH) with biotinylated oligonucleotide probes is utilized for the ultrastructural visualization of pituitary hormone mRNA in rat pituitary cells. EM-ISH is an important tool for clarifying the intracellular localization of mRNA and the exact site of specific hormone synthesis on the rough endoplasmic reticulum. The simultaneous visualization of mRNA and encoded protein in the same cell using preembedding EM-ISH and subsequent postembedding immunoreaction with protein A colloidal gold complex can provide an important clue for elucidating the intracellular correlation of mRNA translation and secretion of translated protein. Another focus of this review is the utilization of a recently developed imaging system of confocal laser scanning microscopy (CLSM). The combination of CLSM and image analysis system (IAS) enables us to visualize an individual dimensional image of the intracellular distribution of mRNA and subcellular organelles successfully at any optional cross sections of light microscopic ISH studies, and can be another useful tool for the ultrastructural ISH study of mRNA.     

Oncocytomas and null cell adenomas of the human pituitary: Morphometric and in vitro functional comparison

Summary In this study, light microscopic and ultrastructural morphometric features of oncocytomas and null cell adenomas were compared and the morphometric data were correlated with in vitro endocrine activity. All tumours were unassociated with clinical or biochemical evidence of hormone excess and were diagnosed as oncocytomas or null cell adenomas, using histology, immunohistochemistry and electron microscopy. In oncocytomas, when compared with null cell adenomas, light microscopic morphometry revealed that total cell areas were significantly larger and nuclear cytoplasmic ratios were smaller due to an increase in cytoplasmic areas. Ultrastructural morphometry disclosed an abundance of mitochondria in oncocytomas. Absolute volumes of cytoplasmic organelles per cell were not reduced in oncocytomas compared with those of null cell adenomas. These results indicate that accumulating mitochondria do not replace other cytoplasmic organelles, and furthermore that the functional potential of oncocytomas is not lost. In vitro study demonstrated the production of pituitary hormones, primarily gonadotropins in oncocytomas and null cell adenomas. It can be concluded that oncocytomas, which represent the final stage of oncocytic transformation, have a close relationship with null cell adenomas based on morphometric comparison as well as in vitro studies.     

Virtual electron microscopy: a simple implementation creating a new paradigm in ultrastructural examination

Ultrastructural examination is a time-consuming and tiring process, requiring search for diagnostic features on a low-contrast screen in a dim environment. This article describes a method to circumvent these problems through the creation of a virtual ultrathin slide. This can be achieved by automated capturing of hundreds of images at high magnification and stitching them together into a digital image with a resolution of 4 nm/pixel. The pathologist can then navigate the virtual slide at his/her workstation computer. The image shows good contrast and resolution for diagnostic purposes, and most important, the pathologist can precisely note where the specific ultrastructural features are located. The setup required to implement virtual electron microscopy includes a transmission electron microscope equipped with motorized stage and automated digital image capture function, 2 free software components, self-developed software, and a desktop-grade computer. Besides use in daily diagnosis, virtual electron microscopy can open up many new applications such as undergraduate teaching, pathology resident training, external quality assurance program, and expert consultation.     

Investigation of basic imaging properties in digital radiography. 7. Noise Wiener spectra of II-TV digital imaging systems

We used Wiener spectral analysis in order to investigate the different noise sources and the effects of various parameters such as pixel size, image intensifier (II) field size, and exposure level on the noise in an II-TV digital system. The digital Wiener spectra in terms of relative x-ray intensity were determined directly from the digital noise data in terms of pixel values, by use of the characteristic curve of the imaging system. From averaged, subtracted, and/or combination images, the amount of structure mottle relative to the amount of quantum mottle was estimated. We found that a substantial amount of structure mottle was included in our II-TV digital subtraction angiography system, whereas the electronic noise of the TV system was quite small relative to the quantum and structure mottle. The effects of time jitter on the noise in single-frame images (consisting of multiple video frames) and in subtracted and averaged images were also investigated.     

Digitizing an Analog Radiography Teaching File Under Time Constraint: Trade-Offs in Efficiency and Image Quality

We digitized the radiography teaching file at Black Lion Hospital (Addis Ababa, Ethiopia) during a recent trip, using a standard digital camera and a fluorescent light box. Our goal was to photograph every radiograph in the existing library while optimizing the final image size to the maximum resolution of a high quality tablet computer, preserving the contrast resolution of the radiographs, and minimizing total library file size. A secondary important goal was to minimize the cost and time required to take and process the images. Three workers were able to efficiently remove the radiographs from their storage folders, hang them on the light box, operate the camera, catalog the image, and repack the radiographs back to the storage folder. Zoom, focal length, and film speed were fixed, while aperture and shutter speed were manually adjusted for each image, allowing for efficiency and flexibility in image acquisition. Keeping zoom and focal length fixed, which kept the view box at the same relative position in all of the images acquired during a single photography session, allowed unused space to be batch-cropped, saving considerable time in post-processing, at the expense of final image resolution. We present an analysis of the trade-offs in workflow efficiency and final image quality, and demonstrate that a few people with minimal equipment can efficiently digitize a teaching file library.     

Application of virtual microscopy in clinical cytopathology.

Virtual microscopy (VM) refers to the use of an automated microscope and digital imaging technology to scan, store, and view glass slides. VM systems allow the user to view a scanned image of the entire slide at multiple magnifications on a computer screen. We tested VM to evaluate its possible utility in diagnostic cytopathology. Ten cervical-vaginal monolayered preparations (AutoCyte preparation) were scanned using a BLISS (Bacus Laboratories Inc. Slide Scanner) system. Approximately 20-30% of the cellular area of each slide was imaged. The cases were randomly chosen to include examples ranging from benign cellular changes (BCC) to high-grade squamous intraepithelial lesions (HSIL). The computer performed image tiling and fusing of multiple JPEG images to create a high-quality VM slide. Six examiners (two each of cytopathologists, senior residents, and cytotechnologists) blindly evaluated the VM slides using an image server program (WebSlide Browser thin client software). The cytopathologic diagnoses made on the VM slide were then compared to the original glass slide diagnoses. BLISS took 36-100 min (avg. 58.4 min) to scan the selected fields in a glass slide with file sizes ranging from 23.1-83.6 MB. Time taken by the examiners to render a diagnosis ranged from 1-15 min (avg. 4.1 min) per case. The combined diagnostic accuracy was 98.3%. Only one case of LSIL was missed by one examiner. VM is a promising new tool, which gives a user the feel and simulated experience of an actual microscopic examination and provides a useful alternative to a glass slide in diagnostic cytopathology. Possible applications include: 1) second opinion consultation without transporting the glass slide, 2) education, 3) VM proficiency tests / board exams, and 4) telepathology. Shortcomings include 1) expensive initial setup, 2) inability to maintain an adequate focus in a thick smear with multiple levels, 3) large storage size of the VM slide, and 4) relatively long time needed to scan a slide.     

Virtual microscopy: high resolution digital photomicrography as a tool for light microscopy simulation

Recent advances in microcomputers and high resolution digital video cameras provide pathologists the opportunity to combine precision optics with digital imaging technology and develop new educational and research tools. We review recent advances in virtual microscopy and describe techniques for viewing digital images using a microcomputer-based workstation to simulate light microscopic examination, including scanning at low power to select features of interest and zooming to increase magnification. Hardware and software components necessary to acquire digital images of histological and cytological slides, and closely simulate their examination under a light microscope are discussed. The workstation is composed of a MicroLumina digital scanning camera (Leaf Systems, Southborough, MA), light microscope (Olympus Optical Co., Lake Success, NY), Pentium (Intel Corp., Santa Clara, CA) 166 MHz microcomputer configured with 64 megabytes of random access memory (RAM), a MGA Millenium Powerdesk graphics card (Matrox Graphics, Inc., Montreal, Canada) and Photoshop software (Adobe Systems Inc., San Jose, CA) running in a Windows 95 (Microsoft Corp., Redmond, WA) environment. Images with spatial resolutions of up to 2700 x 3400 pixels in 36-bit color, can be displayed simultaneously as distinct images in a montage, or merged into a single composite image file to highlight significant features of a histological or cytological slide. These image files are saved in Joint Photographers Experts Group (JPEG) format using compression ratios of up to 80:1 without detectable visual degradation. The advantages and technical limitations of various workstation components are addressed and applications of this technology for pathology education, proficiency testing, telepathology, and database development are discussed.     

Evaluation of a commercial mammography image-enhancement system

A commercial mammography image-enhancement system manufactured by Damon Corporation (Needham, MA) is evaluated. Using a dedicated computer, the system implements a real-time video local adaptive image processing algorithm based on the Wallis equation. Radiographs of a mammographic QA phantom (Nuclear Associates Model 76-001-4) containing five groups of simulated breast microcalcifications ranging in diameter from 0.12 to 0.35 mm were viewed by four investigators under three viewing conditions: on a light box with the unaided eye, on the image enhancer in magnified "bypass" (unenhanced) mode, and on the enhancer using all features for optimum enhancement. A mammogram was then overlaid on the radiographs, and the composite images were viewed under the same three conditions. Using the enhancer, as compared to using a light box alone, average increases of 1.4 and 1.1 microcalcifications per radiograph were observed for the phantom and phantom-with-mammogram radiographs, respectively. High-contrast resolution and spatial distortion were also measured.