Cost-effective gel documentation using a web-cam.

In search for a cost effective gel documentation system applicable for different fields of molecular biology, we analyzed the capabilities of a cheap CCD-camera originally designed to capture images for transmission through the internet (web-cam) with regard to gel documentation. The camera was connected to a personal computer by universal serial bus (USB) and used for the documentation of DNA separated on agarose gels and stained by ethidium-bromide using the software provided with the camera. The web-cam provided digital images of sufficient quality for routine documentation and combined the low set-up costs of a Polaroid system with the low running costs of video capture systems, hence is ideal as a start-up system and as augmentation to existing equipment.     

Digital two-dimensional photogrammetry: a comparison of three techniques of obtaining digital photographs.

Three methods of obtaining digital photographs in a clinical setting were compared using direct anthropometry as a reference standard. The methods included a digital camera, scanning negatives from a 35-mm single-lens reflex camera, and scanning Polaroid photographs taken with a Polaroid camera designed for medical documentation. A total of 11 angular and linear anthropometric measurements obtained from 30 healthy volunteers were used for the comparison. The data were analyzed using a variance covariance approach to repeated measures. The analysis revealed that the three cameras were not statistically different from each other. Other advantages and disadvantages, such as cost and ease of use, are discussed.     

Quantifying fungal infection of plant leaves by digital image analysis using Scion Image software

A digital image analysis method previously used to evaluate leaf color changes due to nutritional changes was modified to measure the severity of several foliar fungal diseases. Images captured with a flatbed scanner or digital camera were analyzed with a freely available software package, Scion Image, to measure changes in leaf color caused by fungal sporulation or tissue damage. High correlations were observed between the percent diseased leaf area estimated by Scion Image analysis and the percent diseased leaf area from leaf drawings. These drawings of various foliar diseases came from a disease key previously developed to aid in visual estimation of disease severity. For leaves of Nicotiana benthamiana inoculated with different spore concentrations of the anthracnose fungus Colletotrichum destructivum, a high correlation was found between the percent diseased tissue measured by Scion Image analysis and the number of leaf spots. The method was adapted to quantify percent diseased leaf area ranging from 0 to 90% for anthracnose of lily-of-the-valley, apple scab, powdery mildew of phlox and rust of golden rod. In some cases, the brightness and contrast of the images were adjusted and other modifications were made, but these were standardized for each disease. Detached leaves were used with the flatbed scanner, but a method using attached leaves with a digital camera was also developed to make serial measurements of individual leaves to quantify symptom progression. This was successfully applied to monitor anthracnose on N. benthamiana leaves. Digital image analysis using Scion Image software is a useful tool for quantifying a wide variety of fungal interactions with plant leaves.     

Non-mydriatic Polaroid photography in screening for diabetic retinopathy: evaluation in a clinical setting

Because of fears that Polaroid colour prints produced with a non-mydriatic fundus camera may not detect important sight threatening lesions in diabetes a study was conducted comparing retinal images obtained on Polaroid prints taken in “field” conditions with those on 35 mm transparencies and fluorescein angiograms. Almost one in five (22/127) Polaroid prints could not be assessed owing to poor quality compared with 3 (2.4%) 35 mm transparencies and 2 (1.6%) fluorescein angiograms. The pick up rate of microaneurysms, haemorrhages, and hard and soft (cotton wool spots) exudates was equivalent for Polaroid prints and 35 mm transparencies of equivalent quality. In two cases with disc new vessels, however, these were not seen on the Polaroid prints.The widespread use of Polaroid colour prints obtained with a non-mydriatic camera without the necessary operative and interpretive skills further limits the usefulness of the technique.     

Automated identification of tubercle bacilli in sputum. A preliminary investigation

OBJECTIVE: To use an automated method to detect tubercle bacilli in sputum specimens. STUDY DESIGN: Using fluorescence microscopy, tubercle bacilli were identified on auramine-stained sputum specimens. Images were then captured with a digital camera and enhanced through imaging processing techniques. The bacilli were recognized using neural network classifiers. RESULTS: This preliminary investigation demonstrated a sensitivity of 94.1% for the identification of individual bacilli. As there are usually fairly numerous tubercle bacilli in the sputum of patients with active pulmonary tuberculosis, the overall diagnostic accuracy of sputum smear-positive patients can be expected to be very high. CONCLUSION: Potential benefits of automated screening for TB bacilli are: rapid, accurate, inexpensive diagnosis; the ability to screen larger numbers of people; increased resources to monitor patients; and reduction in health risks to staff.     

Portable system for microbial sample preparation and oligonucleotide microarray analysis

We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.     

Portable System for Microbial Sample Preparation and Oligonucleotide Microarray Analysis

We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.     

The importance of optical optimization in whole slide imaging (WSI) and digital pathology imaging

In the last 10 years, whole slide imaging (WSI) has seen impressive progress not only in image quality and scanning speed but also in the variety of systems available to pathologists. However, we have noticed that most systems have relatively simple optics axes and rely on software to optimize image quality and colour balance. While much can be done in software, this study examines the importance of optics, in particular optical filters, in WSI.Optical resolution is a function of the wavelength of light used and the numerical aperture of the lens system (Resolution = (f) wavelength/2 NA). When illumining light is not conditioned correctly with filters, there is a tendency for the wavelength to shift to longer values (more red) because of the characteristics of the lamps in common use. Most microscopes (but remarkably few WSI devices) correct for this with ND filter for brightness and Blue filter (depends on the light source) for colour correction.Using H&E slides research microscopes (Axiophot, Carl Zeiss MicroImaging, Inc. NY. Eclipse 50i., Nikon Inc. NY) at 20x, an attached digital camera (SPOT RT741 Slider Color, Diagnosis Instruments., MI USA), and a filter set, we examined the effect of filters and software enhancement on digital image quality. The focus value (as evaluated by focus evaluation software developed in house and SPOT imaging Software v4.6) was used as a proxy for image quality. Resolution of tissue features was best with the use of both the Blue and ND filters (in addition to software enhancement). Images without filters but with software enhancement while superficially good, lacked some details of specimen morphology and were unclear compared with the images with filters.The results indicate that the appropriate use of optical filters could measurably improve the appearance and resolution of WSI images.     

Imaging the propagation of viruses

The propagation of viruses in a growing plaque has been measured using a digital image acquisition and analysis system. Plaques of phage T7 incubated at 37 degrees C and illuminated against a dark field emerged as dark growing spots against a background of host bacteria. Images of the growth were acquired using a charge-coupled device (CCD) camera at 1-h intervals over 24 h. The first 10 h of plaque development coincided with rapid growth of the agar-immobilized Escherichia coli host, measured as a reduction in gray value. Following this period, the average radial velocity of plaque growth remained constant at 0.059 mm/h while the standard deviation about this velocity increased. These results suggest the suitability of the system for spatially resolving the dynamics of viral evolution during plaque growth. (c) 1996 John Wiley & Sons, Inc.     

Comparative 2-DE protein analysis in a 3-D geometry gel

Two-dimensional gel electrophoresis (2-DE) separation has not been considered suitable for large-scale comparative protein expression studies due to its limited throughput. We present a high-throughput analysis method based on three-dimensional (3-D) geometry gel electrophoresis. Following conventional isoelectric focusing (IEF), up to 36 immobilized pH gradient (IPG) strips are arrayed on the top surface of a 3-D gel body, and the samples transferred electrokinetically to the gel. A specific thermal management ensures that sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) occurs under identical electrophoretic and thermal conditions, avoiding gel-to-gel variations and thereby providing immediate comparability of the separation patterns. Proteins are Cy3-labeled for online detection of laser-induced fluorescence (LIF). Images are acquired by a digital camera and recorded as a 3-D image stack during electrophoresis. Image processing software decomposes the 3-D image stack into vertical sections representing conventional 2-DE slab gels, making results immediately accessible without further gel processing. The large number of simultaneously analyzed samples (n = 36) allows treating the sample index as a quasi-continuous experimental parameter (e.g., concentration, time, dose). The method offers a wide range of applications in molecular discovery, clinical diagnosis, pharmacology, and toxicology, like protein monitoring during disease development and screening of drug candidates for their effect on protein expression.     

Chemiluminescent detection of DNA: application for DNA sequencing and hybridization.

A non-radioactive DNA detection chemistry is described and its application is shown for DNA hybridization and standard dideoxy DNA sequencing. The method employes a biotin-streptavidin system which binds an enzyme specifically to a target DNA and upon exposure to substrate, the enzyme catalyzes a chemiluminescent reaction. The image is captured within seconds by a Polaroid or X-ray film. The method is capable of detecting DNA in the hundred attomol range.     

Measurement of green fluorescent protein concentration in single cells by image analysis

The gene encoding the green fluorescent protein (GFP) has been widely used in studies of gene expression. The GFP can be detected nondestructively in living cells or tissues by the green fluorescence of the protein under blue light. Solutions of enhanced GFP (EGFP) of known concentration were filled in glass capillaries and used to calibrate a method for quantitative determination of EGFP or GFP-S65T in plant cells. Images captured by a digital camera were analyzed to determine the linear range for measurement of EGFP expression. The value of the method was illustrated by analysis of the relative levels of GFP expression under control of different promoters in aleurone cells of barley.     

Ground-level spectroscopy analyses and classification of coral reefs using a hyperspectral camera

With the general aim of classification and mapping of coral reefs, remote sensing has traditionally been more difficult to implement in comparison with terrestrial equivalents. Images used for the marine environment suffer from environmental limitation (water absorption, scattering, and glint); sensor-related limitations (spectral and spatial resolution); and habitat limitation (substrate spectral similarity). Presented here is an advanced approach for ground-level surveying of a coral reef using a hyperspectral camera (400–1,000 nm) that is able to address all of these limitations. Used from the surface, the image includes a white reference plate that offers a solution for correcting the water column effect. The imaging system produces millimeter size pixels and 80 relevant bands. The data collected have the advantages of both a field point spectrometer (hyperspectral resolution) and a digital camera (spatial resolution). Finally, the availability of pure pixel imagery significantly improves the potential for substrate recognition in comparison with traditionally used remote sensing mixed pixels. In this study, an image of a coral reef table in the Gulf of Aqaba, Red Sea, was classified, demonstrating the benefits of this technology for the first time. Preprocessing includes testing of two normalization approaches, three spectral resolutions, and two spectral ranges. Trained classification was performed using support vector machine that was manually trained and tested against a digital image that provided empirical verification. For the classification of 5 core classes, the best results were achieved using a combination of a 450–660 nm spectral range, 5 nm wide bands, and the employment of red-band normalization. Overall classification accuracy was improved from 86 % for the original image to 99 % for the normalized image. Spectral resolution and spectral ranges seemed to have a limited effect on the classification accuracy. The proposed methodology and the use of automatic classification procedures can be successfully applied for reef survey and monitoring and even upscaled for a large survey.     

Proliferation and apoptosis measurements by color image analysis based on differential absorption.

Cell proliferation and apoptosis indices are important indicators for the prognosis and treatment of a variety of cancers. A method is described using differential absorption color image analysis to measure proliferation and apoptosis in tumor sections using BrdU (5' bromodeoxyuridine) incorporation and immunohistochemistry and terminal deoxytransferase nick end-labeling (TUNEL). Nuclei were labeled with streptavidin-peroxidase-diaminobenzidine (DAB) secondary detection. The differential absorption method uses a computer-controlled microscope equipped with a tunable filter and digital camera to take advantage of the spectral differences of stained objects of interest. Images collected at defined wavelengths are divided and scaled to form ratio images in which the hematoxylin- or DAB-stained nuclei have intensity ranges far above those of surrounding structures. Using brightness thresholding followed by selection based on nuclear size and shape parameters, binary images were formed of the BrdU/apoptotic-positive tumor and all the tumor nuclei for subsequent counting and calculations of proliferation and apoptotic indices.     

Natural images from the birthplace of the human eye

Here we introduce a database of calibrated natural images publicly available through an easy-to-use web interface. Using a Nikon D70 digital SLR camera, we acquired about six-megapixel images of Okavango Delta of Botswana, a tropical savanna habitat similar to where the human eye is thought to have evolved. Some sequences of images were captured unsystematically while following a baboon troop, while others were designed to vary a single parameter such as aperture, object distance, time of day or position on the horizon. Images are available in the raw RGB format and in grayscale. Images are also available in units relevant to the physiology of human cone photoreceptors, where pixel values represent the expected number of photoisomerizations per second for cones sensitive to long (L), medium (M) and short (S) wavelengths. This database is distributed under a Creative Commons Attribution-Noncommercial Unported license to facilitate research in computer vision, psychophysics of perception, and visual neuroscience.     

Combined confocal and wide-field high-resolution cytometry of fluorescent in situ hybridization-stained cells

BACKGROUND: The recently developed technique of high-resolution cytometry (HRCM) enables automated acquisition and analysis of fluorescent in situ hybridization (FISH)-stained cell nuclei using conventional wide-field fluorescence microscopy. The method has now been extended to confocal imaging and offers the opportunity to combine the advantages of confocal and wide-field modes. METHODS: We have automated image acquisition and analysis from a standard inverted fluorescence microscope equipped with a confocal module with Nipkow disk and a cooled digital CCD camera. The system is fully controlled by a high-performance computer that performs both acquisition and related on-line image analysis. The system can be used either for an automatic two (2D) and three-dimensional (3D) analysis of FISH- stained interphase nuclei or for a semiautomatic 3D analysis of FISH-stained cells in tissues. The user can select which fluorochromes are acquired using wide-field mode and which using confocal mode. The wide-field and confocal images are overlaid automatically in computer memory. The developed software compensates automatically for both chromatic color shifts and spatial shifts caused by switching to a different imaging mode. RESULTS: Using the combined confocal and wide-field HRCM technique, it is possible to take advantage of both imaging modes. Images of some dyes (such as small hybridization dots or counterstain images of individual interphase nuclei) do not require confocal quality and can be acquired quickly in wide-field mode. On the contrary, images of other dyes (such as chromosome territories or counterstain images of cells in tissues) do require improved quality and are acquired in confocal mode. The dual-mode approach is two to three times faster compared with the single-mode confocal approach and the spectrum of its applications is much broader compared with both single-mode confocal and single-mode wide-field systems. CONCLUSIONS: The combination of high speed specific to the wide-field mode and high quality specific to the confocal mode gives optimal system performance.     

Standardizing digital photography: it's not all in the eye of the beholder

Advances in digital photography have made it an efficient and economically appealing alternative to conventional photography. Nevertheless, as objective observers and clinical photographers, we must realize that all digital cameras are not created equal. Different digital cameras frequently used in plastic surgery practices (Olympus 600DL, Olympus 2500, Sony DSC-D700, Nikon Coolpix 950, and Nikon D1) were evaluated, using a subject photographed with each camera in the identical lighting conditions, to determine inherent differences in quality, color, and contrast of the resultant photographs. Three different lighting conditions were examined: single soft-box lighting, dual studio flash boxes, and operating room lighting with on-camera flash. The same digital settings (program mode, ISO camera default setting, high quality setting with JPEG compression) were used. Each camera was digitally color balanced using an 18 percent gray card. Raw and color-balanced images were viewed side-by-side. The macro-image capabilities of each camera were also examined. Conventional 35-mm photographs using a 105 macro-lens on Kodachrome and Ektachrome slide film were obtained for comparison. All of the digital cameras performed with noticeable differences, but they maintained consistency in the three different lighting conditions. Digital photographs differed most greatly with respect to quality and contrast, which was especially obvious once color balancing was performed. Marked differences in quality and ability were observed with respect to macro-image capabilities. Inherent differences in features among digital cameras produce dramatically different photographic results with regard to color, contrast, focus, and overall quality. With the increasing use of digital photography in plastic surgery journals and presentations, it must be recognized that digital cameras do not all display photographs of similar quality, especially when used to evaluate skin appearance. To standardize digital photography, the surgeon must realize that switching digital cameras is akin to switching film types. Standardization of digital photographs should include image resolution between 1.5 and 2.7 million pixels, ISO default setting, color balancing with an 18 percent gray card and software, consistency in focal distance, JPEG compression of medium-to-high quality, and backgrounds of medium blue or 18 percent gray.     

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.     

Control of a Thiobacillus denitrificans bioreactor using machine vision

A PC-based machine vision system has been used to continuously monitor changes in biomass concentration and to control the undesirable production of colloidal elemental sulfer (a reactor upset condition due to an excessive concentration of inhibitory sulfide substrate) in a bioreactor containing Thiobacillus denitrificans. A field of view of a video camera was established which contained regions of different background lighting. Mean values of the distribution of red, green, and blue intensity components within corresponding regions of a digital image image captured from the camera were used to monitr color changes associated with changes in biomass concentration, and to determine if the reactor was in an upset condition. The ration of red to blue intensity components was an important parameter in detecting the formatin of an elemental sulfur precipitant. Using a stepper motor-driven pressure regulator, intelligent process control was performed by altering the hydrogen sulfide feed flow rate setpoint on the vision system measurements.     

A red line not to cross: Evaluating the limitation and properness of gel image tuning procedures

Currently, results of gel electrophoresis are commonly documented in digital formats by image acquisition instruments. In this study, gel images tuned by a common image processing software package, Photoshop, were assessed to understand the transforming algorithms and their impacts on quantitative analysis. TotalLab 100, an electrophoresis gel image analysis software package, was applied for image quantitation and evaluation. The three most frequently used image tuning functions—adjustments of the brightness, contrast, and grayscale span (level) of images—were investigated using both data generated from a standard grayscale tablet and an actual electrophoresis gel image. The influences of these procedures were analyzed for the grayscale transformation between the input and output images. Although all three procedures differentially improved the visualization of the input image, adjusting the contrast of images disrupted the quantitative information because of its nonlinear transforming algorithm. Under certain conditions, adjusting the brightness or the level of images could preserve the quantitative information because of the linear transforming algorithms. It was found that when the minimum and maximum grayscales of a gel image were recognized, using a commercial software package to maximally stretch the level may significantly improve the quality of a gel image without jeopardizing quantitative analysis.