Correction of uneven illumination (vignetting) in digital microscopy images

BACKGROUND: Many digital microscopy images suffer from poor illumination at the peripheries (vignetting), often attributable to factors related to the light path between the camera and the microscope. A commonly used method for correcting this has been to use the illumination of an empty field as a correction filter (white shading correction). AIMS: To develop an alternative shading correction method that does not require this additional image. METHODS:/Results: This report outlines an alternative shading correction method that is based upon the intrinsic properties of the image, which are revealed through Gaussian smoothing. The technique can be implemented within Adobe Photoshop or other graphics editing software and works well with both haematoxylin and eosin and immunohistochemical images. CONCLUSIONS: This technique provides an effective means of optimising digital microscopy image appearances for printing, image analysis, or telepathology.     

How to improve microscopic images obtained with consumer-type digital cameras

AIM: Digital imaging is useful in conventional photography because it immediately provides images, and the image quality can be improved afterwards by the use of computer programs. The major disadvantages of consumer-type digital cameras mounted on microscopes are (i) unequal illumination through the image, and (ii) a coloured background. A computer program was specifically adapted and refined to improve images obtained with consumer-type digital cameras mounted on microscopes. METHODS AND RESULTS: An approach using a division operation between the specimen image and a background image leads to homogeneous illumination throughout the image, with automatically corrected brightness and white background. The correct colour spectrum is preserved by correction of the histogram. This approach was obtained from the freeware computer program 'Image Arithmetic'. In a test, three different consumer-type digital cameras (Sony, Nikon, Olympus) on different microscopes were used to obtain images of different types of histological specimens (cervical smear, bone marrow biopsy, and colonic biopsy). The computer program dramatically improved the quality of images obtained with all tested cameras. CONCLUSION: Using this approach, even low-cost digital cameras mounted on microscopes produce brilliant images with homogeneous illumination and a white background, the image quality being comparable with expensive cameras especially designed for microscopes.     

Effectiveness of colour in displaying radionuclide images

The use of digital technology in medical imaging gives the potential for improving the way in which images are displayed. One possibility is to represent intensity information by false colours rather than the conventional grey shades. Although this has several potential advantages, it has not been widely accepted. The aim of this study was to compare the performance of grey scale coding with three colour scales: the hot-body scale, a blue-green-red scale derived from the Uniform Chromaticity Scale, and a contrasting-colour scale. Two perceptual tasks were studied using computer-simulated images of the type produced in nuclear medicine. The first was to test the ability to perceive changes in image sharpness. The grey scale, hot-body scale and blue-green-red scale performed equally well but the contrasting-colour scale was significantly worse than others. The second pattern measured the ability to detect small changes in image intensity. The contrasting colour scale was best for this task and the grey scale worst. While the best colour scale depends on the information which is to be extracted from the image, both the hot-body scale and the blue-green-red scale appeared to offer a satisfactory compromise.     

Creating low-power photomicrographs by digital scanning of histological sections

The recent introduction of digital film scanners in the medical-scientific field provided everyone dealing with light-microscopy an easy method to obtain low-power photomicrographs from entire histological sections. Direct scanning of the common microscope slide allows the easy creation of high quality images. The digital images obtained may be improved or retouched using dedicated software and then printed on paper or film. The required instrumentation is relatively unexpensive and neither skilled staff nor expertise of photographic techniques is required. This reduces costs and saves time. The obtained images may be employed in histopathology, embryology, histochemistry, image analysis and telepathology, and are suitable for scientific papers, educational purposes and lecturing, as well as for daily reporting. The authors bring their personal experience in this field, making also a complete literature review on the topic.     

Improved digital SEM of cancellous bone: scanning direction of detection,through focus for in-focus and sample orientation

Three-dimensional (3D) study of cancellous bone tissue organization is necessary to understand how modelling and remodelling processes regulate bone structure and connectivity. It requires imaging methods that have both sufficient resolution power and width and depth of field. Since clinical imaging methods fall far short of the first requirement, we can only study prepared tissue in isolation from the body. Scanning electron microscopy (SEM) of macerated plane parallel slices is the most productive method, but we meet special technical problems in imaging porous bone because samples need to be relatively thick to maintain both continuity and context. Problems due to charging under the electron beam can be controlled by imaging with only high-energy backscattered electrons (BSE). This gives an important additional benefit that the direction of apparent illumination can be manipulated by positioning the detector, and multiple detector positions can be employed strategically to generate images in which colour is used to help in coding surface morphology. However, we next confront the difficulty of the limited depth of field. This can be improved by taking series of images, moving the sample along the electron optic axis, and combining these to generate a single extended-focus image. SEM imaging geometry gives a change in magnification with change of working distance, and it is shown that this must be corrected for each image of the through-focus sequence. Colour coding the lighting direction and increasing the depth of field are approaches that can be combined, and are well matched to the possibilities offered by communication by digital data projection. Finally, the latter means also offer another powerful technique for 3D representation through the display of through tilt image sequences. The novel routines considered here are generally applicable to all classes of microanatomical SEM sample.     

A plating method for preparation of cells for culture and for observation by light or electron microscopy

A plating technique that makes use of small volumes of cell suspensions (1–10 μl) is described. Its purpose is to permit microscopic examination or propagation in culture of the entire contents of the microdroplet. Cells are allowed to settle by gravity on Millipore filters where they rapidly and firmly attach after 20 minutes of incubation at 37°C. Such cells can be used for long- or short-term cultures and can be processed for light microscopy and transmission or scanning electron microscopy. The method is particularly useful for preparing cell suspensions for scanning electron microscopy since it is possible to match light photomicrographs with scanning electron micrographs of identical cells.     

Digital Radiographic Image Denoising Via Wavelet-Based Hidden Markov Model Estimation

This paper presents a technique for denoising digital radiographic images based upon the wavelet-domain Hidden Markov tree (HMT) model. The method uses the Anscombes transformation to adjust the original image, corrupted by Poisson noise, to a Gaussian noise model. The image is then decomposed in different subbands of frequency and orientation responses using the dual-tree complex wavelet transform, and the HMT is used to model the marginal distribution of the wavelet coefficients. Two different correction functions were used to shrink the wavelet coefficients. Finally, the modified wavelet coefficients are transformed back into the original domain to get the denoised image. Fifteen radiographic images of extremities along with images of a hand, a line-pair, and contrast–detail phantoms were analyzed. Quantitative and qualitative assessment showed that the proposed algorithm outperforms the traditional Gaussian filter in terms of noise reduction, quality of details, and bone sharpness. In some images, the proposed algorithm introduced some undesirable artifacts near the edges.     

Measurement of cone beam CT coincidence with megavoltage isocentre and image sharpness using the QUASAR Penta-Guide phantom

For image-guided radiotherapy (IGRT) systems based on cone beam CT (CBCT) integrated into a linear accelerator, the reproducible alignment of imager to x-ray source is critical to the registration of both the x-ray-volumetric image with the megavoltage (MV) beam isocentre and image sharpness. An enhanced method of determining the CBCT to MV isocentre alignment using the QUASARtrade mark Penta-Guide phantom was developed which improved both precision and accuracy. This was benchmarked against our existing method which used software and a ball-bearing (BB) phantom provided by Elekta. Additionally, a method of measuring an image sharpness metric (MTF(50)) from the edge response function of a spherical air cavity within the Penta-Guide phantom was developed and its sensitivity was tested by simulating misalignments of the kV imager. Reproducibility testing of the enhanced Penta-Guide method demonstrated a systematic error of <0.2 mm when compared to the BB method with near equivalent random error (s = 0.15 mm). The mean MTF(50) for five measurements was 0.278 +/- 0.004 lp mm(-1) with no applied misalignment. Simulated misalignments exhibited a clear peak in the MTF(50) enabling misalignments greater than 0.4 mm to be detected. The Penta-Guide phantom can be used to precisely measure CBCT-MV coincidence and image sharpness on CBCT-IGRT systems.     

Automatic analysis of immunocytochemically stained tissue samples

An automatic colour image segmentation and cell counting software system has been developed for immunocytochemical analysis of stained tissue samples. The system was designed to count the total number of positive and negative cells in tissue samples treated with cytokine DNA probes from pigs naturally parasitised with Taenia solium metacestodes, using in situ hybridisation. A reaction index was calculated as the ratio of the number of cells with a positive reaction to the total number of cells (positives plus negatives) for each of five different probes. The objectives of automatic counting were to improve the reproducibility of the analysis and reduce the processing time of large image batches. A fast KNN classifier was used for colour segmentation. Watershed segmentation combined with edge detection was used to isolate individual cells that were then automatically labelled, using the results of the corresponding colour segmented image. Validation was performed on 122 non-training digital images with a total of 1069 positive cells and 1459 negative cells, with the following results: a mean true positive rate of 90.2% for positive cells and a mean true positive rate of 85.4% for negative cells. The corresponding mean false positive rates were 9.6% and 6.6%. The mean reaction index error of the automatic analysis was 5.35%. The processing of each digital image took 10 s on a Pentium IV PC.     

Colour in digital pathology: a review

Colour is central to the practice of pathology because of the use of coloured histochemical and immunohistochemical stains to visualize tissue features. Our reliance upon histochemical stains and light microscopy has evolved alongside a wide variation in slide colour, with little investigation into the implications of colour variation. However, the introduction of the digital microscope and whole‐slide imaging has highlighted the need for further understanding and control of colour. This is because the digitization process itself introduces further colour variation which may affect diagnosis, and image analysis algorithms often use colour or intensity measures to detect or measure tissue features. The US Food and Drug Administration have released recent guidance stating the need to develop a method of controlling colour reproduction throughout the digitization process in whole‐slide imaging for primary diagnostic use. This comprehensive review introduces applied basic colour physics and colour interpretation by the human visual system, before discussing the importance of colour in pathology. The process of colour calibration and its application to pathology are also included, as well as a summary of the current guidelines and recommendations regarding colour in digital pathology.     

基于TDICCD的动态成像实验

目的延迟积分型电荷耦合器件（time delay integration and charge coupled device,TDI CCD）灵敏度高,并在低照度下具有获得高质量图像的特点,已成为齿科全景数字化成像系统开发的关键技术。在成像过程中要想得到清晰图像,必须正确使用TDI CCD。方法本文采用FCFR-USB9825B系列采集卡等搭建的动态成像装置进行实验,通过改变TDI CCD积分方向和积分时间,采集到不同图像。结果通过对比图像,发现在TDI CCD使用过程中,TDI CCD的积分方向必须与成像物体移动方向保持一致。TDI CCD积分时间与物体移动速度必须匹配,即二者的乘积等于像元尺寸。结论本实验为基于TDI CCD的全景齿科成像奠定了成像理论基础。     

Application of Image Compression Ratio Analysis as a Method for Quantifying Complexity of Dental Enamel Microstructure

It is well recognized that enamel microanatomy in mammals reflects biomechanical demands placed upon teeth, as determined by mechanical properties of species' diets, use of teeth as weapons, and so forth. However, there are limited options for researchers wishing to perform large-scale comparisons of enamel microstructure with adaptive questions in mind. This is because to date there has been no efficient method for quantification and statistical analysis of enamel complexity. Our study proposes to apply a method previously developed for quantification of 3D tooth cusp morphology to the problem of quantifying microstructural enamel complexity. Here, we use image compression ratio (ICR) as a proxy variable for enamel complexity in 2D enamel photomicrographs taken using circularly polarized transmitted light microscopy. ICR describes the relationship between a digital image captured in an uncompressed file format and the identical image that has had its file size compressed using computer algorithms; more complex images receive less compression. In our analyses, ICR analysis is able to distinguish between images of teeth with simple, radial enamel and teeth with complex decussating enamel. Moreover, our results show a significant correlation between ICR and enamel complexity ranks assigned via visual assessment. Therefore, our results demonstrate that ICR analysis provides a viable methodology for efficient comparison of overall enamel complexity among dental samples. Anat Rec, 302:2279–2286, 2019. © 2019 American Association for Anatomy     

From the protein to the graph: how to quantify immunohistochemistry staining of the skin using digital imaging

Quantitative immunohistochemistry is needed in order to reliably and accurately assess the expression of cellular proteins in tissue. Skin is a difficult tissue for automated image analysis due to its heterogeneous composition and its architecture. In the present study we used a psoriatic skin model to compare the expression of p53 and bcl-2 before and after treatment with anti-tumor necrosis factor-alpha using digital image analysis. Digital photomicrographs were acquired and analyzed with Scion image software in order to obtain the fraction of p53 and bcl-2 immunoreactive cells' area out of the total area investigated. Statistical analysis with ANOVA revealed a significant increase of p53 expression and a decrease of bcl-2 expression in all 3 epidermal layers during the course of therapy (p<0.001). The results were in line with the conventional histopathological evaluation using an arbitrary scale to grade the extent and intensity of the staining. So, the estimation of volume fraction of immunohistochemically labelled cells in skin tissue can be performed easily and rapidly using commonly available image analysis software and provides reproducible and unbiased numerical estimations of the amount of cell labelling.     

Using a tungsten rollbar to characterize the source spot of a megavoltage bremsstrahlung linac

In photon teletherapy, the size and functional form of the photon source spot affect both the sharpness of the penumbra of treatment fields and the sharpness of portal images. Photon source spot parameters are also used in photon teletherapy dose calculation codes. A simple method for characterizing the source spot would complement the existing, more involved methods that have been described in the medical physics literature. Such a method, using a rollbar made of tungsten or other high-Z metal, is used in industrial radiography. We describe the use of a tungsten rollbar for characterizing the source spot edge spread function (and thereby the source spot size and shape) of a megavoltage bremsstrahlung photon source. We use Monte Carlo simulations to quantify anticipated experimental artifacts of the method, assuming typical spot sizes for circ-function, Gaussian, and Bennett line shapes. We illustrate the use of the rollbar method by characterizing the source spot of a typical 9 MV linac used for industrial radiography. The source spot is analyzed using two approaches: (a) fitting the rollbar image with analytic functions and (b) using Abel inversion to obtain the cylindrically symmetric spot profile consistent with the measured rollbar image. Monte Carlo simulations, based on a 6 MV photon teletherapy accelerator, suggest that aspects of the method are applicable to medical bremsstrahlung sources.     

Virtual microscopy and the Internet as telepathology consultation tools. A study of gastrointestinal biopsy specimens.

Telepathology (TP) is the practice of pathology at a distance using videomicroscopy and telecommunication tools. We explore the use of "virtual microscopy" techniques and the Internet as tools for TP gastrointestinal biopsy consultations. Thirty-five gastrointestinal biopsy specimens were photographed in Los Angeles by using a high-resolution digital camera, a light microscope, and a Pentium 166 microcomputer. Several (2-8) digital photomicrographs were collected at 40x or 100x optical magnification, using 2,700 x 3,400 pixel resolution. The photomicrographs illustrated all the tissue fragments present in 1 of the biopsy levels. They were saved in medium compression JPEG image format. These images can be magnified digitally up to 600% without visible degradation and scrolled at different magnifications on a video monitor, simulating examination under a light microscope. The images files (281 to 3,324 KB) were attached to e-mail messages containing patient information and sent through the Internet to Michigan for interpretation using a Power Macintosh 7100 system. The e-mail process was successful in 100% of instances; 2 files were corrupted owing to user error and had to be resent. Additional photos were requested in 1 case. In 33 of 35 cases, there was diagnostic concordance between the original and the TP diagnoses. The 2 discrepancies were due to diagnostic disagreement. This technology offers pathologists relatively inexpensive and effective tools for gastrointestinal TP consultations.     

A novel image quality index using Moran I statistics

Measurement of image quality is very important for various applications such as image compression, restoration and enhancement. Conventional methods (e.g., mean squared error; MSE) use error summation to measure quality change pixel by pixel and do not correlate well with subjective quality measurement. This is due to the fact that human eyes extract structural information from the viewing field. In this study a new quality index using a Moran I statistics is proposed. The Moran statistic that measures the sharpness from a local area is a good index of quality as most image processing techniques alter the smoothness of the image. Preliminary results show that the new quality index outperforms the MSE significantly under various types of image distortions.     

Determining the Sharpness of Electronic Image Displays: An Evaluation of Three Methods

The authors evaluated 3 methods developed to assess the level of monitor cathode ray tube (CRT) sharpness. Results include a comparison of 2 observer-based methods to a charged coupled device (CCD) digital camera-based method for the purposes of CRT equipment comparison, acceptance testing, and routine CRT quality control. Three methods designed to measure a monitor's sharpness were evaluated on a single 20-inch CRT monitor. We defined signal-to-noise ratio (SNR) to be the overall signal difference measured by each method from the highest to lowest values divided by the average standard deviation of the measurements. Comparing the results of the 3 methods, the authors found that the digital CCD camera-based method provided a much higher SNR than the 2 observer-based methods and, therefore, is the preferred of the 3 methods for measuring the sharpness of CRT displays. The SNR values for the CCD, Cx and line target methods were 151.5, 4.9, and 4.3, respectively. The Cx target observer-based method (a novel target and scoring routine dubbed the "Cx" target because of its appearance) had a higher SNR than the line target observer-based method. The average time and standard deviation required to score the Cx and the line targets were 5.45 +/- 2.15 minutes and 8.34 +/- 2.95 minutes, respectively. The observer-based method results (and variability) versus the camera-based method results (and variability) indicate strong linear relationships. Exploring this finding and the optimization of the camera-based method are the subjects of future research.     

Cephalometry with a video display computer

A new technique to develop cephalometric analyses for orthodontic problems is illustrated. A digital analyzer of images is employed in our laboratory, and this allows us to get the X-ray picture digitized and displayed on both a black and white and a colour TV screen. All the cephalometric points are selected by an operator who watches a coloured cross which can be moved on the colour TV screen by means of a keyboard. A suitable software was developed to calculate linear and angular measurements. The cephalometric coloured lines appear at the top of the X-ray image and the data are displayed on a monitor, from which a print-out is obtained. Such a technique has some advantages: the X-ray image can be processed before the cephalometric analysis to enhance details, thus making the analysis more accurate; the cephalometric lines can be seen on the X-ray image displayed on the colour TV screen and can be photographed or stored on floppy or rigid disc.     

Color Error in the Digital Camera Image Capture Process

The color error in images taken by digital cameras is evaluated with respect to its sensitivity to the image capture conditions. A parametric study was conducted to investigate the dependence of image color error on camera technology, illumination spectra, and lighting uniformity. The measurement conditions were selected to simulate the variation that might be expected in typical telemedicine situations. Substantial color errors were observed, depending on the measurement conditions. Several image post-processing methods were also investigated for their effectiveness in reducing the color errors. The results of this study quantify the level of color error that may occur in the digital camera image capture process, and provide guidance for improving the color accuracy through appropriate changes in that process and in post-processing.     

The shift-effect in the lateral geniculate body of the rhesus monkey

Summary Responses to sudden shifts of a pattern far away from the receptive field (shift-effect) were found in 73 out of 81 cells of the ventral, magnocellular LGN layers, but in only 26 out of 85 cells of the dorsal, parvocellular layers. Most of the former responses were clear and excitatory, and most of the latter were weak and inhibitory. Excitatory responses were stronger for steady receptive field illumination which when turned on also yielded an excitation. No convincing dependence on the colour of the receptive field illumination was observed. The results are discussed with respect to the hypothesis of transmission of steady illumination via the shift-effect (restoration), and with respect to a hypothesis assigning a signalling function of low stimulus specificity to the ventral layers.