Person:

Yagi, Yukako

Introduction: Standardization and validation of the color displayed by digital slides is an important aspect of digital pathology implementation. While the most common reason for color variation is the variance in the protocols and practices in the histology lab, the color displayed can also be affected by variation in capture parameters (for example, illumination and filters), image processing and display factors in the digital systems themselves.
Method: We have been developing techniques for color validation and optimization along two paths. The first was based on two standard slides that are scanned and displayed by the imaging system in question. In this approach, one slide is embedded with nine filters with colors selected especially for H&E stained slides (looking like tiny Macbeth color chart); the specific color of the nine filters were determined in our previous study and modified for whole slide imaging (WSI). The other slide is an H&E stained mouse embryo. Both of these slides were scanned and the displayed images were compared to a standard. The second approach was based on our previous multispectral imaging research. Discussion: As a first step, the two slide method (above) was used to identify inaccurate display of color and its cause, and to understand the importance of accurate color in digital pathology. We have also improved the multispectral-based algorithm for more consistent results in stain standardization. In near future, the results of the two slide and multispectral techniques can be combined and will be widely available.

We have been conducting a series of researches and developing projects to improve image quality to establish Image Quality Standardization. This paper discusses one of most important aspects of image quality – color.

This study is an initial investigation on the capability of multispectral imaging to capture subtle spectral information that would enable the automatic delineation between the eosinophilic esophagitis and other eosin stained tissue components, especially the RBCs. In the method, a principal component analysis (PCA) was performed on the spectral transmittance samples of the different tissue components, excluding however the transmittance samples of the eosinophilic esophagitis. From the average spectral error configuration of the eosinophilic esophagitis transmittance samples, i.e. the difference between the actual transmittance and the estimated transmittance using m PC vectors, we indentified two spectral bands by which we can localize the eosinophils. Initial results show the possibility of automatically localizing the eosinophilic esophagitis by utilizing spectral information.

Epithelial cancers account for substantial mortality and are an important public health concern. With the need for earlier detection and treatment of these malignancies, the ability to accurately detect precancerous lesions has an increasingly important role in controlling cancer incidence and mortality. New optical technologies are capable of identifying early pathology in tissues or organs in which cancer is known to develop through stages of dysplasia, including the esophagus, colon, pancreas, liver, bladder, and cervix. These diagnostic imaging advances, together as a field known as optical endomicroscopy, are based on confocal microscopy, spectroscopy-based imaging, and optical coherence tomography (OCT), and function as “optical biopsies,” enabling tissue pathology to be imaged in situ and in real time without the need to excise and process specimens as in conventional biopsy and histopathology. Optical biopsy techniques can acquire high-resolution, cross-sectional images of tissue structure on the micron scale through the use of endoscopes, catheters, laparoscopes, and needles. Since the inception of these technologies, dramatic technological advances in accuracy, speed, and functionality have been realized. The current paradigm of optical biopsy, or single-area, point-based images, is slowly shifting to more comprehensive microscopy of larger tracts of mucosa. With the development of Fourier-domain OCT, also known as optical frequency domain imaging or, more recently, volumetric laser endomicroscopy, comprehensive surveillance of the entire distal esophagus is now achievable at speeds that were not possible with conventional OCT technologies. Optical diagnostic technologies are emerging as clinically useful tools with the potential to set a new standard for real-time diagnosis. New imaging techniques enable visualization of high-resolution, cross-sectional images and offer the opportunity to guide biopsy, allowing maximal diagnostic yields and appropriate staging without the limitations and risks inherent with current random biopsy protocols. However, the ability of these techniques to achieve widespread adoption in clinical practice depends on future research designed to improve accuracy and allow real-time data transmission and storage, thereby linking pathology to the treating physician. These imaging advances are expected to eventually offer a see-and-treat paradigm, leading to improved patient care and potential cost reduction.

This study examined the effect of tissue section thickness and consistency – parameters outside the direct control of the imaging devices themselves – on WSI capture speed and image quality. Preliminary data indicates that thinner, more consistent tissue sectioning (such as those produced by automated tissue sectioning robots) result in significantly faster WSI capture times and better image quality. A variety of tissue types (including human breast, mouse embryo, mouse brain, etc.) were sectioned using an (AS-200) Automated Tissue Sectioning System (Kurabo Industries, Osaka Japan) at thicknesses from 2 – 9 μm (at one μm intervals) and stained with H&E by a standard method. The resulting slides were imaged with 5 different WSI devices (ScanScope CS, Aperio, CA, iScan, BioImagene, CA, DX40, DMetrix, AZ, NanoZoomer, Hamamatsu Photonics K.K., Japan, Mirax Scan, Carl Zeiss Inc., Germany) with sampling periods of 0.43 – 0.69 μm/pixel. Slides with different tissue thicknesses were compared for image quality, appropriate number of focus points, and overall scanning speed. Thinner sections (ie 3 μm sections versus 7 μm) required significantly fewer focus points and had significantly lower (10–15%) capture times. Improvement was seen with all devices and tissues tested. Furthermore, a panel of experienced pathologist judged image quality to be significantly better (for example, with better apparent resolution of nucleoli) with the thinner sections. Automated tissue sectioning is a very new technology; however, the AS-200 seems to be able to produce thinner, more consistent, flatter sections than manual methods at reasonably high throughput. The resulting tissue sections seem to be easier for a WSI system's focusing systems to deal with (compared to manually cut slides). Teaming an automated tissue-sectioning device with a WSI device shows promise in producing faster WSI throughput with better image quality.

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 20×, 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.

The objective is to find a practical balance between quality and performance for daily high volume whole slide imaging. We evaluated whole slide images created by various scanners at different compression factors to determine the best suitable quality factor (QF) needed for pathological images of special stains. Method: We scanned two sets of eight special stains slides each at 0.50 μm/pixel resolution in Hamamatsu scanner at six and five QF levels respectively to generate 72 images which were observed at a calibrated monitor by imaging specialists, a histo-technician, and a pathologist to find the most suitable QF level for special stains in digital slides. Results: Most special stains images were acceptable at QF 30 except for the stain Reticulin where the lowest acceptable QF was 50. The compression of images from QF 90 to QF 50 reduced the size of the images by 62.73%. Conclusion: 0.50 μm/pixel images at QF 50 or above were found suitable 12 special stain.

Background:: Three-dimensional (3D)-reconstruction from paraffin embedded sections has been considered laborious and time-consuming. However, the high-resolution images of large object areas and different fields of view obtained by 3D-reconstruction make one wonder whether it can add a new insight into lung adenocarcinoma, the most frequent histology type of lung cancer characterized by its morphological heterogeneity. Objective:: In this work, we tested whether an automated tissue sectioning machine and slide scanning system could generate precise 3D-reconstruction of microanatomy of the lung and help us better understand and define histologic subtypes of lung adenocarcinoma. Methods:: Four formalin-fixed human lung adenocarcinoma resections were studied. Paraffin embedded tissues were sectioned with Kurabo-Automated tissue sectioning machine and serial sections were automatically stained and scanned with a Whole Slide Imaging system. The resulting stacks of images were 3D reconstructed by Pannoramic Viewer software. Results:: Two of the four specimens contained islands of tumor cells detached in alveolar spaces that had not been described in any of the existing adenocarcinoma classifications. 3D-reconstruction revealed the details of spatial distribution and structural interaction of the tumor that could hardly be observed by 2D light microscopy studies. The islands of tumor cells extended into a deeper aspect of the tissue, and were interconnected with each other and with the main tumor with a solid pattern that was surrounded by the islands. The finding raises the question whether the islands of tumor cells should be classified into a solid pattern in the current classification. Conclusion:: The combination of new technologies enabled us to build an effective 3D-reconstruction of resected lung adenocarcinomas. 3D-reconstruction may help us refine the classification of lung adenocarcinoma by adding detailed spatial/structural information to 2D light microscopy evaluation.

In this paper we proposed a multispectral enhancement scheme in which the spectral colors of the stained tissue-structure of interest and its background can be independently modified by the user to further improve their visualization and color discrimination. The colors of the background objects are modified by transforming their N-band spectra through an NxN transformation matrix, which is derived by mapping the representative samples of their original spectra to the spectra of their target colors using least mean square method. On the other hand, the color of the tissue structure of interest is modified by modulating the transformed spectra with the sum of the pixel’s spectral residual-errors at specific bands weighted through an NxN weighting matrix; the spectral error is derived by taking the difference between the pixel’s original spectrum and its reconstructed spectrum using the first M dominant principal component vectors in principal component analysis. Promising results were obtained on the visualization of the collagen fiber and the non-collagen tissue structures, e.g., nuclei, cytoplasm and red blood cells (RBC), in a hematoxylin and eosin (H&E) stained image.

Background:: Digital staining can be considered as a special form of image enhancement wherein the concern is not only to increase the contrast between the background objects and objects of interest, but to also impart the colors that mark the objects’ unique reactions to a specific stain. In this paper, we extended the previously proposed multispectral enhancement methods such that the colors of the background pixels can also be changed. Methods:: In the previous multispectral enhancement methods a shifting factor is provided to the original spectrum. To implement digital staining, a spectral transformation process is introduced prior to spectral shifting. Results:: The enhancement method is applied to multispectral images of H&E stained liver tissue. The resulting digitally stained images show good correlation with the serial-section images of the tissue which are physically stained with Masson's trichrome. Conclusion:: We have presented a multispectral enhancement method that can be adjusted to produce digitally stained-images. The current experimental results show the viability of the method. However, to achieve robust enhancement performance issues that arise from variations in staining conditions has to be addressed as well. This would be part of our future work.

Despite the prognostic importance of mitotic count as one of the components of the Bloom – Richardson grade [3], several studies ([2, 9, 10]) have found that pathologists’ agreement on the mitotic grade is fairly modest. Collecting a set of more than 4,200 candidate mitotic figures, we evaluate pathologists' agreement on individual figures, and train a computerized system for mitosis detection, comparing its performance to the classifications of three pathologists. The system’s and the pathologists’ classifications are based on evaluation of digital micrographs of hematoxylin and eosin stained breast tissue. On figures where the majority of pathologists agree on a classification, we compare the performance of the trained system to that of the individual pathologists. We find that the level of agreement of the pathologists ranges from slight to moderate, with strong biases, and that the system performs competitively in rating the ground truth set. This study is a step towards automatic mitosis count to accelerate a pathologist's work and improve reproducibility.