Color standardization and optimization in Whole Slide Imaging

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.

     

基于中分辨率TM数据的湿地水生植被提取

利用湿地水生植被生长旺盛、光谱反射较强、光谱信息比较丰富的8月份中分辨率Landsat TM和ETM+多光谱遥感影像,采用面向对象的分类方法,进行野鸭湖湿地水生植被的提取。研究表明:在提取过程中,通过对原始影像进行主成分变换和穗帽变换,将主要信息与噪声分离,不仅减小了数据冗余和波段间的相关性,而且增大了影像上湿地水生植被与其他地物类型光谱和空间信息的差异性,并结合野外水生植被光谱特征分析,选择归一化植被指数NDVI与归一化水体指数NDWI辅助分类,构建特征波段或波段组合,然后,确定适当的隶属度函数和阈值范围,构建分类决策树,完成湿地水生植被的自动分类,提高了影像分割与面向对象分类的精度,取得了较为理想的湿地水生植被提取结果。2002年和2008年两景影像的总体分类精度分别达到86.5%和85.44%,表明中分辨率TM影像可以满足湿地水生植被提取的需要,又因为其具有较高的波谱分辨率、极为丰富的信息量、相对较低的价格、长时间序列,可以作为近20a湿地水生植被提取和动态变化监测的主要数据源。     

Graphical methods for quantifying macromolecules through bright field imaging

Bright field imaging of biological samples stained with antibodies and/or special stains provides a rapid protocol for visualizing various macromolecules. However, this method of sample staining and imaging is rarely employed for direct quantitative analysis due to variations in sample fixations, ambiguities introduced by color composition and the limited dynamic range of imaging instruments. We demonstrate that, through the decomposition of color signals, staining can be scored on a cell-by-cell basis. We have applied our method to fibroblasts grown from histologically normal breast tissue biopsies obtained from two distinct populations. Initially, nuclear regions are segmented through conversion of color images into gray scale, and detection of dark elliptic features. Subsequently, the strength of staining is quantified by a color decomposition model that is optimized by a graph cut algorithm. In rare cases where nuclear signal is significantly altered as a result of sample preparation, nuclear segmentation can be validated and corrected. Finally, segmented stained patterns are associated with each nuclear region following region-based tessellation. Compared to classical non-negative matrix factorization, proposed method: (i) improves color decomposition, (ii) has a better noise immunity, (iii) is more invariant to initial conditions and (iv) has a superior computing performance.     

Differentiation of Wheat Diseases and Pests Based on Hyperspectral Imaging Technology with a Few Specific Bands

Hyperspectral imaging technique is known as a promising non-destructive way for detecting plants diseases and pests. In most previous studies, the utilization of the whole spectrum or a large number of bands as well as the complexity of model structure severely hampers the application of the technique in practice. If a detection system can be established with a few bands and a relatively simple logic, it would be of great significance for application. This study established a method for identifying and discriminating three commonly occurring diseases and pests of wheat, i.e., powdery mildew, yellow rust and aphid with a few specific bands. Through a comprehensive spectral analysis, only three bands at 570, 680 and 750 nm were selected. A novel vegetation index namely Ratio Triangular Vegetation Index (RTVI) was developed for detecting anomalous areas on leaves. Then, the Support Vector Machine (SVM) method was applied to construct the discrimination model based on the spectral ratio analysis. The validating results suggested that the proposed method with only three spectral bands achieved a promising accuracy with the Overall Accuracy (OA) of 83%. With three bands from the hyperspectral imaging data, the three wheat diseases and pests were successfully detected and discriminated. A stepwise strategy including background removal, damage lesions recognition and stresses discrimination was proposed. The present work can provide a basis for the design of low cost and smart instruments for disease and pest detection.     

Analysis of stained objects in histological sections by spectral imaging and differential absorption.

We describe a new light microscopic imaging system and method to perform high through put color image analysis on histological tissue sections. The system features a computer-controlled, random-access liquid crystal tunable filter and high-resolution digital camera on a conventional brightfield microscope. For any combination of stains, the method determines the spectral transmittance of each stain on the slide and selects two or more wavelengths at which the differential absorption between stain and counterstain is greatest and the exposure time is reasonably short. Flatfield corrected digital images at these wavelengths are acquired and divided to produce a gray scale ratio image. The ratio image is calculated such that the stained features of interest are highlighted above a uniform background and the counterstained features are highlighted below background. Image threshold procedures using either visual inspection or a threshold value determined by the image mean intensity and standard deviation are used to segment the stained features of interest for subsequent morphometry. Results are presented for peroxidase-AEC-labeled tumor tissue and trichrome-stained biomaterial implant tissues. In principle, the method should work for any combination of colored stains. (J Histochem Cytochem 47:1307-1313, 1999)     

Multispectral fluorescence and reflectance imaging at the leaf level and its possible applications

Images taken at different spectral bands are increasingly used for characterizing plants and their health status. In contrast to conventional point measurements, imaging detects the distribution and quantity of signals and thus improves the interpretation of fluorescence and reflectance signatures. In multispectral fluorescence and reflectance set-ups, images are separately acquired for the fluorescence in the blue, green, red, and far red, as well as for the reflectance in the green and in the near infrared regions. In addition, 'reference' colour images are taken with an RGB (red, green, blue) camera. Examples of imaging for the detection of photosynthetic activity, UV screening caused by UV-absorbing substances, fruit quality, leaf tissue structure, and disease symptoms are introduced. Subsequently, the different instrumentations used for multispectral fluorescence and reflectance imaging of leaves and fruits are discussed. Various types of irradiation and excitation light sources, detectors, and components for image acquisition and image processing are outlined. The acquired images (or image sequences) can be analysed either directly for each spectral range (wherein they were captured) or after calculating ratios of the different spectral bands. This analysis can be carried out for different regions of interest selected manually or (semi)-automatically. Fluorescence and reflectance imaging in different spectral bands represents a promising tool for non-destructive plant monitoring and a 'road' to a broad range of identification tasks.     

Sensitivity measurement and compensation in spectral imaging.

BACKGROUND: The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented. METHODS: Multicolored fluorescent beads were run on the ImageStream and a flow cytometer. Four single color fluorescent control samples of cells were run to quantify spectral overlap. An additional sample, labeled with all colors was run and compensated in six spectral channels. RESULTS: Analysis of empirical data for sensitivity and dynamic range matched theoretical predictions. The ImageStream system demonstrated fluorescence sensitivity comparable to a PMT-based flow cytometer. A methodology for addressing spectral overlap, individual pixel anomalies, and multiple imaging modalities was demonstrated for spectral compensation of K562 cells. Imagery is shown pre- and post-compensation. CONCLUSIONS: Unlike intensity measurements made with conventional flow cytometers, object size impacts both dynamic range and fluorescence sensitivity in systems that utilize pixilated detection. Simultaneous imaging of alternate modalities can be employed to increase fluorescent sensitivity. Effective compensation of complex multimode imagery spanning six spectral bands is accomplished in a semi-automated manner.     

Dual light‐emitting diode‐based multichannel microscopy for whole‐slide multiplane,multispectral and phase imaging

We report the development of a multichannel microscopy for whole‐slide multiplane, multispectral and phase imaging. We use trinocular heads to split the beam path into 6 independent channels and employ a camera array for parallel data acquisition, achieving a maximum data throughput of approximately 1 gigapixel per second. To perform single‐frame rapid autofocusing, we place 2 near‐infrared light‐emitting diodes (LEDs) at the back focal plane of the condenser lens to illuminate the sample from 2 different incident angles. A hot mirror is used to direct the near‐infrared light to an autofocusing camera. For multiplane whole‐slide imaging (WSI), we acquire 6 different focal planes of a thick specimen simultaneously. For multispectral WSI, we relay the 6 independent image planes to the same focal position and simultaneously acquire information at 6 spectral bands. For whole‐slide phase imaging, we acquire images at 3 focal positions simultaneously and use the transport‐of‐intensity equation to recover the phase information. We also provide an open‐source design to further increase the number of channels from 6 to 15. The reported platform provides a simple solution for multiplexed fluorescence imaging and multimodal WSI. Acquiring an instant focal stack without z‐scanning may also enable fast 3‐dimensional dynamic tracking of various biological samples.      

alpha-Cyclodextrin-modified infrared chemical sensor for selective determination of tyrosine in biological fluids

In this paper, we propose an evanescent wave-based infrared (IR) spectroscopic sensing method for the selective and sensitive detection of tyrosine in aqueous solution. In this approach, alpha-cyclodextrin (alpha-CTD) was chemically immobilized onto the surface of an IR-sensing element to attract tyrosine specifically to the surface of the sensing element. Theoretical equations were developed for the quantitative analysis of tyrosine. Based on its IR spectra, the synthesized alpha-CTD phase was stable in water. Optimal detection with this system occurred when the pH of the solution was ca. 10.5. Based on the absorption bands, we confirmed that alpha-CTD was most effective at attracting tyrosine under basic conditions. Using the unique absorption band of tyrosine at 1500 cm(-1), the alpha-CTD phase allowed the detection of tyrosine selectively from among a range of potentially interfering amino acids and other species commonly present in biological samples. For quantitative analysis, this CTD-modified phase was most suitable for sensing tyrosine at concentrations below 100 microM because of limits in the surface adsorption mechanism. The detection times were, in some instances, lower than 5 min. For a detection time of 10 min, the detection limit of tyrosine was ca. 0.4 microM.     

Spectral karyotyping analysis of human and mouse chromosomes

Classical banding methods provide basic information about the identities and structures of chromosomes on the basis of their unique banding patterns. Spectral karyotyping (SKY), and the related multiplex fluorescence in situ hybridization (M-FISH), are chromosome-specific multicolor FISH techniques that augment cytogenetic evaluations of malignant disease by providing additional information and improved characterization of aberrant chromosomes that contain DNA sequences not identifiable using conventional banding methods. SKY is based on cohybridization of combinatorially labeled chromosome-painting probes with unique fluorochrome signatures onto human or mouse metaphase chromosome preparations. Image acquisition and analysis use a specialized imaging system, combining Sagnac interferometer and CCD camera images to reconstruct spectral information at each pixel. Here we present a protocol for SKY analysis using commercially available SkyPaint probes, including procedures for metaphase chromosome preparation, slide pretreatment and probe hybridization and detection. SKY analysis requires approximately 6 d.     

Spectral imaging of multi-color chromogenic dyes in pathological specimens.

We have investigated the use of spectral imaging for multi-color analysis of permanent cytochemical dyes and enzyme precipitates on cytopathological specimens. Spectral imaging is based on Fourier-transform spectroscopy and digital imaging. A pixel-by-pixel spectrum-based color classification is presented of single-, double-, and triple-color in situ hybridization for centromeric probes in T24 bladder cancer cells, and immunocytochemical staining of nuclear antigens Ki-67 and TP53 in paraffin-embedded cervical brush material (AgarCyto). The results demonstrate that spectral imaging unambiguously identifies three chromogenic dyes in a single bright-field microscopic specimen. Serial microscopic fields from the same specimen can be analyzed using a spectral reference library. We conclude that spectral imaging of multi-color chromogenic dyes is a reliable and robust method for pixel color recognition and classification. Our data further indicate that the use of spectral imaging (a) may increase the number of parameters studied simultaneously in pathological diagnosis, (b) may provide quantitative data (such as positive labeling indices) more accurately, and (c) may solve segmentation problems currently faced in automated screening of cell- and tissue specimens.     

细菌群体感应信号分子的光电检测技术

群体感应（quorum sensing，QS）是一种依赖菌群密度的细菌交流系统。在探究细菌群体感应系统的调控机制中，对QS信号分子的鉴别和检测是不可或缺的环节，其对生命科学、药学等领域涉及细菌等微生物的相互作用、高效检测和作用机制解析等具有重要的参考意义。本文在总结不同类型细菌QS信号分子来源和结构的基础上，对QS信号分子的光电检测方法和技术进行了综述，重点对光电传感检测的敏感介质、传感界面、传感机制及测试效果进行探讨，同时关注了将微流控芯片分析技术应用于细菌QS信号分子原位监测的相关研究进展。     

Temperature-Regulated Surface Plasmon Resonance Imaging System for Bioaffinity Sensing

We describe a temperature-regulated surface plasmon resonance (SPR) imaging biosensor in this article. The sample temperature can be regulated for specific requirements of the bioaffinity sensing, and stabilized to suppress the measurement noise caused by temperature fluctuations. The water thermo optic coefficient is measured to test the temperature regulation performance. The protein interaction is monitored to demonstrate the feasibility of this system for real-time biomolecular interaction analysis. This temperature-regulated SPR imaging biosensor can be readily implemented by adding the common water path and peristaltic pump to the conventional SPR imaging system, which may provide an economical and convenient scheme to improve the analysis accuracy and quality of bioaffinity sensing using SPR sensing platform.     

Three-dimensional FRET reconstruction microscopy for analysis of dynamic molecular interactions in live cells

Analysis of cellular pathways requires concentration measurements of dynamically interacting molecules within the three-dimensional (3D) space of single living cells. Förster resonance energy transfer (FRET) microscopy from widefield, from confocal, and potentially from superresolution microscopes can access this information; however, these measurements are distorted by the inherent 3D blurring of optical imaging, spectral overlap of fluorophores, and detection noise. We propose a mathematical model of these processes and demonstrate, through simulation, how these distortions limit the dynamic range and sensitivity of conventional FRET microscopy. Using this model, we devise and validate a new approach (called 3D-FRET stoichiometry reconstruction, 3DFSR) for reconstructing 3D distributions of bound and free fluorescent molecules. Previous attempts to reconstruct 3D-FRET data relied on sequential spectral unmixing and deconvolution, a process that corrupts the detection statistics. We demonstrate that 3DFSR is superior to these approaches since it simultaneously models spectral mixing, optical blurring, and detection noise. To achieve the full potential of this technique, we developed an instrument capable of acquiring 3D-FRET data rapidly and sensitively from single living cells. Compared with conventional FRET microscopy, our 3D-FRET reconstruction technique and new instrumentation provides orders of magnitude gains in both sensitivity and accuracy wherein sustained high-resolution four-dimensional (x,y,z,t) imaging of molecular interactions inside living cells was achieved. These results verify previous observations that Cdc42 signaling is localized to the advancing margins of forming phagosomes in macrophages.     

Simultaneous Live Cell Imaging Using Dual FRET Sensors with a Single Excitation Light

Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca²⁺ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.     

SPR imaging-based monitoring of caspase-3 activation

The activation of caspase-3 plays an important role in the apoptotic process. In this study, we describe a novel method by which caspase-3-dependent proteolytic cleavage can be monitored, using a surface plasmon resonance (SPR) imaging protein chip system. To the best of our knowledge, this is the first report regarding the SPR imaging-based monitoring of caspase-3 activation. In order to evaluate the performance of this protocol, we constructed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the cleavage of the artificial caspase-3 substrate in response to caspase-3 using an SPR imaging sensor. The purified GST:DEVD:EGFP protein was initially immobilized onto a glutathionylated gold chip surface, and subsequently analyzed using an SPR imaging system. As a result, caspase-3 activation predicated on the proteolytic properties inherent to substrate specificity could be monitored via an SPR imaging system with a detection performance similar to that achievable by the conventional method, including fluorometric assays. Collectively, our data showed that SPR imaging protein chip system can be effectively utilized to monitor the proteolytic cleavage in caspase-3, thereby potentially enabling the detection of other intracellular protease activation via the alteration of the protease recognition site in the linker peptides.     

An Innovative Method for Obtaining Consistent Images and Quantification of Histochemically Stained Specimens

Obtaining digital images of color brightfield microscopy is an important aspect of biomedical research and the clinical practice of diagnostic pathology. Although the field of digital pathology has had tremendous advances in whole-slide imaging systems, little effort has been directed toward standardizing color brightfield digital imaging to maintain image-to-image consistency and tonal linearity. Using a single camera and microscope to obtain digital images of three stains, we show that microscope and camera systems inherently produce image-to-image variation. Moreover, we demonstrate that post-processing with a widely used raster graphics editor software program does not completely correct for session-to-session inconsistency. We introduce a reliable method for creating consistent images with a hardware/software solution (ChromaCal™; Datacolor Inc., NJ) along with its features for creating color standardization, preserving linear tonal levels, providing automated white balancing and setting automated brightness to consistent levels. The resulting image consistency using this method will also streamline mean density and morphometry measurements, as images are easily segmented and single thresholds can be used. We suggest that this is a superior method for color brightfield imaging, which can be used for quantification and can be readily incorporated into workflows.     

Analysis of protein interactions on protein arrays by a novel spectral surface plasmon resonance imaging

We presented a novel surface plasmon resonance (SPR) imaging method for analysis of protein arrays based on a wavelength interrogation-based SPR biosensor. The spectral imaging was performed by the combination of position control and resonance wavelengths calculated from SPR reflectivity spectra. The imaging method was evaluated by analyzing interactions of glutathione S-transferase-fusion proteins with their antibodies. Antigen-antibody interactions were successfully analyzed on glutathione S-transferase-fusion protein arrays by using the spectral imaging method, and the results were confirmed by a parallel analysis using a previously used spectral SPR biosensor based on wavelength interrogation. Specific binding of anti-Rac1 and anti-RhoA to Rac1 and RhoA on the protein arrays was qualitatively and quantitatively analyzed by the spectral SPR imaging. Thus, it was suggested that the novel spectral SPR imaging was a useful tool for the high-throughput analysis of protein-protein interactions on protein arrays.     

Utility of multispectral imaging for nuclear classification of routine clinical histopathology imagery

Background

We present an analysis of the utility of multispectral versus standard RGB imagery for routine H&;E stained histopathology images, in particular for pixel-level classification of nuclei. Our multispectral imagery has 29 spectral bands, spaced 10 nm within the visual range of 420–700 nm. It has been hypothesized that the additional spectral bands contain further information useful for classification as compared to the 3 standard bands of RGB imagery. We present analyses of our data designed to test this hypothesis.

Results

For classification using all available image bands, we find the best performance (equal tradeoff between detection rate and false alarm rate) is obtained from either the multispectral or our "ccd" RGB imagery, with an overall increase in performance of 0.79% compared to the next best performing image type. For classification using single image bands, the single best multispectral band (in the red portion of the spectrum) gave a performance increase of 0.57%, compared to performance of the single best RGB band (red). Additionally, red bands had the highest coefficients/preference in our classifiers. Principal components analysis of the multispectral imagery indicates only two significant image bands, which is not surprising given the presence of two stains.

Conclusion

Our results indicate that multispectral imagery for routine H&;E stained histopathology provides minimal additional spectral information for a pixel-level nuclear classification task than would standard RGB imagery.