Optical quality assessment of whole slide imaging systems for digital pathology.

Whole Slide Imaging (WSI) systems are high-throughput automated microscopes for digital pathology applications. We present a method for testing and monitoring the optical quality of WSI-systems using a measurement of the through-focus Optical Transfer Function (OTF) obtained from the edge response of a custom made resolution target, composed of sagittal and tangential edges. This enables quantitative analysis of a number of primary aberrations. The curvature of the best focus as a function of spatial frequency is indicative for spherical aberration, the argument of the OTF quantifies for coma, and the best focus as a function of field position for sagittal and tangential edges allows assessment of astigmatism and field curvature. The statistical error in the determined aberrations is typically below 20 mλ. We use the method to compare different tube lens designs and to study the effect of objective lens aging. The results are in good agreement with direct measurement of aberrations based on Shack-Hartmann wavefront sensing with a typical error ranging from 10 mλ to 40 mλ.

Color accuracy and reproducibility in whole slide imaging scanners.

We propose a workflow for color reproduction in whole slide imaging (WSI) scanners, such that the colors in the scanned images match to the actual slide color and the inter-scanner variation is minimum. We describe a new method of preparation and verification of the color phantom slide, consisting of a standard IT8-target transmissive film, which is used in color calibrating and profiling the WSI scanner. We explore several International Color Consortium (ICC) compliant techniques in color calibration/profiling and rendering intents for translating the scanner specific colors to the standard display (sRGB) color space. Based on the quality of the color reproduction in histopathology slides, we propose the matrix-based calibration/profiling and absolute colorimetric rendering approach. The main advantage of the proposed workflow is that it is compliant to the ICC standard, applicable to color management systems in different platforms, and involves no external color measurement devices. We quantify color difference using the CIE-DeltaE2000 metric, where DeltaE values below 1 are considered imperceptible. Our evaluation on 14 phantom slides, manufactured according to the proposed method, shows an average inter-slide color difference below 1 DeltaE. The proposed workflow is implemented and evaluated in 35 WSI scanners developed at Philips, called the Ultra Fast Scanners (UFS). The color accuracy, measured as DeltaE between the scanner reproduced colors and the reference colorimetric values of the phantom patches, is improved on average to 3.5 DeltaE in calibrated scanners from 10 DeltaE in uncalibrated scanners. The average inter-scanner color difference is found to be 1.2 DeltaE. The improvement in color performance upon using the proposed method is apparent with the visual color quality of the tissue scans.

Real-time deformable registration of multi-modal whole slides for digital pathology.

Digital pathology provides new ways to visualize tissue slides and enables new workflows for analyzing these slides. Analogous to radiology, adjacent tissue sections prepared with different stains or biomarkers (e.g. H&E, IHC, special stains, or ISH; chromogenic or fluorescent) may be seen as different modalities, each representing different structural and/or functional information. Today, the anatomic pathologist views multiple glass slides using an optical microscope and then combines the information in their head to reach a (diagnostic) opinion. Moreover, due to the nature of the slide preparation and digitization process, the tissue and its features do not have the exact same morphology, appearance, or spatial alignment, making it difficult to find the same region on adjacent slides. To address such concerns, this paper presents a method for the spatial alignment of multi-modal whole slide digital microscopy images. To remain practical, the described method employs a two-step registration strategy designed to reduce computation time: the first step computes a B-spline deformable transform on low-resolution images prior to visualization, the second step applies the precomputed transformation only to the high-resolution region currently being viewed. The proposed method is demonstrated using a number of cases comprising H&E and IHC stained slides. These results indicate the feasibility of deformable registration for spatial alignment of multi-modal whole slide digital microscopy images within practical time constraints.

Real-time single-molecule imaging of oxidation catalysis at a liquid-solid interface.

Many chemical reactions are catalysed by metal complexes, and insight into their mechanisms is essential for the design of future catalysts. A variety of conventional spectroscopic techniques are available for the study of reaction mechanisms at the ensemble level, and, only recently, fluorescence microscopy techniques have been applied to monitor single chemical reactions carried out on crystal faces and by enzymes. With scanning tunnelling microscopy (STM) it has become possible to obtain, during chemical reactions, spatial information at the atomic level. The majority of these STM studies have been carried out under ultrahigh vacuum, far removed from conditions encountered in laboratory processes. Here we report the single-molecule imaging of oxidation catalysis by monitoring, with STM, individual manganese porphyrin catalysts, in real time, at a liquid-solid interface. It is found that the oxygen atoms from an O2 molecule are bound to adjacent porphyrin catalysts on the surface before their incorporation into an alkene substrate.

Highly oriented self-assembled monolayers as templates for epitaxial calcite growth.

The lateral alignment of [012] habit-modified calcite crystals with respect to a carboxylic acid terminated self-assembled monolayer (SAM) of thiols has been determined. The crystals were grown from a Kitano solution (pH 5.6-6.0), and the samples were investigated with scanning electron microscopy, X-ray diffraction, and polarization microscopy. For the first time, a lattice match in one direction, which is the nearest neighbor direction of the SAM and the calcite <100> direction, has been experimentally shown. The experimental results are in good agreement with the theoretical models proposed in previous work, and it is expected that this method can be applied to similar systems where inorganic crystals nucleate with a preferred orientation to a SAM.