Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffinembedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.cancer diagnostics | high-content cellular analysis | image analysis | mTOR | multiplexing
A simple image-based autofocusing scheme for digital microscopy is demonstrated that uses as few as two intermediate images to bring the sample into focus. The algorithm is adapted to a commercial inverted microscope and used to automate brightfield and fluorescence imaging of histopathology tissue sections.
Accurate focusing is a critical challenge of whole slide imaging, primarily due to inherent tissue topography variability. Traditional line scanning and tile-based scanning systems are limited in their ability to acquire a high degree of focus points while still maintaining high throughput. This review examines limitations with first-generation whole slide scanning systems and explores a novel approach that employs continuous autofocus, referred to as independent dual sensor scanning. This “second-generation” concept decouples image acquisition from focusing, allowing for rapid scanning while maintaining continuous accurate focus. The technical concepts, merits, and limitations of this method are explained and compared to that of a traditional whole slide scanning system.
Many pathology laboratories are implementing digital pathology systems. The image resolution and scanning (digitization) magnification can vary greatly between these digital pathology systems. In addition, when digital images are compared with viewing images using a microscope, the cellular features can vary in size. This article highlights differences in magnification and resolution between the conventional microscopes and the digital pathology systems. As more pathologists adopt digital pathology, it is important that they understand these differences and how they ultimately translate into what the pathologist can see and how this may impact their overall viewing experience.
We have described a novel platform for evaluating biomarker co-localization. Expression in multiplexed images is consistent with conventional immunohistochemistry. Automation reduces inconsistencies in staining and positional shifts, while the fluorescent dye cycling approach dramatically expands the number of biomarkers which can be visualized and quantified on a single tissue section.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.