Cytology and histology slides. (a) Superficial squamous cells in a cervical smear: individual cells, spread out of their tissue context. (b) A Pap test showing normal cervicovaginal cytology: scattered cells. (c) Rectal mucosa with radiation proctitis, H&E, high magnification. (d) A whole transverse section of the left colon with diverticulosis: the tissue architecture of the entire section. Images from Wikimedia Commons: (a) Manuel Medina (Patólogo), “Células superficiales”, public domain (via Flickr); (b) Cagliostro, “Pap test wnl.jpg”, public domain; (c) Nephron, “Radiation proctitis - 2 alt -- high mag.jpg”, CC BY-SA 4.0; (d) Alex_brollo, “Colon diverticulosis whole slide.jpg”, CC BY-SA 3.0.
Radiology, covered in 2.2 to 2.7, images the body from the outside and resolves structures down to about a millimeter. The microscopes of 2.8 and 2.9 resolve features down to a few hundred nanometers, but mostly on cultured cells, in fields of view a fraction of a millimeter wide. Diagnosing disease at the cellular level requires both microscopic resolution and patient tissue samples. These samples are made visible with dyes that absorb light and are examined in bright-field, the transmitted-light setup of 2.8.
The samples are mounted on glass slides in one of two ways. In cytology, loose cells are spread on the slide, as in a cervical smear. In histology, tissue is sliced into thin sections, preserving the spatial distribution of cells; its use in diagnosis is known as histopathology. Pathologists read both types of slide to make a diagnosis. In cancer, for instance, this modality can help establish whether a lesion is malignant, its type, and grade.
Increasingly, these glass slides are also digitized. A whole-slide scanner records an entire slide at cellular resolution, producing a whole-slide image (WSI) of several billion pixels. WSIs are the main data source of computational pathology, and they differ from the images of 2.8 and 2.9 in three ways: they are color images of absorbing dyes rather than sets of fluorescence channels; they are far too large to fit into a network at full resolution; and their labels usually describe a whole slide or patient, not individual cells or pixels.
In this page, we first look at what cytology and histology have in common: dyes that create color by absorbing light, and scanners that record it. After a brief look at cytology, we turn to histology and whole-slide images, following the tissue from preparation to the digital file and the models that analyze it.
In cytology, cells are collected through less invasive procedures, such as brushing the cervix during a screening test or drawing cells through a thin needle from a lump. In histology, a piece of tissue is removed, either by biopsy or during surgery, for instance to remove a tumor. A smear therefore shows cells out of their context, scattered alone or in small clusters, while a histology section preserves how the cells are organized in space. This matters because many diseases are recognized as much by how cells are arranged as by how they look.
A tissue section may also show extracellular matrix, blood vessels, connective tissue, and immune cells, arranged in patterns characteristic of each organ. This arrangement is known as tissue architecture. In healthy epithelium, for instance, cells form continuous layers resting on a basement membrane; in a gland, they line a lumen and are surrounded by supporting stroma.
Disease disrupts this pattern at several scales. At the scale of the cell, nuclei in cancer become larger, darker, and more variable in size and shape, and mitoses become more frequent. At the scale of the tissue, glands lose their regular shape, layers lose their regular orientation, and cells cross the boundaries that normally contain them. Whether a tumor has spread through the basement membrane separates an in situ lesion from an invasive cancer, with very different consequences for the patient.
A pathologist therefore reads a slide at several magnifications. A low-power objective shows the whole section: which tissues are present, where the lesion is, and how it relates to its surroundings. Higher magnifications show glands, then individual cells and nuclei. The diagnosis combines both views, and the same group of cells can mean different things in different contexts.
One H&E section at three scales. (a) At low magnification, the section shows the architecture of the tumor: nodules separated by fibrous bands, with a capsule on the right. (b) At intermediate magnification, sheets of tumor cells around a blood vessel. (c) At high magnification, individual nuclei varying in size, shape, and darkness. Boxes mark the region shown in the next panel; a diagnosis usually combines all three scales. Adrenocortical carcinoma, TCGA-OR-A5J1.
As we discussed in 2.8, unstained cells and thin sections are nearly transparent. Phase contrast and DIC solve this optically. Cytology and histology solve it chemically: dyes that bind to particular cell and tissue components absorb light, and the image shows contrast directly.
The routine stain used on most slides made worldwide is hematoxylin and eosin (H&E). Its two dyes bind to different components according to their charge:
The result is the familiar image of purple nuclei on a pink background. H&E shows nuclear morphology and tissue architecture in a single image, which is exactly what a pathologist needs to read at both scales.