Serial Two-photon Tomography

The focused infrared laser excites fluorescent labels near the exposed tissue surface rather than throughout the optical path. This localized excitation reduces out-of-focus fluorescence, helping preserve spatial information in the imaged region. As a result, labeled immune cells, pathogens, and surrounding tissue features can be distinguished more clearly within the larger volume reconstructed from successive sections.

Serial sectioning extends imaging beyond a single exposed surface by removing thin tissue layers and presenting a new surface for repeated imaging. Each image contributes information from a successive depth, allowing the collected data to be assembled into a three-dimensional map. This approach supports analysis of structures distributed across large tissue volumes rather than only one focal region.

The method can show where immune cells, pathogens, and tissue microenvironments are located relative to one another across intact organs. These spatial relationships provide context for examining cellular interactions and infection-associated tissue remodeling. Such organization may connect local events observed in tissue to broader tissue-scale outcomes of host-pathogen responses.

The workflow uses fluorescent labels, a focused infrared laser, and automated physical sectioning. First, the exposed tissue surface is imaged with two-photon fluorescence microscopy. Automated sectioning then removes a thin layer, after which the newly exposed surface is imaged again. Repeating these cycles generates the image series needed for volumetric reconstruction.

Researchers can use it when they need tissue-scale maps of immune cells, pathogens, and their surrounding microenvironments within intact organs. The approach is particularly relevant when spatial organization matters alongside molecular measurements. It can help investigate how infection-associated remodeling and cellular interactions are distributed through tissue, rather than interpreting those processes from isolated locations alone.

Molecular analyses provide information about biological components or processes, while this imaging approach adds their three-dimensional tissue context. By mapping labeled structures across large volumes, researchers can relate cellular distributions and infection-associated remodeling to tissue-scale organization. Combining these perspectives supports more complete models of host-pathogen responses than either spatial imaging or molecular analysis considered separately.