Obtaining multiplexed molecular readouts from large numbers of single cells in situ is an important technological goal to facilitate scientific discoveries in basic and translational research. Various methods have been developed in recent years to achieve spatially resolved multiplexed measurements of the abundance of large sets of mRNAs or proteins in biological samples. These technologies have brought the promise that, through large-scale efforts, all functionally relevant cell types of an organism will emerge from such multiplexed data in an unbiased manner. Furthermore, these cell types will be able to be mapped within their physical context within a tissue.

The involvement of an mRNA or protein (state) in cellular function depends on its specific intracellular location and interaction with other molecules and cellular structures. Moreover, the phenotype of an individual cell is determined by the functional state, abundance, morphology, and turnover of its organelles and cytoskeletal structures. To functionally interpret molecular multiplexed information, such measurements will thus need to resolve the intracellular length scale.

We report a simple, robust, and nondegrading protocol that achieves 40-plex protein staining in the same biological sample using off-the-shelf antibodies called iterative indirect immunofluorescence imaging (4i). In conjunction with high-throughput automated microscopy and computer vision, 4i allows highly reproducible multiplexed measurements from surface areas of several mm² subsampled by pixels of 165 nm by 165 nm. This approach simultaneously captures functionally relevant properties that emerge at the cell population, cellular, and intracellular level. 4i can thus quantify the influence of local cell crowding on protein abundance, the effect of cell cycle position on protein phosphorylation, patterns of protein subcompartmentalization, and organelle morphology all in the same single cell and across thousands of cells.

We developed a data-driven computer vision approach that generates multiplexed protein maps (MPMs). MPMs comprehensively quantify intracellular protein composition with high spatial detail in large numbers of single cells. They are not confounded by the specific relative geometry and orientation of an individual cell. Thus, MPMs allow systematic comparisons of subcellular spatial protein distribution between single cells that experience different cell cycle states, microenvironments, growth conditions, or exposure to drugs. Using the example of subcellular relocalization of epidermal growth factor (EGF) receptor upon exposure to EGF, we demonstrate that MPMs allow the systematic definition of cellular states undetectable by multiplexed whole-cell measurements. The findings are functionally relevant and can be connected to multiple molecular and phenotypic properties apparent at the cellular and cell population scale.

By preventing photocrosslinking during imaging, 4i enables multiplexed immunofluorescence with off-the-shelf antibodies, both in small-scale and high-throughput experiments. 4i datasets cover multiple length scales, eliminating the need for extrapolation or inference in the interpretation of results. Integration of these length scales in one dataset reveals a richness of scale-crossing connections that current models of biological processes do not yet consider. These connections determine how gene expression is adapted to the cellular state, how a cell type is determined, how a pathological cellular phenotype emerges, or how a tumor cell responds to a drug.

4i obtains 40-plexed …