A major obstacle to visualizing biological structures in all their complexity is spectral overlap between fluorophores. This 'color barrier' practically limits most experiments to the examination of three or four targets. But what if we could see cells in many colors? Multiplexed imaging could make it possible to view structures in a more meaningful context and allow better analysis of how multicomponent complexes form and change in cells. It is poised to transform our understanding of biological processes.

Imaging in many colors. Credit: Samuel Kenny, Zhengyang Zhang & Ke Xu

Several strategies seek to address current issues with multiplexed imaging. Developers are designing better probes; these include probes that span the visible region and beyond for more color options, as well as probes such as intracellular lasers (Nat. Photonics 9, 572–576, 2015; Nano Lett. 15, 5647–5652, 2015) that have very narrow spectra and are therefore relatively easy to resolve from one another. These improvements could enable better multiplexed imaging with standard microscopes.

Researchers are also developing optical setups to detect overlapping fluorophores. For example, Ke Xu and colleagues reported spectrally resolved stochastic optical reconstruction microscopy (SR-STORM) in which both the position and the spectra of individual labeled molecules are measured for true-color super-resolution imaging (Nat. Methods 12, 935–938, 2015). With such methods, even fluorophores with high spectral overlap can be readily discriminated, opening the door to imaging of numerous targets.

Methods for highly multiplexed immunofluorescence imaging are also emerging. In these methods, a subset of targets is labeled with antibodies and imaged. These probes are then rendered invisible by bleaching or stripping followed by additional rounds of the same labeling process against different targets. This allows highly multiplexed images to be built up over multiple rounds.

A related strategy has been used for highly multiplexed transcriptome imaging in which, over multiple rounds of imaging, individual transcripts are identified by a unique barcode (Nat. Methods 11, 360–361, 2014; Science 348, aaa6090, 2015). In the case of the MERFISH approach from the Zhuang lab, thousands of transcripts are imaged using only a single color.

Although these methods are powerful, improved methods are still needed for multiplexed imaging in live cells. Live-cell approaches have the same issues as fixed-cell approaches, but they also have other challenges, such as a smaller range of useful fluorescent dyes and probes, the need for rapid image acquisition, and sensitivity to light exposure. Methods to achieve massively multiplexed imaging will continue to be developed, enabling the study of biological processes as they occur in cells.