Time Gated Luminescence Imaging of Immunolabeled Human Tissues.

Multiplexed immunofluorescence imaging of formalin-fixed, paraffin-embedded tissues is a powerful tool for investigating proteomic profiles and diagnosing disease. However, conventional immunofluorescence with organic dyes is limited in the number of colors that can be simultaneously -visualized, is made less sensitive by tissue autofluorescence background, and is usually incompatible with commonly used hematoxylin and eosin staining. Herein, we demonstrate the comparative advantages of using time-gated luminescence microscopy in combination with an emissive M(Ill) complex, Lumi4-Tb, for tissue imaging in terms of sensitivity, multiplexing potential, and compatibility with common immunohistochemistry protocols. We show that time-gated detection of millisecond-scale Tb(III) emission increases signal-to-noise ratio relative to conventional steady-state detection of organic dye fluorescence and permits visualization of low-abundance tissue markers such as Bcl-6 or MSH-6. In addition, temporal separation of long- and short-lifetime (nanosecond) signals adds a second dimension for multiplexing and also permits detection of intermolecular Tb(III)-to-dye Forster resonance energy transfer. Furthermore, we demonstrate that the Lumi4-Tb complex is compatible with tyramide signal amplification and, unlike conventional organic dyes, can be reliably used on tissue stained with hematoxylin and eosin. Our results indicate that time-gated luminescence microscopy using Tb(III) labels can provide a sensitive and robust method to perform multiplexed immunofluorescence on archived or clinical tissue specimens.