Spatiotemporal multiplexed immunofluorescence imaging of living cells and tissues with bioorthogonal cycling of fluorescent probes

Abstract

Cells in complex organisms undergo frequent functional changes, but few methods allow comprehensive longitudinal profiling of living cells. Here, we introduce scission-accelerated fluorophore exchange (SAFE), a method for multiplexed temporospatial imaging of living cells with immunofluorescence. SAFE uses a rapid bioorthogonal click chemistry to remove immunofluorescent signals from the surface of labeled cells, cycling the nanomolar-concentration reagents in seconds and enabling multiple rounds of staining of the same samples. It is nontoxic and functional in both dispersed cells and intact living tissues. We demonstrate multiparameter

Multiplexed analysis of a single EV (MASEV) reveals unique biomarker composition with diagnostic impact

“…Likewise, for the 555 and 647 channels, stained (vs. de-stained) S/B were 7.4±1.8 (vs. 1.2±0.4) and 6.0±0.4 (vs. 1.1±1.1), respectively. These values are consistent with the >95% scission observed in other contexts 26,27 .…”

“…In the current study, we demonstrate the feasibility of performing a highly multiplexed analysis of proteins in single EVs. This analysis was enabled by the recent development of a bioorthogonal, immolating click chemistry that allows rapid cleavage of fluorochromes from antibodies 26, 27 . The fast kinetics and exceptionally complete cleavage allow cycling and repeat staining of individual vesicles in simple microscope slide flow chambers.…”

“…Commercially-available antibodies ( Table S1 ) were purchased carrier-free for in-lab modification with Dye-C 2 TCO-NHS linker SAFE probes. Probes were synthesized and activated according to 27 and stored at -80 °C until use. Antibodies were exchanged into 0.1 M PBS-bicarbonate buffer (pH 8.4) using a 40 k Zeba column (87765 Thermo Fisher) and incubated with a 5- to 12-fold molar excess of the SAFE probe with 10% DMSO for 25 minutes at room temperature in the dark.…”

“…Over 90% de-staining is complete within 1-2 minutes at these reaction kinetics, and EV can then be stained for subsequent rounds. The cyclic staining chemistry is also compatible with live cells and tissues 27 and does not affect the properties of EV. We developed a specially constructed flow chamber to facilitate rapid cycling and preservation of reagents, with no need for harsh conditions (e.g., paraformaldehyde to fix and adhere EVs or hydrogen peroxide to bleach fluorochromes), thus allowing unequivocal analyses of a single EV.…”

Tetrazine-Triggered Bioorthogonal Cleavage of trans-Cyclooctene-Caged Phenols Using a Minimal Self-Immolative Linker Strategy

“…As a result, click in head-to-head orientation is favored, thereby improving the efficiency of the overall bond-cleavage process. In addition, the release step upon click with ammoniumfunctionalized Tz was reported to be instantaneous (when NH3 + is in head-to-head position) [5,14] and independent from pH. [13] We exploited those explorations by using acidic HPLC conditions to achieve better separation of the intermediates (all bearing an NH3 + moiety) formed upon reaction of 6 with PymK ( 9) without provoking any pseudo-release (for details see the Supporting Information).…”