Reversible Chemical Reactions for Single‐Color Multiplexing Microscopy

Abstract

Recent developments in biology demand an increasing number of simultaneously imaged structures with standard fluorescence microscopy. However, the number of multiplexed channels is limited for most multiplexing modalities, such as spectral multiplexing or fluorescence-lifetime imaging. We propose extending the number of imaging channels by using chemical reactions, controlling the emissive state of fluorescent dyes. As proof of concept, we reversibly switch a fluorescent copper sensor to enable successive imaging of two different structures in the same spectral channel. We also show that this chemical multiplexing is orthogonal to existing methods. By using two different dyes, we combine chemical with spectral multiplexing for the simultaneous imaging of four different structures with only two spectrally different channels. We characterize and discuss the approach and provide perspectives for extending imaging modalities in stimulated emission depletion microscopy, for which spectral multiplexing is technically demanding.

Super‐resolved insights into human immunodeficiency virus biology

“…Super‐resolution imaging of up to three colors with the same STED laser line is possible by employing excitation multiplexing, long Stokes‐shift dyes or life‐time separation of fluorophores . Activation of a dye by the addition of Cu 2+ could also be used for imaging with two colors . In STORM, multiple dyes are more easily implemented , and three colour SMLM (with 488‐, 532‐, and 647‐nm excitation light) is routinely applied .…”

Copper(II)‐induced Fluorescence Quenching of a BODIPY Fluorophore

“…In previous studies of similar probes, we have observed comparable effects and could exclude energy transfer as possible quenching mechanism. We found that the overlap between dye emission and ligand/complex absorption was too small to enable energy transfer , . However, as complex and chromophoric centers were always in close proximity, as is the case for BODIPY‐BPY 1 , photo‐induced electron transfer (PET) remains as possible quenching mechanism.…”

“…Beyond this, functionalization of fluorescent metal indicators have an interesting perspective in the field of microscopy, e.g. as probes for super‐resolutions microscopy or as additional approach for multiplexing in microscopy to increase the number of simultaneously observable targets , . Moreover, functionalized probes could be used for implementing sensory layers in nano‐structured materials adding function to certain regions.…”

“…In previous studies, the bidentate bipyridine was found a suitable ligand for designed Cu II ‐dependent switchable fluorescent probes , . Therefore, we designed a bipyridine functionalized turn‐off probe with the shortest possible distance to the chromophore to maximize quenching upon coordination of Cu II .…”

Monitoring hydroquinone–quinone redox cycling by single molecule fluorescence spectroscopy

“…Due to the reversible nature of the reaction, the controlled on-off switching may have potential for applications requiring the chemical control of emissive and dark molecular states, like in super-resolution fluorescence microscopy, or for enabling new ways of multiplexing in fluorescence microscopy. 29,30 However, for such applications, the dynamic switching between the oxidized and reduced states still needs to be established and reliably defined by experimental parameters. Nevertheless, the advantage of the probe is given by its flexibility towards structural tuning of its electrochemical redox-potential by variation of residues attached to the hydroquinone unit thus allowing for combinations with different redox-systems.…”