Abstract
Recent developments in fluorescence microscopy call for novel small‐molecule‐based labels with multiple functionalities to satisfy different experimental requirements. A current limitation in the advancement of live‐cell single‐molecule localization microscopy is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live‐cell experiments. At the same time, quality of super‐resolution imaging depends on high label specificity, making removal of excess dye essential. Approaching both hurdles, we present the design and synthesis of a small‐molecule label comprising both fluorogenic and self‐blinking features. Bioorthogonal click chemistry ensures fast and highly selective attachment onto a variety of biomolecular targets. Along with spectroscopic characterization, we demonstrate that the probe improves quality and conditions for regular and single‐molecule localization microscopy on live‐cell samples.
Highlights
Small-molecule fluorophores are essential tools for biological imaging, which is a key method in modern life sciences
We set out to test the feasibility of this strategy for the synthesis of the simple unfunctionalized HMSiR without tetrazine (Figure 1) and found that this four step route using methoxymethyl (MOM) protection group chemistry enabled the synthesis of HM-silicon rhodamine (SiR) at 22 % overall yield (Supporting Information, Scheme S2)
Our attention turned to the synthesis of the tetrazinemodified hydroxymethyl SiR (HM-SiR) derivative, in the following termed fluorogenic HM-SiR (f-HM-SiR)
Summary
Small-molecule fluorophores are essential tools for biological imaging, which is a key method in modern life sciences. DAinv enables labeling various biomolecules other than proteins like nucleotides,[12] sugars,[13] and lipids[14] as well as to exploit small-molecule-mediated targeting.[15] tetrazine-based labeling can be used to reduce unspecific signal; Carefully designed tetrazine probes have been shown to undergo an increase in fluorescence when the tetrazine is consumed in DAinv, that is, when the dye label is covalently linked to its target structure.[15a,16] This is a valuable additional feature for live-cell fluorescence microscopy because it obviates the need for extensive excess dye wash-out and dramatically reduces background signal.[17] This is advantageous for SMLM as the localization precision is affected by background signal.[18] Overall, we reason that a broadly applicable probe for SMLM would comprise all of the above-mentioned features It should be as small as possible, self-blinking, fluorogenic, readily suited for bioconjugation, and equipped with generally favorable photophysical properties, such as high brightness and photostability. The fluorogenicity of f-HM-SiR allows for minimal- or no-wash procedures in live-cell imaging while its self-blinking feature minimizes phototoxicity in SMLM experiments
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