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, 59 (2), 804-810

Live-Cell Localization Microscopy With a Fluorogenic and Self-Blinking Tetrazine Probe


Live-Cell Localization Microscopy With a Fluorogenic and Self-Blinking Tetrazine Probe

Philipp Werther et al. Angew Chem Int Ed Engl.


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.

Keywords: bioorthogonal chemistry; click chemistry; fluorescent probes; super-resolution imaging; tetrazines.

Conflict of interest statement

The authors declare no conflict of interest.


Figure 1
Figure 1
f ‐HM‐SiR, a fluorogenic and spontaneously blinking fluorophore for bioorthogonal DAinv. a) Spiro‐cyclization equilibrium of spontaneously blinking and fluorogenic f ‐HM‐SiR and its Diels–Alder cycloadduct (DAinv‐Product). b) Fluorescence enhancement of f ‐HM‐SiR upon conversion in DAinv at pH 4 in aqueous buffer. c) pH‐dependent fluorescence of f ‐HM‐SiR (blue) and DAinv‐Product (red).
Figure 2
Figure 2
Cell‐permeable f ‐HM‐SiR can be specifically conjugated in live cells by DAinv. a) Prior to reaction with dienophile, f ‐HM‐SiR resides in quenched form. Target‐bound f ‐HM‐SiR spontaneously switches between fluorescent on‐ and off‐form. b) HeLa cells transiently expressing H2A‐HaloTag were incubated with (upper row) or without (lower row) HTL‐BCN (10 μm) for 30 min, washed, and labeled with f ‐HM‐SiR (2 μm, magenta) prior to imaging. Reference images (green) were generated by labeling with HTL‐TMR after incubation with HTL‐BCN. c) HeLa cells transiently expressing TOM20‐HaloTag were incubated with TPP‐BCN (10 μm) and HTL‐TMR (green), washed, and labeled with f ‐HM‐SiR (2 μm, magenta) prior to imaging. d) Background intensity of HeLa cells incubated with various SiR derivatives (2 μm), n=20. Medium was replaced once after incubation with dyes. Note the broken y‐axis and change of scale. e) Representative images for diagram in (d). Settings for image acquisition, processing, and display were identical for all shown images. Outlines of cells indicated by white line. Scale bars: b,c,e) 10 μm.
Figure 3
Figure 3
Spontaneous blinking of f ‐HM‐SiR enables SMLM with improved resolution and reduced background. a) COS‐7 cells transiently expressing TOM20‐mCherry‐HaloTag were incubated with HTL‐TCO* (10 μm), washed, and labeled with f ‐HM‐SiR (2 μm) for SMLM imaging. See Movie S1 in the Supporting Information. b) Average projection of 500 raw data frames used for reconstruction in (a). c) Merged zoom‐ins of boxed regions in (a) (red) and (b) (green). d) Line profile along boxed region in (c) comparing normalized intensities in averaged raw data (green) and SMLM reconstruction (red). e) Averaged cross‐sectional profiles of labeled mitochondria ±1 standard deviation, n=16. Individual profiles were aligned to minimum between peaks (see methods and Figure S11 in the Supporting Information for all profiles). f) Peak‐to‐peak distances in cross‐sectional profiles shown in (e). g) Long‐term stability of f ‐HM‐SiR labeled TOM20‐HaloTag in fixed COS‐7 cells. Mean localizations per frame normalized to number of localizations in first frame (black line) ±1 standard deviation, n=8. h) Background localization rate for HM‐SiR and f ‐HM‐SiR in non‐transfected HeLa cells, n=20. Scale bars: a,b) 5 μm, c) 1 μm.
Figure 4
Figure 4
f ‐HM‐SiR reveals cellular dynamics in live HeLa cells with improved resolution. a) HeLa cells transiently expressing H2A‐HaloTag were incubated with HTL‐BCN (10 μm), washed and labeled with f ‐HM‐SiR (2 μm). A reconstruction from 333 frames corresponding to 6.67 seconds acquisition time is shown. b) Zoom‐in of boxed region in (a). c) Corresponding averaged image for boxed region in (a). d) HeLa cells were incubated with TPP‐BCN (10 μm), washed, and labeled with f ‐HM‐SiR (2 μm). Reconstruction from 500 frames (10 seconds) is shown. e) Averaged cross‐sectional profiles from mitochondrial tubules after alignment, ±1 standard deviation, n=11. f) Width of individual profiles shown in (e). See Figure S11 in the Supporting Information for position off all profiles. g) Zoom‐in of boxed region in (d). Average (left) and reconstructions from 500 frames at different time points (right). Arrowhead indicates mitochondrial fusion event. h) Average (top left) and reconstructions of boxed region in (g). Localizations are colored with respect to their relative time of appearance within a single reconstruction. Scale bars: a) 5 μm, d) 10 μm, g) 2 μm, h) 1 μm.

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