Synthesis and in vitro characterization of pcHoechst. A) Hoechst33258 binds to the minor groove of DNA (PDB ID: 8BNA), showing a disordered, partly solvent‐exposed piperazine. B) pcHoechst is obtained by benzylation of Hoechst33342 with an o‐nitro benzyl photocage that can be removed with UV light to re‐obtain Hoechst33342. C) Chromatograms of Hoechst33342, pcHoechst and a sample of pcHoechst that was irradiated for 5 min with white light show that the appearance of Hoechst33342 (top) matches the occurrence of Hoechst after pcHoechst irradiation by retention time and molecular weight (enlargement below). D) Concentration‐response curve for Hoechst33342 and pcHoechst against hairpin (hp) DNA. No fluorescence can be detected for pcHoechst. E) UV/Vis spectra of Hoechst33342, pcHoechst and o‐nitrobenzylbromide 1. Mathematical addition of Hoechst33342 plus compound 1 is drawn as a dashed line. F) UV/Vis spectra of Hoechst33342 and pcHoechst in the presence of 0.1 mM hpDNA (top) with enlargement (bottom). G) Fluorescence emission spectra of Hoechst33342 and pcHoechst in the presence of 0.1 mM hpDNA. H) Quantum yield measurements of Hoechst33342 and pcHoechst in the absence (control) and presence of 0.1 mM hpDNA.

In cellulo uncaging of pcHoechst. A) Epifluorescence imaging of live HeLa cells incubated with Hoechst33342 or pcHoechst (10 μM each) before and after 4 s of UV light irradiation shows an increase in nuclear fluorescence for pcHoechst‐treated cells. Scale bar: 20 μm. B) As in (A), but phase contrast images. C) Cells from (A) were imaged over 24 h to determine single cell viability to progress through a complete cell cycle during compound and UV treatment, numbers indicate absolute cell division events. D) Confocal images of live HeLa cells incubated with pcHoechst (10 μM). Between each image, UV light was applied with higher intensity to uncage pcHoechst. Scale bar: 20 μm. E) Fluorescence increase over time upon pcHoechst uncaging (n=15 cells). F) Line scans result in fluorescence increase over time upon pcHoechst uncaging (from dashed line in D). G) Colocalization of cytosolic signals from pcHoechst with the lysosomal system labeled with LysoTracker Red. Scale bar: 5 μm. H) Pearson's correlation R value for cytosolic colocalization is positive for co‐applied pcHoechst and LysoTracker Red, and negative in controls, thus arguing for extranuclear signals stemming from acidic compartments; n=6 cells.

In vivo imaging of cell nuclei in zebrafish with pcHoechst. A) General schematic workflow for experiments in Figures 3, 4, S11, and S12. H2B‐RFP mRNA is injected into single‐cell‐stage zebrafish embryos for correlative nuclear staining of pcHoechst with a red fluorescent DNA‐associated fusion protein (Histone 2B fused to mRFP). Hoechst33342 or pcHoechst is applied (between 32–56 hpf), with 4–16 hours′ incubation in the dark before targeted uncaging and subsequent imaging. B) Agarose‐embedded H2B‐RFP‐expressing zebrafish embryos were incubated with 10 μM Hoechst 33342 (upper row) or 100 μM pcHoechst (lower row) overnight at 32 hpf. Spot irradiation using a 405 nm UV laser targeted at a large cluster of cells was performed for uncaging pcHoechst at 52 hpf. Confocal imaging of the targeted area at the zebrafish tail fin at 54 hpf shows colocalization of pcHoechst (blue, bottom row) with mRFP‐tagged H2B (red) in comparison to Hoechst33342 (blue, top row). Merge includes bright‐field image. The shown images are representative of three uncaging experiments using the same parameters.

In vivo uncaging of pcHoechst with optimized light intensity at single‐cell resolution. H2B‐mRFP (red) injected zebrafish embryos were incubated with 100 μM pcHoechst in the dark overnight and activated by using a 405 nm UV laser at 0.07 mW and at 74 hpf. The arrow indicates a nucleus targeted with two bleach points. A pre‐bleach image was recorded prior to illumination, post bleach images 7.5 min and 1.5 h after bleaching. The white rectangles depict magnified areas. Uncaged pcHoechst (blue) can be observed 7.5 min and 1.5 h after UV illumination and colocalizes with H2B‐mRFP (red) in a single nucleus. Representative images of two experiments with the same intensity, and ten experiments in total.

pcHoechst uncaging allows cell targeting and subnuclear visualization of DNA. A) Confocal imaging of live HeLa cells incubated with pcHoechst (100 nM) using a FRAP bleach point with UV light in the indicated area (white circle) shows nuclear staining. High laser power (110–1100 μW for 10–15 s) activates the entire cell. Scale bar: 10 μm. B) As for (A) but with lower laser power (1.42 μW for 10–15 s) allows more precise uncaging in a fraction of the nucleus C) with a resolution of 1.4–1.7 μm that D) remained stable over 90 s. Representative images from three repetitions. E) Confocal images of live HeLa cells incubated with pcHoechst (10 μM) targeting a bleach point with a two‐photon laser (720 nm, 19.7 mW) in the indicated area (white circle) shows subnuclear staining in the illuminated periphery. Representative images of n>15 cells. Scale bar: 10 μm.