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. For MaP555-CA the combination of HaloTag9, HaloTag10 and HaloTag11 shows both a large circumference and an α close to 90°. However, other factors are also of importance (see c). Separation of four components using the phasor approach solely based on lifetime information only becomes possible using transformation into higher harmonics 3.. Live-Cell Fluorescence Lifetime Multiplexing Using Synthetic .. MaP555-BG, MaP555-CA, and MaP618-CA were prepared according to literature procedures by B. Réssy or D. Schmidt (MPI-MR). All other probes were purchased from commercial vendors (Supporting Table S1), or obtained from Spirochrome

Fluorophores were prepared as stock solutions in dry DMSO and diluted in imaging medium such that the final .. Exchangeable HaloTag Ligands for Super-Resolution Fluorescence .. A panel of fluorogenic xHTLs (MaP555-T5, MaP618-S5, JF 635-S5, SiR-S5, and SiR-T5) was characterized in multiframe STED imaging of mitochondria in live U2OS cells. Overall, the STED-frame numbers with >50% signal intensity (τ 1/2 ) was increased by 3- to 5-fold compared to covalent labeling for all fluorophores tested ( Figure S12C-H ).. Reversible Live‐Cell Labeling with Retro‐engineered HaloTags Enables .. In turn, the MaP555 signal remained almost unchanged with a decrease largely caused by photobleaching map555. In contrast, efficient exchange of HTL-MaP555 by HTL-MaP618 was observed for the reHaloTags (Supplementary Figure S1). Interestingly, we could see that this exchange was substantially faster for reHaloTagF as compared to reHaloTagS.. Reversible Live-Cell Labeling with Retro-engineered HaloTags Enables .. 200 nM HTL-MaP555. After the initial frame, 800 nM HTL-MaP618 was added to the microscopy chamber map555. For the HaloTag7, only a slight fluorescence increase in the MaP618 channel was observed, which was largely at the level of unspecific background (Supplementary Figure S1B)

In turn, the MaP555 signal remained almost unchanged with a