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Self‐labeling protein tags can introduce advanced molecular motifs to specific cellular proteins. Here we introduce the third‐generation covalent TMP‐tag (TMP‐tag3) and showcase its comparison with HaloTag and SNAP‐tag. TMP‐tag3 is based on a proximity‐induced covalent Michael addition between an engineered Cys of E. coli dihydrofolate reductase (eDHFR) and optimized trimethoprim (TMP)–acrylamide conjugates with minimal linkers. Compared to previous versions, the TMP‐tag3 features an enhanced permeability when conjugated to fluorogenic spirocyclic rhodamines. As a small protein, the 18‐kD eDHFR is advantageous in tagging selected mitochondrial proteins which are less compatible with bulkier HaloTag fusions. The proximal N−C termini of eDHFR also enable facile insertion into various protein loops. TMP‐tag3, HaloTag, and SNAP‐tag are orthogonal to each other, collectively forming a toolbox for multiplexed live‐cell imaging of cellular proteins under fluorescence nanoscopy. [ABSTRACT FROM AUTHOR]

Multi-functional florescent dyes capable of acting as molecular probes in living systems under two-photon microscopy, as well as super-resolution nanoscopy, are of great interest. Herein, a novel donor–π–acceptor (D–π–A) type pyrimidine imidazole derivative with a flexible alkyl chain molecule (EX-1) has been efficiently synthesized through improved Knoevenagel condensation with high yield and relatively large 2PA cross-sections. Furthermore, a cytotoxicity assay and photo-resistance evaluation indicated that such small organic dyes could not only effectively target the endoplasmic reticulum in living cells, but also reveal its ultrastructure under stimulated emission depletion (STED) nanoscopy. [ABSTRACT FROM AUTHOR]

Fluorescent dyes synergize with advanced microscopy for researchers to investigate the location and dynamic processes of biomacromolecules with high spatial and temporal resolution. However, the instability of fluorescent dyes, including photobleaching and photoconversion, represent fundamental limits for super-resolution and time-lapse imaging. In this review, we discuss the latest advances in improving the photostability of fluorescent dyes. We summarize the primary photobleaching processes of cyanine and rhodamine dyes and highlight a range of strategies developed in recent years to strengthen these fluorophores. Additionally, we discuss the influence of protein microenvironments and labeling methods on the photostability of fluorophores. We aim to inspire next-generation robust and bright fluorophores that ultimately enable the routine practice of time-lapse super-resolution imaging of live cells. [ABSTRACT FROM AUTHOR]