A Photochromic Bioconjugate with Photoactivable Fluorescence for Superresolution Imaging

JANET CUSIDO; SHERIF SHABAN RAGAB; EK RAJ THAPAPALIVA; SUBRAMANI SWAMINATHAN; JAUME GARCIA AMOROS; M. JULIA ROBERTI; BEATRIZ ARAOZ; MERCEDEZ M. A. MAZZA; SHIORI YAMAZAKI; AMY M. SCOTT; FRANCISCO M. RAYMO; MARIANO L. BOSSI

JOURNAL OF PHYSICAL CHEMISTRY C

AMER CHEMICAL SOC

Lugar: Washington; Año: 2016 vol. 120 p. 12860 - 12870

1932-7447

A coumarin fluorophore and an oxazine photochrome can be integrated within the same molecular skeleton and connected covalently to a secondary antibody. Illumination of the antibody?dyad conjugate at an appropriate activation wavelength opens the oxazine ring reversibly and shifts bathochromically the ground-state absorption of the coumarin component. Selective excitation of the photochemical product then produces significant fluorescence and allows the detection of activated bioconjugates at the single-molecule level. Such fluorescence activation events can be exploited to resolve temporally individual emitters and reconstruct images of immunolabeled cells with subdiffraction resolution. Relying on a similar conjugation protocol, a model compound, incorporating the same chromophore of the photochemical product, can also be connected covalently to a secondary antibody. Stimulated emission can be exploited to deplete the excited state of the bioconjugated chromophores and switch their fluorescence off. These operating principles for fluorescence switching also permit the imaging of immunolabeled cells with subdiffraction resolution. Thus, these photoswitchable molecules, in combination with the labeling ability of antibodies, can evolve into valuable probes for bioimaging with superresolution.