Novel photochromic probes for enhanced contrast and super-resolution imaging

FLORENCIA GILLANDERS; ANTHONY DE VRIES; THOMAS JOVIN; NATHAN COOK; VAISHALI CHAKRABARTI; DONNA ARNDT-JOVIN; PEDRO ARAMENDÍA

Conferencia; Quantitative Bioimaging Conference; 2016

Photomodulatable fluorescent probes are key elements in many modalities for superresolution (PALM, STORM) and enhanced contrast ?lock-in? [Marriott] microscopies. Fluorescent photochromic (pc) diheteroarylethenes are of particular interest because they feature reversible photoswitching between a dark and a bright isomer via ring-closure induced by near UV light and cycloreversion by exposure to visible light ([1], Fig. 1). Certain substituents also confer a pronounced environmental sensitivity (solvatochromism) on the fluorescent closed form [1]. Unfortunately, diheteroarylethenes are poorly soluble in water ? a drawback for applications in cellular microscopy. We have explored various methods for increasing solubility while maintaining the desired emission and photoswitching properties. One approach is based on the covalent coupling of mono and bis-reactive maleimide derivatives to cysteine containing proteins. Two successful examples are alpha-synuclein and a goat anti-rabbit secondary antibody. The latter (denoted pcAb) is readily photoconvertible and constitutes to our knowledge, the first photochromic antibody suitable for immunocytochemistry. Optical sectioning with the Programmable Array Microscope (PAM, [2]; other presentations) yielded excellent optically-sectioned images of cytoskeletal elements (actin, tubulin) in various cell types and of b-tubulin in neuronal cells (see figure below). Reversible photoconversion is demonstrated by checkerboard patterns of successive UV and visible irradiation. The combination of pc probes and structured illumination provide the basis for novel superresolution strategies.Acknowledgments FG acknowledges the DAAD, the Ministerio de Educación of the Republic of Argentina, the Fundación Bunge y Born, the Max Planck Society and the CONICET for financial support of her PhD. [1] F. Gillanders, L. Giordano, S. A. Díaz, T. M. Jovin and E. A. Jares-Erijman, 'Photoswitchable fluorescent diheteroarylethenes: substituent effects on photochromic and solvatochromic properties,' Photochem. Photobio. Sci., 13, 603 (2014).