Morales Alma R, Schafer-Hales Katherine J, Marcus Adam I, Belfield Kevin D
Department of Chemistry, The College of Optics and Photonics, University of Central Florida, P.O. Box 162366, Orlando, Florida 32816-2366, USA.
Bioconjug Chem. 2008 Dec;19(12):2559-67. doi: 10.1021/bc800415t.
With the increasing demand for confocal and two-photon fluorescence imaging, the availability of reactive probes that possess high two-photon absorptivity, high fluorescence quantum yield, and high photostability is of paramount importance. To address the demand for better-performing probes, we prepared two-photon absorbing amine-reactive fluorenyl-based probes 2-(9,9-bis(2-(2-methoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)benzothiazole (1) and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)vinyl)phenyl)benzothiazole (2), incorporating the isothiocyanate as a reactive linker. Probe design was augmented by integrating high optical nonlinearities, increased hydrophilicity, and coupling with reactive functional groups for specific targeting of biomolecules, assuring a better impact on two-photon fluorescence microscopy (2PFM) imaging. The isothiocyanate (NCS) derivatives were conjugated with cyclic peptide RGDfK and Reelin protein. The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described. The conjugates displayed high chemical stability and photostability. The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially "lighting up" after conjugation. Conventional and 2PFM imaging and fluorescence lifetime imaging (FLIM) of HeLa, NT2, and H1299 cells, incubated with two-photon absorbing amine-reactive probe (1), RGDfK-dye conjugate (7), and Reelin-dye conjugate (6), was demonstrated.
随着对共聚焦和双光子荧光成像的需求不断增加，拥有高双光子吸收率、高荧光量子产率和高光稳定性的活性探针的可用性至关重要。为了满足对性能更好的探针的需求，我们制备了双光子吸收胺反应性芴基探针2-(9,9-双(2-(2-甲氧基乙氧基)乙基)-2-异硫氰酸酯基-9H-芴-7-基)苯并噻唑(1)和2-(4-(2-(9,9-双(2-(2-乙氧基乙氧基)乙基)-2-异硫氰酸酯基-9H-芴-7-基)乙烯基)苯基)苯并噻唑(2)，将异硫氰酸酯作为反应性连接基。通过整合高光学非线性、增加亲水性以及与用于生物分子特异性靶向的反应性功能基团偶联来增强探针设计，确保对双光子荧光显微镜(2PFM)成像有更好的影响。异硫氰酸酯(NCS)衍生物与环肽RGDfK和Reelin蛋白共轭。描述了新标记试剂以及共轭物的化学和光物理性质的研究。共轭物表现出高化学稳定性和光稳定性。NCS衍生物的荧光量子产率较低，而它们的生物共轭物表现出高荧光量子产率，基本上在共轭后“点亮”。展示了用双光子吸收胺反应性探针(1)、RGDfK-染料共轭物(7)和Reelin-染料共轭物(6)孵育的HeLa、NT2和H1299细胞的传统和2PFM成像以及荧光寿命成像(FLIM)。
