ABSTRACT
We report the synthesis and characterization of two amine reactive fluorescent dyes with efficient two-photon absorption (2PA) properties and high fluorescence quantum yields. Bioconjugation of these dyes with the DC-101 antibody proved to be useful for selectively imaging the vascular endothelial growth factor receptor 2 (VEGFR-2) in cells expressing this receptor in vitro and in "whole" mounted excised tumors (ex vivo) by two-photon fluorescence microscopy (2PFM). The penetration depths reached within the tumors by 2PFM was over 800 µm. In addition, the concentration of dye required for incubation of these bioconjugates was in the picomolar domain, the probes possessed very good photostability, and the 2PFM setup did not require any additional means of increasing the collection efficiencies of fluorescent photons to achieve the relatively deep tissue imaging that was realized, due, in large part, to the favorable photophysical properties of the new probes.
Subject(s)
Antibodies, Monoclonal/chemistry , Carcinoma, Lewis Lung/diagnosis , Fluorescent Dyes/chemistry , Immunoconjugates/chemistry , Vascular Endothelial Growth Factor Receptor-2/analysis , Animals , Antibodies, Monoclonal/immunology , Cell Line , Humans , Immunoconjugates/immunology , Male , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Models, Molecular , Molecular Imaging , Swine , Vascular Endothelial Growth Factor Receptor-2/immunologyABSTRACT
We report two-photon fluorescence microscopy (2PFM) imaging and in vitro cell viability of a new, efficient, lysosome-selective system based on a two-photon absorbing (2PA) fluorescent probe (I) encapsulated in Pluronic® F-127 micelles. Preparation of dye I was accomplished via microwave-assisted synthesis, resulting in improved yields and reduced reaction times. Photophysical characterization revealed notable 2PA efficiency of this probe.
Subject(s)
Fluorescence , Fluorescent Dyes/chemical synthesis , Lysosomes/metabolism , Micelles , Molecular Imaging/methods , Animals , Cell Survival , Humans , PoloxamerABSTRACT
The synthesis, linear photophysical, and photochemical parameters, two-photon absorption (2PA), and superfluorescence properties of 2,2'-(5,5'-(9,9-didecyl-9H-fluorene-2,7-diyl)bis(ethyne-2,1-diyl)bis(thiophene-5,2-diyl))dibenzo[d]thiazole (1) were investigated, suggesting its potential as an efficient fluorescent probe for bioimaging applications. The steady-state absorption, fluorescence, and excitation anisotropy spectra of 1 were measured in several organic solvents and aqueous media. Probe 1 exhibited high fluorescence quantum yield (~0.7-0.8) and photochemical stability (photobleaching quantum yield ~(3-7) × 10(-6)). The 2PA spectra were determined over a broad spectral range (640-920 nm) using a standard two-photon induced fluorescence method under femtosecond excitation. A well-defined two-photon allowed absorption band at 680-720 nm with corresponding 2PA cross sections δ(2PA) ≈ 800-900 GM was observed. The use of probe 1 in bioimaging was shown via one- and two-photon fluorescence imaging of HCT-116 cells. An amplification of the stimulated emission of 1 was demonstrated in organic solvents and thin polystyrene films, which potentially can be used for the development of new fluorescent labels with increased spectral brightness.
Subject(s)
Fluorescent Dyes/chemistry , Photons , Thiazoles/chemistry , Thiophenes/chemistry , Absorption , Endosomes/metabolism , Fluorescence Polarization , Fluorescent Dyes/chemical synthesis , HCT116 Cells , Humans , Lasers , Lysosomes/metabolism , Microscopy, Fluorescence , Solvents/chemistry , Thiazoles/chemical synthesis , Thiophenes/chemical synthesisABSTRACT
The synthesis, structural, and photophysical characterization of a series of new fluorescent donor-acceptor and acceptor-acceptor molecules, based on the fluorenyl ring system, with two-photon absorbing properties is described. These new compounds exhibited large Stokes shifts, high fluorescence quantum yields, and significantly, high two-photon absorption cross sections, making them well suited for two-photon fluorescence microscopy imaging. Confocal and two-photon fluorescence microscopy imaging of COS-7 and HCT 116 cells incubated with probe I showed endosomal selectivity, demonstrating the potential of this class of fluorescent probes in multiphoton fluorescence microscopy.
Subject(s)
Fluorenes/chemistry , Fluorescent Dyes/chemical synthesis , Molecular Imaging , Photons , Absorption , Fluorescence , Fluorescent Dyes/chemistry , HCT116 Cells , Humans , Microscopy, Confocal , Molecular Structure , PhotochemistryABSTRACT
Microwave-assisted synthesis of triarylsulfonium salt photoacid generators (PAGs) afforded reaction times 90 to 420 times faster than conventional thermal conditions, with photoacid quantum yields of new sulfonium PAGs ranging from 0.01 to 0.4.
ABSTRACT
We report the photochemistry and development of a fluorescence readout-based, nonlinear absorption, three-dimensional optical data storage system. In this system, writing was achieved by acid generation upon two-photon absorption (2PA) of a photoacid generator (PAG; at 710 or 730 nm). Readout was then performed by interrogating two-photon-absorbing dyes, after protonation, at 860 nm. Linear and nonlinear photophysical characterization of 2PA PAGs and acid-sensitive fluorescent dyes demonstrates good spectral resolution between the PAG and protonated 2PA dye and relatively high two-photon absorptivity. Solution spectroscopic studies confirm photoacid generation and dye protonation. Two-photon recording and readout of voxels were demonstrated in five and eight consecutive, crosstalk-free layers within a polymer matrix, generating a data storage capacity of up to 1.8 x 10(13) bits/cm(3).
