Deoxyribonucleoside-modified squaraines were synthesized by Sonogashira coupling reactions using an unsymmetrical, terminal alkynylated benzothiazolium squaraine dye. These non-natural nucleosides exhibited fluorescent ‘turn-on’ properties in viscous conditions with an enhancement of >300-fold. The viscosity-dependent fluorescence enhancement was attributed to a combination of hampering both molecular aggregation and intramolecular bond rotation of the squaraine probe. Fluorescence microscopy allowed visualization of highly viscous regions during various stages of cellular mitosis.

The synthesis, linear photophysical, two-photon absorption (2PA), femtosecond transient absorption, and superfluorescence properties of a new symmetrical squaraine derivative (1) are reported. Steady-state linear spectral and photochemical properties, fluorescence lifetimes, and excitation anisotropy of 1 were investigated in various organic solvents. High fluorescence quantum yields (≈0.7) and very high photostability (photodecomposition quantum yields ≈10⁻⁶–10⁻⁸) were observed. An open-aperture Z-scan method was used to obtain 2PA spectra of 1 over a broad spectral range (maximum 2PA cross section ≈1000 GM). Excited-state absorption (ESA) and gain was observed by femtosecond transient absorption spectroscopy, in which both reached a maximum at approximately 500 fs. Squaraine 1 exhibits efficient superfluorescence. The quantum chemical study of 1 revealed the simulated vibronic nature of the 1PA and 2PA spectra were in good agreement with experimental data; this may provide the ability to predict potential advanced photonic materials.

With the extensive use of two-photon fluorescence microscopy (2PFM) in the biomedical field, the need for development of fluorescent probes with improved two-photon fluorescence (2PF) properties has triggered extensive studies in the synthesis of new probes that undergo efficient two-photon absorption (2PA). In order to provide a more comprehensive comparison of fluorophores for 2PF bioimaging, a figure of merit (F_M) was developed by normalizing the 2PA action cross-section, a commonly used parameter for characterizing bioimaging 2PF probes, by the photodecomposition quantum yield. Another important aspect of developing 2PA fluorophores is hydrophilicity. Although hydrophilic fluorophores are generally preferred in 2PFM bioimaging, hydrophobic fluorophores are typically easier to synthesize and purify, and have been used successfully in 2PFM bioimaging. The methodologies of dispersing hydrophobic fluorophores into aqueous media, such as in a DMSO/water mixture, micelles, silica nanoparticles, or forming polymer nanoparticles, are reviewed. The design and synthesis of hydrophilic 2PA fluorophores, achieved by introducing polyethylene glycol (PEG), anionic acid groups, cationic ammonium salt, and PAMAM dendrimers, is presented. Introduction of specificity to target certain biomarkers via labeling of antibodies, DNA, smallbioactive molecules, and peptides, and for the sensing of sepcific cations and pH, is also reviewed.

The synthesis, comprehensive linear photophysical characterization, two-photon absorption (2PA), steady-state and time-resolved stimulated emission depletion properties of a new fluorene derivative, (E)-1-(2-(di-p-tolylamino)-9,9-diethyl-9H-fluoren-7-yl)-3-(thiophen-2-yl)prop-2-en-1-one (1), are reported. The primary linear spectral properties, including excitation anisotropy, fluorescence lifetimes, and photostability, were investigated in a number of aprotic solvents at room temperature. The degenerate 2PA spectra of 1 were obtained with open-aperture Z-scan and two-photon induced fluorescence methods, using a 1 kHz femtosecond laser system, and maximum 2PA cross-sections of ∼400–600 GM were obtained. The nature of the electronic absorption processes in 1 was investigated by DFT-based quantum chemical methods implemented in the Gaussian 09 program. The one- and two-photon stimulated emission spectra of 1 were measured over a broad spectral range using a femtosecond pump–probe-based fluorescence quenching technique, while a new methodology for time-resolved fluorescence emission spectroscopy is proposed. An effective application of 1 in fluorescence bioimaging was demonstrated by means of one- and two-photon fluorescence microscopy images of HCT 116 cells containing dye encapsulated micelles.

Two-photon stimulated emission depletion (STED) cross sections were determined over a broad spectral range for a novel two-photon absorbing organic molecule, representing the first such report. The synthesis, comprehensive linear photophysical, two-photon absorption (2PA), and stimulated emission properties of a new fluorene-based compound, (E)-2-{3-[2-(7-(diphenylamino)-9,9-diethyl-9H-fluoren-2-yl)vinyl]-5-methyl-4-oxocyclohexa-2,5-dienylidene} malononitrile (1), are presented. Linear spectral parameters, including excitation anisotropy and fluorescence lifetimes, were obtained over a broad range of organic solvents at room temperature. The degenerate two-photon absorption (2PA) spectrum of 1 was determined with a combination of the direct open-aperture Z-scan and relative two-photon-induced fluorescence methods using 1 kHz femtosecond excitation. The maximum value of the 2PA cross section ∼1700 GM was observed in the main, long wavelength, one-photon absorption band. One- and two-photon stimulated emission spectra of 1 were obtained over a broad spectral range using a femtosecond pump–probe technique, resulting in relatively high two-photon stimulated emission depletion cross sections (∼1200 GM). A potential application of 1 in bioimaging was demonstrated through one- and two-photon fluorescence microscopy images of HCT 116 cells incubated with micelle-encapsulated dye.