Near-Infrared-Emitting Squaraine Dyes with High 2PA Cross-Sections for Multiphoton Fluorescence Imaging

Abstract

Designed to achieve high two-photon absorptivity, new near infrared (NIR) emitting squaraine dyes, (E)-2-(1-(2-(2-methoxyethoxy)ethyl)-5-(3,4,5-trimethoxystyryl)-1H-pyrrol-2-yl)-4-(1-(2-(2-methoxyethoxy)ethyl)-5-(3,4,5-trimethoxystyryl)-2H-pyrrolium-2-ylidene)-3-oxocyclobut-1-enolate (1) and (Z)-2-(4-(dibutylamino)-2-hydroxyphenyl)-4-(4-(dibutyliminio)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1-enolate (2) were synthesized and characterized. Their linear photophysical properties were investigated via UV-visible absorption spectroscopy and fluorescence spectroscopy in various solvents, while their nonlinear photophysical properties were investigated using a combination of two-photon induced fluorescence and open aperture z-scan methods. Squaraine 1 exhibited a high two-photon absorption (2PA) cross section (δ2PA), ~ 20,000 GM at 800 nm, and high photostability with the photochemical decomposition quantum yield one order of magnitude lower than Cy 5, a commercially available pentamethine cyanine NIR dye. The cytotoxicity of the squaraine dyes were evaluated in HCT 116 and COS 7 cell lines to assess the potential of these probes for biomedical imaging. The viability of both cell lines was maintained above 80% at dye concentrations up to 30 μM, indicating good biocompatibility of the probes. Finally, one-photon fluorescence microscopy (1PFM) and two-photon fluorescence microscopy (2PFM) imaging was accomplished after incubation of micelle-encapsulated squaraine probes with HCT 116 and COS 7 cells, demonstrating their potential in 2PFM bioimaging.

Linear Photophysics and Femtosecond Nonlinear Spectroscopy of a Star-Shaped Squaraine Derivative with Efficient Two-Photon Absorption

“…25 −27 In particular, the majority of symmetrical squaraines exhibit sharp, intense long wavelength one-photon absorption (1PA) bands, 28,29 high two-photon absorption (2PA) efficiency (maxima cross sections ∼1·10 4 GM), 30,31 high fluorescence quantum yields (∼0.4−1.0), 32,33 and high photochemical stability, 11,34,35 that make them promising candidates for twophoton fluorescence microscopy (2PFM) applications, including bioimaging. 18,30 It is worth mentioning that some of the specific nonlinear optical properties such as superluminescence 36,37 (or amplified spontaneous emission) and lasing abilities were recently reported for symmetrical squaraines, 11 and can be employed in the development of a new generation of fluorescent labels with increased brightness and high spectral resolution. 38,39 Here we report the synthesis and comprehensive characterization of the linear spectroscopic, photochemical, and nonlinear optical properties of a three arm star-shaped squaraine derivative (1), including excitation anisotropy, two-photon absorption (2PA), femtosecond pump−probe spectroscopy, superluminescence, and symmetry breaking phenomena.…”

Superfluorescent Squaraine with Efficient Two‐Photon Absorption and High Photostability

“…Most symmetrical squaraines possess efficient two‐photon absorption (2PA) properties, which are typically exhibited in the two‐photon‐allowed short‐wavelength absorption bands under double‐resonance excitation conditions, whereby the effect of intermediate state resonance enhancement is realized 10. The intensive, linear absorption bands (typically the maximum of the extinction coefficient, ε max ≈1–4×10 5 M −1 cm −1 ;6b, 10b, 11) and large 2PA cross sections [ δ 2PA ≈ (1–3)×10 4 GM,12] are suitable for absorption in the biological transparency window and the emission spectral range;13 high fluorescence quantum yields— Φ fl of approximately 0.5 – 1.04b,c, 11—and high photostability5b, 14 strongly suggest that squaraine derivatives have good potential for applications in bioimaging,15 which includes two‐photon fluorescence microscopy (2PFM) 12c. In contrast to conventional (one‐photon) excited fluorescent labels, 2PA squaraine‐based fluorescent molecules have received less attention for two‐photon NIR excitation and need further research.…”

Bovine Serum Albumin Nanoparticles with Fluorogenic Near-IR-Emitting Squaraine Dyes

“…20-22 Normally, their absorption and emission spectra lie in the visible red and near-IR regions; as for fluorescence imaging studies, they are typically outside the self-absorption and autofluorescence regions of biological matrices. 23 These unique chemical and physical properties of squaraine molecules have led to studies of their application as near-IR fluorescent chromophores and environmental sensors, 24 for bioimaging and biochemical labeling, 25-28 chromo/fluorogenic probes, 29 pH responsive probes, 30 and metal ion recognition. 31-33 Despite optical advantageous, studies using squaraine dye as chromophores for bioimaging are scarce.…”

“…31-33 Despite optical advantageous, studies using squaraine dye as chromophores for bioimaging are scarce. 23,26,27,34-36 The low fluorescence quantum yield in aqueous solution as well as the strong tendency to form aggregates 21 hinders the performance of this potentially important class of materials. To address this problem, encapsulation of squaraine dyes with rotaxanes 25,26 and mesoporous silica nanoparticles 36 has been developed to provide protection of the squaraine core and inhibit aggregation.…”