Two-photon STED spectral determination for a new V-shaped organic fluorescent probe with efficient two-photon absorption

Abstract

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.

Figure 1

Normalized steady-state absorption (1–6) and fluorescence (1′-6′) spectra of 1 in CHX (1, 1′), TOL (3, 2′), CHCl₃ (5, 3′), THF (2, 4′), ODCB (6, 5′) and CH₂Cl₂ (4, 6′).

Figure 2

(a) Lippert plot for 1. (b) Excitation anisotropy spectra of 1 in pTHF (—), CHX (—), ODCB (—), CH₂Cl₂ (—), THF (—), TOL (—), CHCl₃ (—) and normalized absorbance in CHX (1) and ODCB (2).

Figure 3

Calculated electronic spectrum of 1 in vacuum (vertical black lines, value of each) corresponds to the oscillator strength) and normalized absorption spectrum of 1 in CHX (blue).

Figure 4

Spectroscopic data of 1 in CHCl₃: 2PA spectrum obtained by open aperture Z-scan (1) and 2PF (1′) methods; one- (2) and two-photon (3) stimulated emission spectra; normalized steady-state absorption (4) and fluorescence (5) spectra.

Figure 5

Dependences 1–I_F / I_F0 = f (^qE_P) and $1-I_F/I_{F0}=f({{}^{q}E}_P^2)$ for fluorescence quenching of 1 in CHCl₃. (a) One-photon STED at 710 nm (blue circles), 750 nm (green circles), and 830 nm (red circles). (b) Two-photon STED at 1280 nm (blue circles), 1360 nm (green circles), and 1400 nm (red circles). Solid black lines are linear fittings.

Figure 6

Images of HCT 116 cells incubated with micelle-encapsulated 1 (50 μM, 2 h). (A) DIC, (B) one-photon fluorescence microscopy image, (C) 3D reconstruction from overlaid two-photon fluorescence microscopy images (Ex: 940 nm; power: 120 mW; Em. short-pass filter 800 nm); 5 μm grid.

Figure 7

Schematic diagram of the experimental setup: M - 100 % reflection mirrors; B - beam splitters; SM - spectrometer; BBO - 1 mm crystal; F - set of neutral and/or interferometric filters; SF - space filters; WP - wave plate λ / 2 ; P - polarizer; DL - optical delay line with retro-reflector; Sample - 1 mm quartz cuvette containing sample; FSM - fiber optic spectrometer HR4000; PD -calibrated Si and/or InGaAs photodetectors; L - focusing lenses; BD - beam dump.

Scheme 1

Synthesis of fluorenyl-probe 1