A new series of D-
Fluorescence biomarker has become a powerful tool for the monitoring and investigating of cellular processes in live tissues, as well as
Compared to single-photon imaging, two-photon imaging shows significant benefits, such as deep tissue penetration and less photobleaching [
See Scheme
Synthesis routes for compounds
All chemicals were commercially available and used without further purification. The solvents were purified by conventional methods before being used. 4-(1H-Imidazol-1-yl)benzaldehyde was synthesized according to the methods reported. The 1H-NMR and 13C-NMR spectra recorded at 25°C using Bruker Avance 400 spectrometer were reported as parts per million (ppm) from TMS. Mass spectra were determined with a Micromass GCT-MS (EI source).
X-ray diffraction data of single crystals were collected on CCD diffractometer. The determination of unit cell parameters and data collections were performed with Mo-K
Electronic absorption spectra were obtained on a UV-265 spectrophotometer. Fluorescence measurements were performed using a Hitachi F-7000 fluorescence spectrophotometer. For time-resolved fluorescence measurements, the fluorescence signals were collimated and focused onto the entrance slit of a monochromator with the output plane equipped with a photomultiplier tube (HORIBA HuoroMax-4P). The decays were analyzed by “least-squares.” The quality of the exponential fits was evaluated by the goodness of fit (
4-(1H-Imidazol-1-yl)benzaldehyde was prepared according to the literature method [
The single crystals of
The UV-vis absorption spectra of the three chromophores
Photophysical properties of
Compound | Solvent |
|
|
|
|
|
Stokes’ shift (cm−1)[e] |
---|---|---|---|---|---|---|---|
|
Benzene | 299, 377 | 4.39, 4.55 | 428 | 0.61 | 1.71 | 10080, 3160 |
DCM | 296, 375 | 4.37, 4.52 | 458 | 0.67 | 2.28 | 11949, 4832 | |
Ethanol | 294, 371 | 4.42, 4.56 | 454 | 0.59 | 2.33 | 11987, 4927 | |
Ethyl acetate | 296, 371 | 4.43, 4.54 | 444 | 0.37 | 1.97 | 11261, 4431 | |
DMF | 297, 374 | 4.37, 4.53 | 466 | 0.59 | 2.54 | 12210, 5278 | |
|
|||||||
|
Benzene | 297, 383 | 4.46, 4.46 | 448 | 0.37 | 1.66 | 11348, 3788 |
DCM | 296, 389 | 4.47, 4.55 | 499 | 0.28 | 1.86 | 13743, 5666 | |
Ethanol | 294, 381 | 4.50, 4.64 | 494 | 0.18 | 1.37 | 13770, 6003 | |
Ethyl acetate | 294, 375 | 4.50, 4.56 | 455 | 0.35 | 1.84 | 12035, 4688 | |
DMF | 296, 380 | 4.51, 4.65 | 505 | 0.23 | 2.14 | 13981, 6513 | |
|
|||||||
|
Benzene | 298, 389 | 4.15, 4.19 | 460 | 0.69 | 1.53 | 11817, 3967 |
DCM | 295, 393 | 4.18, 4.27 | 509 | 0.42 | 1.97 | 14251, 5798 | |
Ethanol | 294, 381 | 4.27, 4.40 | 494 | 0.33 | 1.37 | 13770, 6003 | |
Ethyl acetate | 294, 377 | 4.36, 4.45 | 469 | 0.47 | 1.92 | 12691, 5203 | |
DMF | 296, 381 | 4.16, 4.30 | 505 | 0.45 | 2.21 | 13981, 6444 |
The normalized UV-vis spectra (a) and single-photon fluorescence spectra (b) of
The single-photon fluorescence spectra of
The chromophores exhibit high quantum yield (>0.1) in various solvents. Importantly, the quantum yields (
The Lippert-Mataga equation (shown in Supplementary Material) is widely used to evaluate the dipole moment changes of the dyes with photoexcitation [
Lippert-Mataga plots for
2PA cross sections of the compounds were determined by two-photon excited fluorescence (2PEF) method in near-IR (NIR) range from 700 to 1000 nm. Experiments revealed that linear absorption did not exist from 500 nm to 900 nm for all the chromophores, indicating that there were no molecular energy levels corresponding to an electron transition in this spectral range. Hence, upon excitation from 700 nm to 1000 nm at intervals of 10 nm (similarly hereinafter), it was impossible to produce single-photon excited upconverted fluorescence. If frequency upconverted fluorescence appeared upon excitation with a tunable laser in this range, it should be safely attributed to multiphoton excited fluorescence.
By tuning the pump wavelengths incrementally from 700 to 880 nm, keeping the input power fixed, the 2PEF intensities were recorded. As shown in Figure S5 all of the chromophores exhibit fluorescence in the wavelength range of 720–880 nm. Figure S4 shows the linear dependence on the square of input laser power which suggests a two-photon excitation mechanism at 760 nm for the chromophores. The normalized 2PEF peak positions of
The normalized two-photon excited fluorescence spectra of
The two-photon absorption (2PA) cross section (
Two-photon absorption cross sections of
Due to their high quantum yield, long fluorescence lifetime, and large 2PA cross section, these three chromophores potentially can be utilized in bioimaging.
Before exploring their biological applications, cytotoxicities of the chromophores were firstly measured toward the human cervical carcinoma cells (HepG2) by MTT assay, a standard method to probe cell survival fraction. Figure
Cytotoxicity data results of
Afterwards, fluorescent images of confocal microscopy and two-photon microscopy of HepG2 cells labeled with the three fluorescent probes were captured, along with differential interference contrast (DIC) micrographs. As shown in Figure
(a) One-photon image of HepG2 cells incubated with 20
Confocal fluorescence imaging reveals that
For the imidazolium salts (
In this contribution, three imidazole derivatives were designed and synthesized. UV-vis absorption, single-photon fluorescence, two-photon absorption characters, and two-photon fluorescence microscopy (2PFM) are systematically investigated. It was discovered that there is almost no influence on the UV-vis absorption of the chromophores while varying with the polarity of the solvents. However, an obvious influence on their fluorescent spectra can be observed. On account of large 2PA cross sections in the near-IR region, the imidazolium derivatives can selectively stain cell well which make them potential bioimaging applications in the future.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Yingzhong Zhu and Lufei Xiao contributed equally to this work.
This work was supported by a grant for the National Natural science Foundation of China (21271004, 51372003, 51432001, and 51271003), Ministry of Education Funded Projects Focus on Returned Overseas Scholar, Program for New Century Excellent Talents in University (China), Doctoral Program Foundation of Ministry of Education of China (20113401110004), Department of Education of Anhui province (KJ2014A190).