Photostability Enhancement of Fluorenone-Based Two-Photon

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Photostability Enhancement of Fluorenone-Based Two-Photon Fluorescent Probes for Cellular Nucleus Monitoring and Imaging Shuheng Chi, Liang Li, and Yiqun Wu J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b02827 • Publication Date (Web): 06 Jun 2016 Downloaded from http://pubs.acs.org on June 11, 2016

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The Journal of Physical Chemistry

Photostability Enhancement of Fluorenone-Based Two-Photon Fluorescent Probes for Cellular Nucleus Monitoring and Imaging Shuheng Chi1,2,a, Liang Li1,2,b and Yiqun Wu*1,3,c 1

Key Laboratory of Material Science and Technology for High Power Lasers,

Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, NO.390 Qinghe Road, Jiading District, Shanghai 201800, PR China 2

3

University of Chinese Academy of Sciences, Beijing 100049, China

Key Lab of Functional Inorganic Material Chemistry, Heilongjiang University, NO.74 Xuefu Road, Nangang District, Harbin 150080, PR China a

[email protected], [email protected], *[email protected] *

Corresponding author. Tel.: +8602169918592

ACS Paragon Plus Environment


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ABSTRACT A series of fluorenone-based two-photon fluorescent probes with high photostability for nucleus imaging are prepared and developed. The one- and two-photon photophysical properties exhibit these new probes possess 0.448–0.634 of fluorescence quantum yields and 469–495 GM of two-photon absorption cross-sections at 800-nm femtosecond laser excitation. The luminescence “turn-on” experiment in buffer solutions indicates that 35-fold of fluorescence intensity and 68-fold fluorescence quantum yield enhancement appear between new probes and calf thymus DNA. In the nuclear double-staining experiment, the high mean co-localization coefficients of 0.92–0.96 between new probes and nuclear labeling dye Hoechst 33342 are acquired, demonstrating excellent nuclear localization in 3T3 cells. The counterstain studies by introducing commercial mitochondrial staining reagent MTR and nuclear staining dye DAPI further show good membrane permeability and counterstain compatibility in multicolor cell labeling application. The photostability studies show that 3000 s of observation time and 0.028%/s–0.03%/s of mean fluorescence attenuation rates under persistent laser irradiation in two-photon nuclear imaging are achieved. Meanwhile, the cause of fluorescence attenuation in the photostability test for cellular nuclei monitoring are discussed as well.


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1. INTRODUCTION The biological fluorescence imaging of living cells and tissues has attracted considerable attention for selective labeling and tracking. In particular, the study of novel biological fluorescent probes is currently a focus of research in the bioimaging field.1 Traditional one-photon excitation fluorescent (OPEF) microscopic technologies, whose excitation wavelength is mainly in the ultraviolet or visible region, are limited regarding fluorescence bioimaging owing to the photodamage, two-dimensionality, high Rayleigh scattering and poor spatial resolution. However, the excitation wavelength of two-photon fluorescence microscope is two times of the one-photon excitation, so it is located from the near-infrared to infrared wave band, which is considered to be the main biological optical window. Thus, the photon energy is much lower than that of the one-photon absorption. Meanwhile, the linear absorption and Rayleigh scattering of light energy in the material system are smaller, which could



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solve the tomographic problems of deep substances in biological tissues.2–4 Furthermore, due to the long distance between two-photon fluorescence wavelength and excitation wavelength, the dark field imaging can be realized. In the real-time monitoring and tracking of living cells and tissues, two-photon excitation could diminish the problems of photoinduced toxicity to a great extent.5–7 In addition, two-photon transtion has a strong selective excitation, which is conducive to the high-resolution imaging of some special sites in biological tissues.8–10 Therefore, with the continuous development of the two-photon confocal fluorescence microscope—which is considered to have excellent performance because of its three-dimensional spatial selectivity, high penetration depth, low Rayleigh scattering, and femtosecond laser source operating—two-photon excitation fluorescent (TPEF) probes have gradually gained attention in scientific research.11,12 Compared with one-photon fluorescent probes, two-photon fluorescent probes can label deep sites in biogical samples by virtue of three dimensional imaging capabilities of two-photon absorption. Moreover, the imaging can possess higher resolution and contrast ratio than that of one-photon absorption. Two-photon fluorescent probes with various morphologies, structures, and functions have been applied for bioimaging in cell organelles and living tissues, including graphitic-C3N4 quantum dots, ruthenium complexes, gold nanopatterns, acidic pH probes, and cyclometalated platinum.13–18 However, deficiencies such as the high cytotoxicity of the labels used for metalized complexes and the low photostability and high photobleaching of organic dyes seriously restrict the application of TPEF probes for the imaging of in vivo tissues and photodynamic therapy in biomedical science. The cellular nucleus is the major bioenvironment where the storage, replication, and transcription of genetic material, i.e., DNA, occurs.19 As an indispensable method in two-photon fluorescence bioimaging, the long-term persistent real-time detection of cellular nuclei is vitally important for the proliferation, migration, apoptosis, and morphological differentiation of living cells.20 So far, a number of two-photon probes have been studied for cellular DNA or other organelles targeting and imaging.21-25 However, studies involving organic small-molecule probes targeting cellular nucleus for the long-term monitoring and tracking are rarely reported.26,27 Thus, extreme attention should be paid to nucleus monitoring for the enhancement of photostability in biomedical imaging. Recently, some two-photon fluorescent labels for two-photon bioimaging in cells with relatively good photostability were reported. Xu et al.14 prepared a series of ruthenium complexes with polypyridyl groups for two-photon fluorescence cellular imaging. The fluorescence decay amplitude of one representative complex reached 38% when the irradiation time was in the 300 s range. Liu et al.28 developed two fluorescent probes for two-photon fluorescence monitoring of RNA in living cells. After 300 s of persistent irradiation, the fluorescence intensities of these two probes decreased by 8% and 28%, respectively. Miao et al.29 reported a pair of novel carbazole-based derivatives with unilateral pyridine moieties to selectively target mitochondria. The time-dependent fluorescence cells imaging was conducted within


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275 s, and the fluorescence intensity attenuation reached approximately 10–20%. These above observation time was not long enough especially for long-term detection in two-photon cell labeling and imaging. In addition, the reported fluorescence attenuation amplitude of these two-photon fluorescent probes did not seem very low, thus gradually influencing the imaging observation effects. Therefore, aiming at the above-mentioned problems, we decided to fill up the deficiency, and further expanded and optimized the results of scientific research. Herein, we report a series of fluorenone-based cationic derivatives for the first time (Scheme 1). The fluorenone moieties functioned as the central π-conjugated bridge, which were connected to the bilateral symmetric 4-vinyl pyridine rings with the unilateral cations. This rigid and stable structure effectively enlarged the π-conjugated delocalized plane as the length of the molecule chains increased. We employed two-photon fluorescent probes whose optimal excitation wavelengths were all in near-infrared biological optical window. The one-photon photophysical performance including fluorescence quantum yields by measuring absorption and fluorescence spectra were studied. The two-photon absorption (TPA) cross-sections in the excitation wavelength ranging from 750 to 840 nm under femtosecond laser system were caculated based on two-photon fluorescence spectra. The luminance “turn-on” effect was researched by exploring the affinities between new probes and DNA in Tris-HCl buffer solutions. The one- and two-photon fluorescence imaging and the capability of new probes to excusively label cellular nucleus were successfully exhibited. The photostability under persistent femtosecond laser irradiation in the process of cellular nucleus imaging was studied, meanwhile, the fluorescence attenuation mechanism in the photostability test was also analyzed and discussed.

2. EXPERIMENTAL SECTION 2.1 Materials and chemicals 2,7-Dibromo-9-fluorenone was purchased from Alfachem Co. (Zhengzhou, China). 4-Vinylpyridine was purchased from Alfa Aesar (USA). Reagents for the chemical reaction, ethiodide (C2H5I), iodobutane (C4H9I) and iodine hexane (C6H13I) were purchased from J&K Chemicals (Beijing, China). Other raw materials and solvents for the chemical reaction were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 4′,6-Diamidino-2-phenylindole (DAPI) was purchased from Sigma Co. (USA). Hoechst 33342 and MitoTracker Red CMXRos (MTR) were purchased from Invitrogen Co. (USA). Calf thymus DNA (ct-DNA) was purchased from Biodee Biotechnology Co., Ltd (Beijing, China). All reagents used in the chemical reactions were of analytical grade. The Tris-HCl buffer solution (10 mmol Tris, 100 mmol NaCl, pH = 7.4) was formulated and stored at 4 °C before use. The concentration of samples/DAPI and ct-DNA in buffer solutions was determined to be 1×10-5 M and 0–1.4×10-3 M, respectively. 2.2 Preparation procedures



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2.2.1 Synthesis of 2,7-bis((E)-2-(pyridin-4-yl)vinyl)-9H-fluoren-9-one (FOVPC) Firstly, 2,7-dibromo-9-fluorenone (4.06 g, 0.012 mol), 4-vinylpyridine (2.63 g, 0.025 mol), tri(o-tolyl)phosphine (0.73 g, 2.4 mmol), palladium acetate (0.11 g, 0.5 mmol), anhydrous potassium carbonate, and N-methyl-2-pyrrolidone (30 ml) were added into an airtight container that was filled with a certain pressure of high-purity nitrogen. Secondly, the reaction temperature was gradually increased to 125 °C under nitrogen, with a holding time of 20 h. Finally, the major product 2,7-bis((E)-2-(pyridin-4-yl)vinyl)-9H-fluoren-9-one (FOVPC) was purified by extraction using dichloromethane, followed by vacuum distillation and silica-gel column chromatography in the order mentioned. 2.2.2 Synthesis of FOVPC2, FOVPC4 and FOVPC6 FOVPC2: FOVPC (0.75 g, 1.94 mmol), ethiodide (0.61 g, 3.95 mmol) and acetone (40 ml) were added into a round-bottom flask, in which quaterisation was conducted with heating at 85 °C and stirring for 48 h. After cooling to room temperature, the turbid liquid was purified by vacuum filtration, and then the residue collected on the filter paper was washed with methanol followed by drying under vacuum. Finally, orange-red FOVPC2 was obtained as the major product via column chromatography by acetonitrile-dichloromethane (4:1) on silica gel. Yield: 75 %, 1H NMR (400 MHz, DMSO-d6), δ (ppm): 9.02 (s, 2H), 8.78 (d, 2H), 8.25 (d, 2H), 8.12 (s, 2H), 8.07 (d, 2H), 7.99 (m, 4H), 7.73 (d, 2H), 7.68 (d, 2H), 4.55 (m, 2H), 1.54 (t, 3H). 13C NMR (125 MHz, DMSO-d6), δ (ppm): 16.1, 55.3, 122.3, 122.5, 123.9, 124.3, 134.4, 135.5, 136.8, 139.0, 144.0, 144.3, 152.3, 191.8. MALDI-TOF-MS: m/z calcd [M-I]+ for C29H23IN2O, 415.18; found, 415.24. Anal. calcd. for C29H23IN2O (%): C 64.22, H 4.27, N 5.16, O 2.95; found: C 64.35, H 4.40, N 5.12, O 2.98. FOVPC4: FOVPC (0.74 g, 1.93 mmol), iodobutane (0.72 g, 3.92 mmol) and acetone (40 ml) were added into a round-bottom flask, in which quaterisation was conducted with heating at 85 °C and stirring for 48 h. After cooling to room temperature, the turbid liquid was purified by vacuum filtration, and then the residue collected on the filter paper was washed with methanol followed by drying under vacuum. Finally, orange-red FOVPC4 was obtained as the major product via column chromatography by acetonitrile-dichloromethane (4:1) on silica gel. Yield: 77 %, 1H NMR (400 MHz, DMSO-d6), δ (ppm): 8.98 (m, 2 H), 8.60 (d, 2H), 8.24 (d, 2 H), 8.09 (m, 2 H), 7.96 (m, 4H), 7.68 (m, 4H), 7.48 (d, 2H), 4.50 (d, 2H), 1.91 (s, 2H), 1.26 (d, 2H), 0.88 (d, 3H). 13C NMR (125 MHz, DMSO-d6), δ (ppm): 13.2, 18.7, 32.4, 59.5, 122.3, 122.5, 123.9, 124.4, 134.5, 135.5, 136.8, 139.2, 144.2, 144.4, 152.3, 191.8. MALDI-TOF-MS: m/z calcd [M-I]+ for C31H27IN2O, 443.21; found, 443.23. Anal. calcd. for C31H27IN2O (%): C 65.27, H 4.77, N 4.91, O 2.80; found: C 65.33, H 4.82, N 4.88, O 2.87. FOVPC6: FOVPC (0.76 g, 1.96 mmol), iodine hexane (0.83 g, 3.94 mmol) and acetone (45 ml) were added into a round-bottom flask, in which quaterisation was


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conducted with heating at 85 °C and stirring for 48 h. After cooling to room temperature, the turbid liquid was purified by vacuum filtration, and then the residue collected on the filter paper was washed with methanol followed by drying under vacuum. Finally, orange-red FOVPC6 was obtained as the major product via column chromatography by acetonitrile-dichloromethane (2:1) on silica gel. Yield: 69 %, 1H NMR (400 MHz, DMSO-d6), δ (ppm): 9.00 (d, 2H), 8.65 (d, 2H), 8.25 (d, 2H), 8.10 (m, 2H), 8.06 (s, 2H), 8.00 (t, 2H), 7.96 (s, 2H), 7.93 (s, 2H), 7.76 (m, 2H), 4.51 (s, 2H), 1.96 (d, 2H), 1.23 (m, 6H), 0.88 (d, 3H). 13C NMR (125 MHz, DMSO-d6), δ (ppm): 13.7, 21.7, 25.0, 30.4, 59.7, 121.1, 121.8, 122.3, 122.5, 123.9, 124.1, 124.3, 126.7, 127.0, 132.4, 132.5, 134.2, 134.5, 135.5, 136.4, 136.8, 137.4, 137.9, 139.2, 143.0, 143.3, 144.2, 144.4, 148.8, 149.0, 152.4, 192.3. MALDI-TOF-MS: m/z calcd [M-I]+ for C33H31IN2O, 471.24; found, 471.29. Anal. calcd. for C33H31IN2O (%): C 66.22, H 5.22, N 4.68, O 2.67; found: C 66.35, H 5.45, N 4.59, O 2.72. 2.3 Spectroscopic measurements Ultraviolet absorption spectra were recorded on a HITACHI UH5300 spectrophotometer. One-photon excitation fluorescence spectra were recorded on a FP-6500 fluorescence spectrometer at room temperature. The fluorescence quantum yield was determined using Rhodamine B in ethanol at 25 °C as the reference. In the one-photon photophysical performance experiment, the concentrations of all samples in DMF solutions were determined to be 10-5 M. Two-photon excitation fluorescence spectra were excited by a mode-locked Ti:sapphire laser system at a pulse duration of 80 fs with a repetition rate of 80 MHz. The TPEF spectroscopic data were recorded using a 7ISW301 spectrometer equipped with a 7IDA1 data acquisition unit and a 7IP1100 high voltage-regulated power supply. The TPA cross-sections were calculated according to the two-photon induced fluorescence method by using Rhodamine B in ethanol as the reference at a concentration of 10−4 M,30,31 which can be determined by Eq.(1):

σ s = σ r (Fsφr cr nr / Frφs cs ns ) Here, “s” and “r” refer to the synthesized and reference samples, respectively. σ, F, Ф, c, and n represent the TPA cross section, integral TPEF intensity, quantum yield, solution concentration, and refractive index of the solution, respectively. 2.4 Cell culture and biological fluorescence imaging 3T3 mouse myoblasts (0.2 µl) were added into petri dishes containing coverslips and culture solution by using pipettes. Cells were allowed to gradually grow in a medium under 5% CO2 for 13 h. Thereafter, the medium was removed from cells, followed by addition of 1 ml fixative solution into the petri dishes and incubation for 15 min at 25 °C. The fixative solution was removed, and cells were washed with PBS for approximately 5 min. The permeabilization buffer was added, followed by further incubation for 15 min at 25 °C. The procedures described for removal of the



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permeabilization solution and washing with PBS buffer were repeated 3 times. Thereafter, the blocking solution was added and cells were incubated for 1 h. Finally, cells were stained with FOVPC2, FOVPC4, FOVPC6 and DAPI for 15 min for subsequent biological fluorescence imaging. The one- and two-photon fluorescence imaging were obtained on a Carl Zeiss LSM 710 confocal scanning microscope. The one-photon excitation wavelength of FOVPC2, FOVPC4 and FOVPC6 was 405 nm, and the two-photon fluorescence emission was recorded between 525 nm and 575 nm upon excitation at 800 nm using a Ti:sapphire femtosecond laser pump source. The one- and two-photon excitation wavelength of DAPI/Hoechst 33342 was respectively selected as 330–380 nm and 740 nm, and two fluorescence emission collective windows were set in the range of 410–490 nm and 580–650 nm for DAPI/Hoechst 33342 and MTR, respectively.

3. RESULT AND DISCUSSION 3.1 One-photon photophysical properties The one-photon property is an important part of the photophysical properties of the fluorescent probe. Thus, we researched the absorption and one-photon fluorescence spectra of FOVPC2, FOVPC4 and FOVPC6 (FOVPC2,4,6) in N,N-dimethylformamide (DMF) solutions. As shown in Fig. 1 and Fig. 2, the absorption wavelengths of three derivatives were all close to 400 nm, and there were almost no absorption in the range of 600–900 nm, reflecting that the wavelength of 800 nm could be used as two-photon excitation test window. Furthermore, the main fluorescence emission peaks in the one-photon fluorescence spectra of FOVPC2,4,6 were approximately 540 nm, indicating large Stokes shift of ~140 nm, which were far greater than that for the commercial nuclear staining reagent DAPI (only ~50 nm). This was influenced by the stable rigid molecular structures and large π-conjugated delocalized planes, on which the intra-molecule charge transfer efficiency was strengthened under the promoting action of the quaterisation. The fluorescence quantum yields of FOVPC2,4,6 were 0.448, 0.558 and 0.634, respectively, exhibiting favorable one-photon photophysical performance. These larger Stokes shifts and fluorescence quantum yields are not only beneficial for two-photon fluorescence imaging but also play important roles in the real-time monitoring process of TPEF bioimaging. 3.2 Two-photon photophysical properties To the best of our knowledge, under tight-focusing conditions, it is beneficial to apply small organic molecules with large π-electron conjugated systems and transition dipole moments to complete the process of electron transition.32–34 According to this theoretical principle and the aforementioned one-photon photophysical properties, we studied the two-photon photophysical characteristics of FOVPC2,4,6 in DMF solutions at an excitation wavelength of 800 nm from a Ti:sapphire femtosecond laser pump source. With the increase of laser power (mw), the fluorescence intensity of all sample solutions (10-4 M) gradually enhanced. The power-dependent logarithmic


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relationships between the laser incident intensity and fluorescence intensity are shown in Fig. 3. The slopes of the straight lines for the three probes after the linear fitting was all close to 2, indicating the occurrence of obvious TPA behavior. Furthermore, the TPA cross-sections—deemed to be indispensable indicators of the two-photon fluorescence bioimaging performance—were calculated, as shown in Fig. 4 and Fig. 5. As indicated by the experimental data, the TPA cross-sections (σ) of FOVPC2,4,6 were estimated to be 495 GM, 473 GM and 469 GM, respectively, i.e., almost 1,000 times higher than that of DAPI (only ~0.46 GM). Notably, the quantum yields (Ф) and action TPA cross-sections (σФ) of FOVPC2,4,6 exhibited a trend of gradual increase as the increase of the terminal alkyl iodide chains, suggesting that this growth of terminal alkyl chains may be beneficial for the improvement of the clarity and resolution in two-photon fluorescence bioimaging (Fig. 5). Because the action TPA cross-section is a truly valuable parameter for researching the levels and effects of two-photon bioimaging, especially in the process of real-time monitoring and tracking of the cells. Hence, the design philosophy of increasing the length of terminal alkane chains by the quaterisation reaction should be significant and feasible for the two-photon fluorescent probes. The electron-transition mechanism can explain this phenomenon. On one hand, the ability to push electrons by enhancing the lengths of conjugated chains can undoubtedly improve the intramolecular charge transfer efficiency. On the other hand, to some extent, the increase in the length of the terminal alkyl chains can effectively improve the operation of the integral π-conjugated system and transition dipole moment.35 To explore the optimal two-photon excitation wavelength, The TPA cross-sections of FOVPC2,4,6 in DMF solutions in the wavelength range 750–840 nm were measured. As shown in Fig. 6, the maximum TPA cross-sections were obtained at an excitation wavelength of 800 nm, indicating that the optimal effects are achieved at an excitation of 800 nm in two-photon biological fluorescence imaging. 3.3 Luminescence “turn-on” test To investigate the fluorescence “turn-on” effects of FOVPC2,4,6 and evaluate the preliminary nuclear-staining ability, the absorption, one- and two-photon luminescence “turn-on” experiment were researched. The variation trends and degrees of the spectra and binding mechanism in the process of the affinities between FOVPC2,4,6 and double stranded ct-DNA were analyzed and discussed in detail. As presented in Fig. 7, the absorption spectra all exhibited obvious hypochromic effects with constant increases of the DNA concentration in Tris-HCl buffer solutions. In contrast, all the one- and two-photon fluorescence spectra exhibited consistent hyperchromic effects until the concentration ratio reached a high degree ([DNA]:[probe] ˃ 130:1]). For the OPEF spectra, when the DNA concentrations were close to saturation, the one-photon fluorescence intensity and fluorescence quantum yields of FOVPC2,4,6 in the Tris-HCl buffer solution were respectively enhanced 27-fold–35-fold and 54-fold–68-fold, indicating an obvious preponderance over that of DAPI (only 5-fold and 10-fold). Moreover, ~400%–500% increases in the TPA cross-sections were also achieved in the TPEF spectra. The aforementioned



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hyperchromism fully describes the occurrence of the fluorescence “turn-on” effect, indicating that the affinities between FOVPC2,4,6 and DNA should be considered as an intercalative binding mechanism. The unified hyperchromism is attributed to the following factors.36,37 When the probes bound with the DNA through intercalative binding, they were protected from the basic groups by the hydrophobic environment, preventing fluorescence quenching due to the water molecules. Thus, the vibrations of the probes molecules orbitals were restricted, leading to enhancement of fluorescence intensity. However, the DAPI molecules were mainly located in the specific A-T rich regions of the DNA molecules owing to minor groove binding. The spatial locations of three base pairs could only be occupied by one DAPI molecule. By contrast, since the terminal alkyl chains can be freely oscillating, the space detection ranges should be much wider, leading to higher detection efficiency and probability for bases pairs in three-dimensional space. Then the probes may attract numerous DNA molecules by virtue of electrostatic effects and longer flexible alkyl chains. In this case, plenty of FOVPC2,4,6 molecules could be embedded in double stranded DNA with their good structural flatness and small sizes. Namely, the intercalative binding possibly yields more probes bound with DNA basic groups under the equal DNA concentration. Hence, when equal numbers of DNA molecules were incorporated into DAPI and these probes in the Tris-HCl buffer solutions, the fluorescence intensities of the FOVPC2,4,6 probes were far higher because of their larger TPA cross-sections, special intercalative binding force, and good hydrophilic and lipophilic properties. As a result, the luminescence “turn-on” performance of FOVPC2,4,6 were much better than that of DAPI, laying a solid foundation for nuclear localization and monitoring in two-photon fluorescence bioimaging. 3.4 The one- and two-photon fluorescence imaging and nucleus-localization studies To investigate the nuclear-targeting properties in practice, the one- and two-photon fluorescence imaging of cellular nuclei staining with FOVPC2,4,6 in 3T3 cells were conducted by confocal fluorescence microscopy. To distinguish the strength of the one- and two-photon fluorescence intensity, the fluorescent colors of OPEF and TPEF were selected as green and red, respectively, via a built-in microscopic imaging software. As shown in Fig. 8, the merged images exhibited relatively obvious yellow fluorescence in the nuclear regions, suggesting no significant differences between the OPEF and TPEF intensity in the process of nuclear staining. Because the fluorescence wavelength (~540 nm) was less than the excitation wavelength (800 nm) for TPA behavior, in this case, the background noises generated by Rayleigh scattering became far lower compared with the OPA behavior, greatly reducing the interference of the scattering. Hence, the contrast ratio of the TPEF microscopic images appeared to be higher than that of OPEF bioimaging. To determine the labeling locations of FOVPC2,4,6 in 3T3 cells, a set of co-localization experiments were performed by employing FOVPC2, FOVPC4 or FOVPC6 double-stained with Hoechst 33342. As shown in Fig. 9, each fluorenone-based fluorescent dye and Hoechst 33342 were all accumulated in the


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nuclear regions, and the bright indigo fluorescence in Fig. 9c demonstrated that their nuclear staining levels reflected a high degree of overlapping. By calculation from the finite number of double-stained cells in each photo, the mean co-localization coefficient of FOVPC2, FOVPC4, FOVPC6 and Hoechst 33342 was 0.93, 0.92 and 0.96, respectively, indicating accurate co-localization levels for nuclear bioimaging. Meanwhile, because Hoechst 33342 is a commercially available dye specifically for nuclear staining, by means of the high degrees of fluorescence overlapping and mean co-localization coefficients, it could be convinced that these fluorenone-based fluorescent probes could be able to exclusively target the nuclear regions. In order to further prove the bio-compatibility in the multicolor-targeting application, a group of counterstain experiments were conducted by employing each probe co-stained with MTR (a commercial red cellular mitochondrial reagent) to stain 3T3 cells. DAPI was selected as the reference sample because of its certified nuclear staining ability. As shown in Fig. 10, both green and blue luminescence were all emitted from the nuclear regions, which were distinguished from the red cytoplasmic regions stained with MTR. The clearly visible staining boundaries and fluorescence distribution between green or blue nucleus and red cytoplasm fully exhibited good counterstain compatibility between FOVPC2,4,6 and MTR. The smooth entrance into the nuclei not only showed that the size of each probe molecule was smaller than that of the nuclear pore but also indicated the excellent solubility and membrane permeability in the intracellular bioenvironment. 3.5 Photostability For improving the dynamic therapy in biomedical fields, the photostability in monitoring and tracking of the cellular nucleus has important practical significance with regard to gene regulation, chromosome translocation, and enzyme dynamics.38 Thus, the time-dependent fluorescence signals of the nuclei in the 3T3 cells stained with FOVPC2, FOVPC4, FOVPC6 and DAPI were conducted. The entire fluorescence processes, from emission to extinction, for FOVPC2,4,6 and DAPI at 800-nm femtosecond laser excitation were observed and examined. The nuclear-staining images were taken at intervals of 30 s by confocal fluorescence microscopy (Fig. S1–Fig. S4 of the Supporting Information). The nuclei observation time for labeling with FOVPC2,4,6 all reached at least 3,000 s, which were far greater than that of DAPI (only ~900 s). Moreover, the two-photon fluorescence intensities of FOVPC2,4,6 from emission to extinction were also much higher than those of DAPI (Fig. 11). Moreover, the mean fluorescence attenuation rates of FOVPC2,4,6 within 3000 s were in the range of 0.028%/s–0.03%/s, and the fluorescence attenuation amplitudes of FOVPC2,4,6 in the 300 s range were 9.6%, 8.7% and 9.3%, respectively. Compared with some previously reported two-photon fluorescenent probes,14,28,29 FOVPC2,4,6 showed relatively higher photostability and better photobleaching resistence in the process of two-photon fluorescence imaging. Notably, with the increase of alkyl chain length, the fluorescence intensity appeared to have slight increase trend, which may be due to the following factors: on one hand, the gradually increasing action TPA cross-sections were conducive to the improvement of intramolecular charge transfer efficiency,



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which possibly led to the enhancement of two-photon fluorescence intensities; on the other hand, the fluorescence increasing multiple for FOVPC2,4,6 binding with DNA gradually enhanced in the fluorescence “turn-on” experiment with the increase of alkyl chain length, reflecting the level differences in detection of DNA molecules in the intracellular environment. However, with the extension of irradiation time, the fluorescence intensities of FOVPC2,4,6 all gradually decreased, revealing a certain degree of photobleaching phenomenon still existed. To the best of our knowledge, this common fluorescence attenuation maybe attributed to DNA injury due to the sustained femtosecond laser excitation, which gradually weakened until the binding force between FOVPC2,4,6/DAPI and the molecular DNA was eliminated. When the DNA-DAPI complexes were excited by a sustained high power laser, a large amount of thermal energy was detained in the molecules DNA for a prolonged time owing to the far slower energy dissipation process among the base pairs. This probably caused injury to the DNA, including base losing and the breakage of single and double strands.39 Then, the dissociation of the DNA-DAPI complexes caused DAPI molecules to separate from the DNA structure. Thus, the TPEF intensity quickly decayed until its disappearance (Scheme 2). However, the action TPA cross-sections of FOVPC2,4,6 were far larger than that of DAPI, therefore, most of the incident laser was absorbed by FOVPC2,4,6 and converted into luminous energy, leaving only small portion of laser energy to transform into thermal energy, which probably remained among the double helix DNA chains. The slower thermal energy dissipation delayed the DNA injury rate and slowed down the destruction of the binding force in the DNA-FOVPC2,4,6 complexes, yielding longer observation time and higher TPEF intensities compared with DAPI.

4. CONCLUSIONS In conclusion, we prepared a series of novel two-photon fluorenone-based cationic fluorescent probes for enhancing photostability in cellular nucleus imaging. The optimal fluorescence quantum yields of 0.634 and TPA cross-sections of 495 GM in DMF showed these new probes possessed good photophysical properties, reflecting high intramolecular electrons donating abilities of terminal alkyl chains. Because of excellent hydrophilic and lipophilic characteristics in fluorescence “off-on” experiment, the new probes have maximum 35-fold fluorescence intensity and 68-fold quantum yield enhancement in buffer solutions, displaying strong binding force with DNA molecules. The maximum mean co-localization coefficient of 0.96 between new probes and Hoechst 33342 revealed accurate targeting ability of cellular nucleus. The good counterstain compatibility between FOVPC2,4,6 and MTR indicated potential multicolor labeling application prospect. Compared to previouly reported typical two-photon fluorescent probes, these new probes possessed longer continuous observation time of 3000 s, slower mean fluorescence attenuation rates of 0.028%/s–0.03%/s and smaller fluorescence attenuation amplitudes of 8.7% within 300 s, effectively improving the photostability in the process of nuclear monitoring and tracking. Thus, these new probes should have potential application value in the


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field of cellular nucleus imaging.

ASSOCIATED CONTENT Supporting Information Time-dependent confocal fluorescence images results of FOVPC2,4,6 and DAPI; 1H NMR, 13C NMR and MALDI-TOF-MS spectra of FOVPC2,4,6.

ACKNOWLEDGEMENTS This research was supported in part by the National Natural Science Foundation of China (Grant Nos. 61137002, 61178059 and 51172253).

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TOC graphic



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Scheme 1. Synthesis of fluorenone-based fluorescent probe 68x34mm (300 x 300 DPI)


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Fig.1 Normalized absorption spectra of FOVPC2,4,6 in DMF, [FOVPC2,4,6]=10-5 M. 52x37mm (300 x 300 DPI)



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Fig.2 Normalized one-photon fluorescence spectra of FOVPC2,4,6 in DMF, [FOVPC2,4,6]=10-5 M. 52x37mm (300 x 300 DPI)


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Fig.3 Quadratic log-linear relationship between fluorescence intensity and input laser intensity of (a) FOVPC2, (b) FOVPC4 and (c) FOVPC6 in DMF, [FOVPC2,4,6]=10-4 M. 224x52mm (300 x 300 DPI)



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Fig.4 Two-photon fluorescence spectra of FOVPC2,4,6 and DAPI at excitation wavelangth of 800 nm and 740 nm in DMF, [FOVPC2,4,6]=[DAPI]=10-4 M. 52x37mm (300 x 300 DPI)


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Fig.5 Two-photon absorption cross-sections and fluorescence quantum yields of FOVPC2,4,6 at excitation wavelangth of 800 nm in DMF, [FOVPC2,4,6]=10-4 M. 52x37mm (300 x 300 DPI)



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Fig.6 Two-photon absorption cross-sections of FOVPC2,4,6 in the excitation wavelength ranging from 750 to 840 nm in DMF, [FOVPC2,4,6]=10-4 M. 52x37mm (300 x 300 DPI)


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Fig.7 Absorption (I), one-photon (II) and two-photon fluorescence (III) spectra of FOVPC2 (a), FOVPC4 (b), FOVPC6 (c) and DAPI (d) as a function of different DNA concentration in Tris-HCl buffer solutions. [DNA]=0– 1.4×10-3 M. [FOVPC2]=[FOVPC4]=[FOVPC6]=[DAPI]=10-5 M, The arrows represent the increasing or decreasing trends of concentration ratios. 115x80mm (300 x 300 DPI)



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Fig.8 One- and two-photon confocal fluorescence images of 3T3 cell nucleus stained with FOVPC2 (Ⅰ, 0.3 µM), FOVPC4 (Ⅱ, 0.3 µM) or FOVPC6 (Ⅲ, 0.3 µM) for 15 min. (a) TPEF biological images, λex = 800 nm, and λem = 525–575 nm; (b) DIC pictures; (c) OPEF biological images, λex = 405 nm, and λem = 525–575 nm; (d) Overlay images of a and c. Scale bar was 50 um. 115x90mm (300 x 300 DPI)


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Fig.9 Confocal fluorescent images of 3T3 cells double-stained with FOVPC2 (Ia, 0.3 µM), FOVPC4 (IIa, 0.3 µM) or FOVPC6 (IIIa, 0.3 µM) and Hoechst 33342 (b, 0.3 mM) for 15 min. (a) images of FOVPC2 (I), FOVPC4 (II) and FOVPC6 (III), λex = 405 nm, and λem = 525–575 nm; (b) images of Hoechst 33342, λex = 330–380 nm, and λem = 410–490 nm; (c) overlay images of a and b. Scale bar was 20 um. 170x170mm (300 x 300 DPI)



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Fig.10 Confocal fluorescence images of 3T3 cells co-stained with FOVPC2 (a, 0.3 µM), FOVPC4 (b, 0.3 µM), FOVPC6 (c, 0.3 µM) or DAPI (d, 0.3 µM) and MTR (a–d, 0.3 µM) for 15 min. For FOVPC2,4,6, λex = 405 nm, and λem = 525–575 nm. For DAPI, λex = 330–380 nm, and λem = 410–490 nm. For MTR, λex = 575 nm, and λem = 580–650 nm. Scale bar was 50 um. 57x57mm (300 x 300 DPI)


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Fig.11 The two-photon fluorescence intensity decay of FOVPC2,4,6 and DAPI as time variation under persistent irradiation. 52x37mm (300 x 300 DPI)



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Scheme 2. Microscopic mechanism of fluorescence decay in imaging of cellular nucleus stained with FOVPC2,4,6 and DAPI. 153x140mm (300 x 300 DPI)


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Photostability Enhancement of Fluorenone-Based Two-Photon

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