USE OF A SUBSTITUTED OR UNSUBSTITUTED POLYCYCLIC AROMATIC HYDROCARBON COMPOUND FOR HIGH-RESOLUTION MICROSCOPY

Abstract

The present invention relates to the use of a compound in single-molecule localization microscopy (SMLM), in stimulated emission depletion microscopy (STED), in minimal emission fluxes microscopy (MINFLUX) or in structured illumination and localization microscopy (SIMFLUX), wherein the compound is a substituted or unsubstituted polycyclic aromatic hydrocarbon comprising six or more substituted and/or unsubstituted aromatic hydrocarbon rings, wherein each of at least six of the six or more substituted and/or unsubstituted aromatic hydrocarbon rings is fused with at least another one of the at least six substituted and/or unsubstituted aromatic hydrocarbon rings.

Claims

1. Use of a compound in single-molecule localization microscopy (SMLM), in stimulated emission depletion microscopy (STED), in minimal emission fluxes microscopy (MINFLUX) or in structured illumination and localization microscopy (SIMFLUX), wherein the compound is a substituted or unsubstituted polycyclic aromatic hydrocarbon comprising six or more substituted and/or unsubstituted aromatic hydrocarbon rings, wherein each of at least six of the six or more substituted and/or unsubstituted aromatic hydrocarbon rings is fused with at least another one of the at least six substituted and/or unsubstituted aromatic hydrocarbon rings.

2. The use in accordance with claim 1, wherein the compound is used in the high-resolution microscopy as fluorescent marker, and wherein the compound is used in photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), ground state depletion individual molecule return (GSDIM), binding activated localization microscopy (BALM) or fluorescence photo-activation localization microscopy (FPALM).

3. The use in accordance with claim 1, wherein the compound comprises six to 91 substituted and/or unsubstituted aromatic hydrocarbon rings, wherein each of at least six and preferably each of all the six to 91 substituted and/or unsubstituted aromatic hydrocarbon rings is fused with at least another one of the at least six substituted and/or unsubstituted aromatic hydrocarbon rings.

4. The use in accordance with claim 1, wherein the compound comprises any of the below units: i) —Ar.sub.1(Ar.sub.2).sub.x—, wherein the residues Ar.sub.1 and Ar.sub.2 are the same or different and independently from each other a substituted and/or unsubstituted aromatic hydrocarbon ring and x is in an integer of 5, 6 in the case that Ar.sub.1 is at least a C.sub.7-ring or 7, ii) —Ar.sub.1(Ar.sub.2).sub.x—Ar.sub.3(Ar.sub.4).sub.y—, wherein the residues Ar.sub.1 to Ara are the same or different and independently from each other a substituted and/or unsubstituted aromatic hydrocarbon ring and x and y are independently from each other integers of 1 to 6, wherein the sum of x and y is at least 4, or iii) —Ar.sub.1(Ar.sub.2).sub.x—Ar.sub.3(Ar.sub.4).sub.y—Ar.sub.5(Ar.sub.6).sub.z—, wherein the residues Ar.sub.1 to Ar.sub.6 are the same or different and independently from each other a substituted and/or unsubstituted aromatic hydrocarbon ring and x, y and z are independently from each other integers of 1 to 7, wherein the sum of x, y and z is at least 3, or iv) —Ar.sub.1—(Ar.sub.2).sub.n—Ar.sub.3—, wherein the residues Ar.sub.1 to Ar.sub.3 are the same or different and independently from each other a substituted and/or unsubstituted aromatic hydrocarbon ring and n is an integer of 4 to 14, wherein the residues Ar.sub.1 and Ar.sub.3 may be bonded or fused with each other to form a ring.

5. The use in accordance with claim 1, wherein the compound comprises a unit with the general formula (1) or the compound has the general formula (1): ##STR00047## wherein in general formula (1) the residues R are the same or different and independently from each other a hydrogen atom, an unsubstituted C.sub.1-20 hydrocarbon residue, a substituted C.sub.1-20 hydrocarbon residue, a halogen, an azide group, a hydroxy group, a nitro group, an amino group, a formyl group, a cyano group or one or more of two adjacent residues R are linked with each other to form C.sub.5-20-aromatic group, a C.sub.4-20-cycloaliphatic group or a C.sub.5-20-heterocyclic group, wherein any of the unsubstituted and/or substituted C.sub.1-20 hydrocarbon residues may be a C.sub.1-20-alkyl group, a C.sub.1-20-alkenyl group, a C.sub.1-20-alkynyl group, C.sub.1-20-alkoxy group, a C.sub.4-20-cycloalkyl group, a C.sub.5-20-aromatic group, a C.sub.4-20-cycloaliphatic group or a C.sub.5-20-heterocyclic group.

6. The use in accordance with claim 1, wherein the compound comprises a unit with the general formula (2) or the compound has the general formula (2): ##STR00048## wherein in general formula (2): residues R.sub.1 to R.sub.8 and Ar are independently from each other selected from the group consisting of hydrogen, unsubstituted alkyl groups, substituted alkyl groups, unsubstituted alkenyl groups, substituted alkenyl groups, unsubstituted alkynyl groups, substituted alkynyl groups, unsubstituted cycloalkyl groups, substituted cycloalkyl groups, unsubstituted aryl groups, substituted aryl groups, unsubstituted aralkyl groups, substituted aralkyl groups, unsubstituted hetaryl groups, substituted hetaryl groups, azide groups, and groups formed in that two of adjacent residues of R.sub.1 to R.sub.8 and Ar are linked with each other to form an aromatic, heteroaromatic, cyclic or heterocyclic group.

7. The use in accordance with claim 6, wherein in general formula (2) the residues R.sub.1 to R.sub.8 and Ar are independently from each other selected from the group consisting of hydrogen, unsubstituted linear or branched C.sub.1-30-alkyl groups, unsubstituted C.sub.3-30-cycloalkyl groups, phenyl groups, naphthyl groups, anthryl groups, pyrenyl groups, azide groups, polyethylene groups with 2 to 20 ethylene moieties, phenyl ethylene, triisopropylsilyl ethynyl, trimethylsilyl ethynyl, and groups formed in that two of adjacent residues of R.sub.1 to R.sub.8 and Ar are linked with each other to form an aromatic, heteroaromatic, cyclic or heterocyclic group.

8. The use in accordance with claim 6, wherein in general formula (2): residue R.sub.1 is hydrogen or a C.sub.1-20-alkyl group, residues R.sub.2 to R.sub.8 are hydrogen and residue Ar is aryl, a C.sub.6-15-alkyl group or a trialkylsilyl alkynyl group.

9. The use in accordance with claim 6, wherein in general formula (2) residue Ar is selected from the group consisting of phenyl, trifluorphenyl, 1,5-dimethylphenyl, mesitylene, triisopropylsilyl ethynyl, trimethylsilyl ethynyl, phenylsilyl ethynyl and C.sub.6-15-alkyl groups.

10. The use in accordance with claim 6, wherein in the general formula (2) at least one of the residues R.sub.1 to R.sub.8 and Ar is a hydrophilic group selected from the group consisting of groups comprising one or more carboxy groups, groups including one or more polyethylene residues with each 2 to 20 alkylene moieties, one or more sulfonate groups, one or more quaternary amine groups, one or more amide groups, one or more imine groups and one or more pyridine groups.

11. The use in accordance with claim 1, wherein the compound comprises a unit with the general formula (3) or the compound has the general formula (3): ##STR00049## wherein in general formula (3) the residues R.sup.a and Rb are the same or different and independently from each other a hydrogen atom, an unsubstituted C.sub.1-20 hydrocarbon residue, a substituted C.sub.1-20 hydrocarbon residue, a halogen, an azide group, a hydroxy group, a nitro group, an amino group, a formyl group, a cyano group or one or more of two adjacent residues R.sup.a and Rb are linked with each other to form an aromatic group, a cycloaliphatic group or a heterocyclic group, each of residues Ar is a cyclic group and m is an integer of 1 to 4.

12. The use in accordance with claim 11, wherein in the general formula (3) at least one of the residues Ar is a heterocyclic group and each of the remaining residues Ar is independently from each other a benzene group, a cycloaliphatic group or a heterocyclic group.

13. The use in accordance with claim 1, wherein the compound comprises a unit with the general formula (33) or the compound has the general formula (33): ##STR00050## wherein in general formula (23) the residues R are the same or different and independently from each other a hydrogen atom, an unsubstituted C.sub.1-20 hydrocarbon residue, a substituted C.sub.1-20 hydrocarbon residue, a halogen, an azide group, a hydroxy group, a nitro group, an amino group, a formyl group, a cyano group or one or more of two adjacent residues R are linked with each other to form C.sub.5-20-aromatic group, a C.sub.4-20-cycloaliphatic group or a C.sub.5-20-heterocyclic group, wherein any of the unsubstituted and/or substituted C.sub.1-20 hydrocarbon residues may be a C.sub.1-20-alkyl group, a C.sub.1-20-alkenyl group, a C.sub.1-20-alkynyl group, a C.sub.1-20-alkoxy group, a C.sub.4-20-cycloalkyl group, a C.sub.5-20-aromatic group, a C.sub.4-20-cycloaliphatic group or a C.sub.5-20-heterocyclic group.

14. The use in accordance with claim 1, wherein the compound comprises a residue with a terminal alkyne group.

15. The use in accordance with claim 1 for high resolution evaluations of biological systems, such as cells, for the detection of material imperfections, such as micro- or nano-cracks, and for monitoring and detecting nano-structure fabrication.

Description

[0070] Subsequently, the present invention is further described by means of illustrative, but not limiting examples and figures.

[0071] FIG. 1 shows a reaction scheme for synthesizing symmetric dibenzo[hi,st]ovalenes to be used in accordance with an embodiment of the present invention.

[0072] FIG. 2 shows a reaction scheme for converting a symmetric dibenzo[hi,st]ovalene into an asymmetric dibenzo[hi,st]ovalene to be used in accordance with another embodiment of the present invention.

[0073] FIG. 3a-d show the absorption and emission spectra of four DBOV derivatives prepared in examples 2, 5, 7 and 8 (Abs: absorbance; PI: PL-emission).

[0074] FIG. 4a-d show the absorption and emission spectra of four polycyclic aromatic hydrocarbons shown in example 10 (Abs: absorbance; PI: PL-emission).

[0075] FIG. 5a-d show a widefield image (FIG. 5a) and the blinking properties of 6,14-bis(dimethylphenyl)dibenzo[hi,st]ovalene embedded in polystyrene (FIG. 5b-d)—single-molecule fluorescence time trace (FIG. 5b), detected photon numbers (FIG. 5c) and duty cycle diagram (FIG. 5d).

[0076] FIG. 6a-b show a gravure printing plate (FIG. 6a), the surface of which has been analyzed with SMLM, and a respective image obtained for a section of this plate (FIG. 6a) and an enlarged cutout thereof (FIG. 6b).

[0077] FIG. 7a-f show a confocal image (FIG. 7a) and corresponding STED image (raw data) (FIG. 7b), the corresponding line profiles of confocal mode (FIG. 7c) and STED mode (FIG. 7d) of the boxed region of FIGS. 7a and 7b as well as the 3D confocal image (FIG. 7e) and the corresponding STED image (FIG. 7f).

[0078] FIG. 8 shows the results of a cytotoxicity test DBOV-Mes-OTEG described in example 13 in living cells.

EXAMPLE 1

Synthesis OF 6,14-didodecyldibenzo[hi,st]ovalene

Synthesis of 6, 6′-diiodo-[5, 5′-bichrysene]-3, 3′-dicarbaldehyde

[0079] In accordance with the reaction scheme shown in FIG. 1, 6,6′-diiodo-[5,5′-bichrysene]-3,3′-dicarbaldehyde, which is compound 6 with all residues R.sub.1 to R.sub.7 being hydrogen atoms, was prepared starting from compound 1, in which all residues R.sub.1 to R.sub.3 are hydrogen atoms. Compound 1 was prepared as described in Nano Lett., 2017, volume 17, pages 5521 to 5525. Then, to a solution of compound 1 (2.0 g, 3.9 mmol) dissolved in anhydrous dichloromethane (240 mL) ICI (8.58 mmol, 8.58 mL, 1 M in dichloromethane) was added. After stirring at room temperature for 2 hours, the excess ICI was quenched by addition of saturated aqueous Na.sub.2S.sub.2O.sub.3 solution (50 mL). The organic phase was separated, washed with brine (50 mL), dried over Na.sub.2SO.sub.4 and evaporated. The residual solid was recrystallized with dichloromethane and methanol.

[0080] After filtration, the product (2.2 g, 76%) was obtained as white solid. The product had the following characteristics:

[0081] Mp: >400° C.; .sup.1H NMR (300 MHz, Methylene Chloride-d.sub.2) δ 9.18 (d, J=9.2 Hz, 2H), 9.00 (d, J=8.5 Hz, 2H), 8.72 (s, 2H), 8.53-8.42 (m, 4H), 8.28 (d, J=9.1 Hz, 2H), 8.04 (d, J=8.3 Hz, 2H), 7.98-7.88 (m, 2H), 7.86-7.74 (m, 4H); .sup.13C NMR (75 MHz, methylene chloride-d.sub.2) δ 191.8, 150.1, 137.4, 135.3, 134.6, 134.5, 133.7, 132.1, 130.9, 130.5, 130.4, 130.2, 129.7, 129.5, 129.3, 125.7, 124.4, 123.7, 114.1; FD-MS (8 kV): m/z 762.2; HRMS (MALDI-TOF): m/z Calcd for C.sub.38H.sub.20I.sub.2O.sub.2: 761.9553 [M]+, found: 761.9553 (error=0 ppm).

[0082] Thus, it was shown that the product had the formula:

##STR00033##

Synthesis of 5,14-diformylbenzo[a]dinaphtho[2,1,8-cde:1′,2′,3′,4′-ghi]perylene

[0083] In accordance with the reaction scheme shown in FIG. 1, 5,14-diformylbenzo[a]dinaphtho[2,1,8-cde:1′,2′,3′,4′-ghi]perylene, which is compound 7 with all residues R.sub.1 to R.sub.7 being hydrogen atoms, was prepared starting from compound 6 prepared as described above. More specifically, to a 3 L cylindrical quartz reactor containing 6,6′-diiodo-[5,5′-bichrysene]-3,3′-dicarbaldehyde (300 mg, 0.394 mmol) was added a mixture of acetone (600 mL) and triethylamine (6 mL). Then the mixture was degassed by bubbling with Ar for 20 minutes. After that, the reaction mixture was stirred and irradiated at room temperature in a photoreactor equipped with six 300 nm wavelength UV lamps with strong stirring for 2 hours.

[0084] After cooling down to room temperature, the solvent was evaporated and the residue was purified by column chromatography (n-hexane:ethyl acetate=4:1) to give the product (170 mg, 86% yield) as red solid. The product had the following characteristics, which was further confirmed by X-ray single crystal structure analysis. Mp: >400° C.; .sup.1H NMR (300 MHz, 1,1,2,2-tetrachloroethane-d.sub.2) δ9.46 (s, 2H), 9.01 (d, J=9.1 Hz, 2H), 8.90 (d, J=8.4 Hz, 2H), 8.52-8.42 (m, 4H), 8.34 (t, J=9.1 Hz, 4H), 7.83-7.72 (m, 2H), 7.64 (dd, J=8.1, 1.1 Hz, 2H); .sup.13C NMR (75 MHz, C.sub.2D.sub.2Cl.sub.4) δ 190.8, 133.7, 131.7, 131.6, 130.6, 128.5, 128.2, 127.9, 127.5, 127.1, 127.1, 125.9, 124.9, 124.4, 124.0, 123.9, 123.2, 121.2, 120.6; FD-MS (8 kV): m/z 506.9; HR MS (MALDI-TOF): m/z Calcd for C38H.sub.18O.sub.2: 506.1307 [M].sup.+, found: 506.1288 (error=−3.7 ppm).

[0085] Thus, it was shown that the product had the formula:

##STR00034##

Synthesis of 6,14-didodecyldibenzo[hi,st]ovalene

[0086] In accordance with the reaction scheme shown in FIG. 1, 4,12-didodecyldibenzo[hi,st]ovalene, which is compound 9 with all residues R.sub.1 to R.sub.7 being hydrogen atoms and with residues Ar being n-dodecyl, was prepared starting from compound 7 prepared as described above. More specifically, to a 50 mL round bottom flask was added 5,14-diformylbenzo[a]dinaphtho[2,1,8-cde:1′,2′,3′,4′-ghi]perylene (10.1 mg, 0.0199 mmol), the flask was evacuated and backfilled with Ar for 3 times before dry tetrahydrofuran (10 mL) was added. To the mixture was injected a solution of n-C.sub.12H.sub.25MgBr (0.30 mmol, 0.30 mL, 1 M) in ether. After stirring at room temperature for 5 hours, the reaction was quenched by addition of saturated NH.sub.4Cl solution (10 mL). The organic phase was extracted with ethyl acetate (15 mL) for three times, washed with brine (30 mL) dried over Na.sub.2SO.sub.4 and evaporated. After drying under vacuum for 2 hours, the residue was dissolved in dry dichloromethane (10 mL) and degassed by bubbling with dichloromethane saturated Ar flow for 10 minutes. BF.sub.3.OEt.sub.2 (0.1 mL) was added and the mixture was stirred overnight at room temperature. After quenching with methanol (1 mL), p-chloranil (10 mg, 0.041 mmol) was added and stirred for 2 hours.

[0087] The crude product was precipitated by addition of methanol (30 mL), which was further purified by column chromatography (n-hexane:tetrahydrofuran=4:1 to 0:1) to give the product (6.5 mg, 41% yield) as blue solid. The product had the following characteristics:

[0088] Mp: >400° C. HRMS (MALDI-TOF): m/z Calcd for C.sub.62H.sub.64: 808.5008 [M].sup.+, found: 808.4978 (error=−3.7 ppm).

[0089] Thus, it was shown that the product had the formula:

##STR00035##

[0090] The maximum absorption wavelength in toluene (10.sup.−5 mol/L) was 611 nm (molar extinction coefficient ε=2.03×10.sup.5 M.sup.−1 cm.sup.−1) with a high fluorescence quantum yield of 0.85.

EXAMPLE 2

(Synthesis of 6,14-bis(dimethylphenyl)dibenzo[hi,sf]ovalene [DBOV-DMEP])

[0091] In accordance with the reaction scheme shown in FIG. 1, 6,14-bis(dimethylphenyl)dibenzo[hi,st]ovalene, which is compound 9 shown in FIG. 1 with all residues R.sub.1 to R.sub.7 being hydrogen atoms and with residues Ar being 2,6-dimethylenpehnyl, was prepared starting from compound 7 prepared as described in example 1. More specifically, to a solution of 5,14-diformylbenzo[a]dinaphtho[2,1,8-cde:1′,2′,3′,4′-ghi]perylene prepared as described in example 1 (63 mg, 0.12 mmol) dissolved in dry THF (63 mL) was added 2,6-dimethylphenylmagnesium bromide solution (1.8 mmol, 1.8 mL, 1 M in THF). After stirring for 3 hours at room temperature, the reaction was quenched with saturated NH.sub.4Cl solution (45 mL). The mixture was extracted with ethyl acetate (50 mL) for three times and the organic solution was combined, washed with brine (30 mL), dried over Na.sub.2SO.sub.4 then evaporated. After drying under vacuum for 2 hours, the residue was dissolved in anhydrous dichloromethane (60 mL) and degassed with dichloromethane vapor saturated argon flow. BF.sub.3 OEt.sub.2 (0.6 mL) was added and the reaction mixture was stirred overnight. After quenching with methanol (2 mL), p-chloranil (30 mg, 0.12 mmol) was added and the mixture was stirred for 2 hours at room temperature.

[0092] The solvent was evaporated and the residue was purified by column chromatography (n-hexane:DCM=3:1) to give the product (59 mg, 72% yield) as blue solid. The product had the following characteristics:

[0093] Mp: >400° C.; .sup.1H NMR (300 MHz, THF-d.sub.8) δ 9.54 (d, J=8.3 Hz, 2H), 9.23 (d, J=7.7 Hz, 2H), 8.59 (d, J=8.2 Hz, 2H), 8.13 (d, J=9.2 Hz, 2H), 8.00 (t, J=7.9 Hz, 2H), 7.87 (d, J=8.1 Hz, 2H), 7.70 (d, J=9.1 Hz, 2H), 7.45 (dd, J=6.1, 2.7 Hz, 4H), 7.40 (s, 3H), 2.00 (s, 12H); .sup.13C NMR (75 MHz, THF-d.sub.8) δ 138.8, 138.7, 135.9, 133.2, 131.8, 131.5, 130.6, 129.9, 129.9, 129.1, 128.9, 128.8, 128.1, 127.0, 126.9, 126.1, 125.5, 125.4, 124.1, 124.1, 122.5, 121.9, 20.9; HRMS (MALDI-TOF): m/z Calcd for C.sub.54H.sub.32: 680.2504 [M].sup.+, found: 680.2487 (error=−2.5 ppm).

[0094] Thus, it was shown that the product had the formula:

##STR00036##

[0095] It was evaluated that this compound has a narrow absorption spectrum as well as a narrow emission spectrum, which are both shown in FIG. 3a (Abs: absorbance; PI: PL-emission).

[0096] The maximum absorption wavelength in toluene (10.sup.−5 mol/L) was 609 nm (molar extinction coefficient c=2.83×10.sup.5 M.sup.−1 cm.sup.−1) with a high fluorescence quantum yield of 0.85.

EXAMPLE 3

Synthesis of triisopropylsilyl ethynyl substituted 6,14-dimesityldibenzo[hi,st]ovalene

Synthesis of dibrominated 6,14-dimesityldibenzo[hi,st]ovalene

[0097] To a solution of 6,14-dimesityldibenzo[hi,st]ovalene (14 mg, 0,020 mmol) dissolved in tetrahydrofuran (70 mL) was added N-bromosuccinimide (NBS) (14 mg, 0,079 mmol). The resulting mixture was stirred at room temperature for 2 h. The solvent was evaporated and the residue was purified by column chromatography to give the product (14 mg, 84%) as blue solid. The product had the following characteristics:

[0098] Mp: >400° C.; .sup.1H NMR (700 MHz, THF-d.sub.8) δ 10.67 (d, J=8.3 Hz, 2H), 8.72 (d, J=8.5 Hz, 2H), 8.34 (d, J=8.8 Hz, 2H), 8.29 (d, J=9.2 Hz, 2H), 7.87 (d, J=9.0 Hz, 2H), 7.85 (d, J=8.8 Hz, 2H), 7.27 (s, 4H), 1.93 (s, 12H); MS (MALDI-TOF): m/z Calcd for C.sub.54H.sub.30Br.sub.2: 864.10 [M]+, found: 864.06.

[0099] Thus, it was shown that the product had the formula:

##STR00037##

Synthesis of triisopropylsilyl ethynyl substituted 6,14-dimesityldibenzo[hi,st]ovalene

[0100] To a Schlenk tube equipped with a stirring bar was added 3,11-dibromo-6,14-dimesityldibenzo[hi,st]ovalene prepared above (4.3 mg, 5.0 μmol), Pd(PPh.sub.3).sub.4 (1.2 mg, 1.0 μmol) and CuI (0.38 mg, 2.0 μmol). The reaction tube was evacuated and backfilled with Argon for three times before addition of tetrahydrofuran (3 mL) and triethyl amine (1 mL). After degassing by three times freeze-pump-thaw cycles, triisopropylsilyl ethynyl (TIPS) acetylene (4.5 mg, 25 μmol) was added using a syringe. The resulting mixture was heated at 80° C. for 16 h.

[0101] After cooling down to room temperature, the solvent was evaporated and the residue was purified by column chromatography (n-hexane:ethyl acetate=10:1) to give product (4.0 mg, 75%) as blue solid. The product had the following characteristics: MS (MALDI-TOF): m/z Calcd for C.sub.78H.sub.76Si.sub.2: 1068.55 [M].sup.+, found: 1068.55

[0102] Thus, it was shown that the product had the formula:

##STR00038##

EXAMPLE 4

Synthesis of 3,11-bis(3,4,5-tris(2,5,8,11-tetraoxatridecan-13-yloxy)phenyl)-6,14-dimesityldibenzo[hi,st]ovalene

[0103] To a Schlenk tube equipped with a stirring bar was added 3,11-dibromo-6,14-dimesityldibenzo[hi,st]ovalene prepared as described in example 3 (3.0 mg, 3.5 μmol), 3,4,5-tris(tetraethylene glycol monomethyl ether)phenyl boronic acid pinacol ester (12 mg, 14 μmol), Pd(PPh.sub.3).sub.4 (1.6 mg, 1.4 μmol) and K.sub.2CO.sub.3 (4.8 mg, 35 μmol). The reaction tube was evacuated and backfilled with Ar for three times before a mixture of toluene/EtOH/H.sub.2O=2 mL/0.5 mL/0.5 mL was added. The mixture was degassed by three time freeze-pump-thaw cycles and heated at 90° C. overnight. After cooling down to room temperature, the reaction solution was extracted with ethyl acetate, washed with brine, dried over Na.sub.2SO.sub.4 and evaporated.

[0104] The residue was purified by column chromatography (ethyl acetate:MeOH=10:1 to 1:1) to give the product (5 mg, 69%) as blue oil. The product had the following characteristics:

[0105] .sup.1H NMR (250 MHz, THF-d.sub.8) δ 8.95 (d, J=8.6 Hz, 1H), 8.24 (d, J=8.7 Hz, 1H), 8.09 (d, J=9.3 Hz, 1H), 7.95 (s, 2H), 7.73 (d, J=9.2 Hz, 1H), 7.26 (s, 2H), 7.01 (s, 2H), 4.31 (t, J=5.2 Hz, 2H), 3.93-3.84 (m, 2H), 3.75 (d, J=5.5 Hz, 8H), 3.68-3.31 (m, 84H), 3.27 (s, 8H), 3.19 (s, 6H), 1.96 (s, 6H). MS (MALDI-TOF): m/z Calcd for C.sub.122H.sub.152O.sub.30: 2097.04 [M].sup.+, found: 2097.06

[0106] Thus, it was shown that the product had the formula:

##STR00039##

[0107] The solubility of this compound in water was evaluated at room temperature and was found to be 0.1 g/l.

EXAMPLE 5

Synthesis of 6,14-di(triisopropylsilyl ethynyl) dibenzo[hi,st]ovalene [DBOV-TIPS]

[0108] To an oven dried 25 mL Schlenk tube was added tetrahydrofuran (1.4 mL) and (triisopropylsilyl)acetylene (182 mg, 0.998 mmol). The solution was cooled down to 0° C. and n-BuLi (0.6 mL, 0.96 mmol, 1.6 M in n-hexane) was added slowly. The mixture was stirred at room temperature for 30 mins and the resulting solution (1.5 mL, 0.73 mmol) was transferred to a solution of 5,14-diformylbenzo[a]dinaphtho[2,1,8-cde:1′,2′,3′,4′-ghi]perylene prepared as described in example 1 (25 mg, 0.049 mmol) in dry THF (25 mL). After stirring for 21 h, the reaction was quenched by addition of saturated aqueous solution of NH.sub.4Cl (20 mL) and extracted with ethyl acetate (20 mL) for three times. The combined organic phase was washed with brine (20 mL), dried over Na.sub.2SO.sub.4 and evaporated. The residue was dissolved in anhydrous dichloromethane (25 mL) and degassed with dichloromethane vapor saturated argon flow. After addition of BF.sub.3 OEt.sub.2 (0.025 mL), the resulting solution was stirred at room temperature for 6 h. The reaction was quenched by addition of methanol (1 mL) and p-chloranil (12 mg, 0.049 mmol) was added. The mixture was stirred for 3 h and the solvent was evaporated.

[0109] The residue was purified by recrystallization from dichloromethane and methanol to give the product (10 mg, 50% yield) as blue solid. The product had the following characteristics:

[0110] Mp: >400° C. HRMS (MALDI-TOF): m/z Calcd for C.sub.60H.sub.56Si.sub.2: 832.3921 [M].sup.+, found: 832.3859 (error=−7.4 ppm).

[0111] Thus, it was shown that the product had the formula:

##STR00040##

[0112] It was evaluated that this compound has a narrow absorption spectrum as well as a narrow emission spectrum, which are both shown in FIG. 3b (Abs: absorbance; PI: PL-emission).

[0113] The maximum absorption wavelength in toluene (10.sup.−5 mol/L) was 647 nm (molar extinction coefficient ε=61511 M.sup.−1 cm.sup.−1) with a fluorescence quantum yield of 0.67.

EXAMPLE 6

Synthesis of 6,14-bis[3,4,5-tris(dodecoxyl)-phenyl]dibenzo[hi,st]ovalene

Synthesis of 5-bromo-1,2,3-tris(dodecyloxy)benzene

[0114] 5-Bromo-1,2,3-trimethoxybenzene (2.5 g, 10 mmol) was dissolved in dry DCM (30 mL), the temperature was cooled down to −78° C. and stirred for 10 minutes before BBr.sub.3 (8.26 g, 33.0 mmol) was added dropwise. After addition, the reaction mixture was gradually warmed up to room temperature and stirred overnight. The reaction mixture was added into a 100 mL of ice water and then extracted with ethylacetate (100 mL) for three times. The organic phase was combined, washed with brine (100 mL) and dried over Na.sub.2SO.sub.4. After evaporation of the solvent under reduced pressure and dried under vacuum pump for 2 hours, crude 5-bromobenzene-1,2,3-triol (2.0 g, 98%) was obtained as white solid. This intermediate was used directly for the next step without characterization and further purification.

[0115] To a 100 mL Schlenk flask was added 5-bromobenzene-1,2,3-triol (2.0 g, 9.8 mmol), 1-bromododecyl (9.92 g, 40.0 mmol) and K.sub.2CO.sub.3 (5.52 g, 40.0 mmol). The flask was evacuated and backfilled with Ar for three times before N,N-dimethylformamide (50 mL) was added. The mixture was heated at 80° C. for 20 hours. After completion of the reaction shown by TLC (n-hexane:ethyl acetate=10:1), the mixture was cooled down to room temperature and diluted with ethyl acetate (200 mL), washed with water (50 mL), brine (50 mL), dried over Na.sub.2SO.sub.4 and evaporated.

[0116] The obtained residue was purified by column chromatography (n-hexane) and recrystallized with ethanol to give 5-bromo-1,2,3-tris(dodecyloxy)benzene (4.5 g, 63% yield). The product had the following characteristics:

[0117] Mp: >400° C.; .sup.1H NMR (300 MHz, Methylene Chloride-d.sub.2) δ 6.68 (s, 2H), 3.97-3.83 (m, 6H), 1.85-1.72 (m, 4H), 1.72-1.62 (m, 2H), 1.51-1.39 (m, 6H), 1.39-1.22 (m, 48H), 0.94-0.82 (m, 9H); .sup.13C NMR (75 MHz, Methylene Chloride-d2) δ 154.3, 137.8, 115.8, 110.3, 73.8, 69.7, 32.4, 30.7, 30.2, 30.2, 30.1, 30.1, 30.1, 30.0, 29.8, 29.8, 29.7, 26.5, 26.5, 23.1, 14.3; FD-MS (8 kV): m/z 709.6; HRMS (MALDI-TOF): m/z Calcd for C.sub.42H.sub.77BrO.sub.3: 708.5056 [M].sup.+, found: 708.5005 (error=−6.5 ppm).

Synthesis of 6,14-bis[3,4,5-tris(dodecoxyl)phenyl]dibenzo[hi,st]ovalene

[0118] To a 25 mL Schlenk tube was added magnesium turnings (27 mg, 1.1 mmol), 12 (3 mg, 0.011 mmol) and tetrahydrofuran (1 mL). The mixture was gently heated while approximately 1 mL of 5-bromo-1,2,3-tris(dodecyloxy)benzene (568 mg, 0.802 mmol) dissolved in tetrahydrofuran (3 mL) was added. As soon as the solution became colorless, the remaining solution was added dropwise under mild reflux and stirring was continued overnight to give Grignard reagent solution. The Grignard reagent solution (4 mL) prepared above was transferred to a solution of 5,14-diformylbenzo[a]dinaphtho[2,1,8-cde:1′,2′,3′,4′-ghi]perylene prepared as described in example 1 (25 mg, 0.049 mmol) in dry THF (30 mL). After stirring at room temperature for 4 h, the reaction was quenched by addition of saturated NH.sub.4Cl solution (15 mL). After stirring for 15 mins, the solution was extracted with ethyl acetate (50 mL) for three times. The combined organic phase was washed with brine, dried with Na.sub.2SO.sub.4 and evaporated.

[0119] The residue obtained above was dried under vacuum for 2 h and dissolved in anhydrous dichloromethane (30 mL). After bubbling with dichloromethane vapor saturated argon flow for 15 mins, BF.sub.3 OEt.sub.2 (0.1 mL) was added and the stirring was continued overnight. After quenching with methanol (2 mL), p-chloranil (12 mg, 0.049 mmol) was added and the mixture was stirred for 2 hours at room temperature.

[0120] The solvent was evaporated and the residue was purified by column chromatography (n-hexane:DCM=3:1 to 0:1) to give the product (72 mg, 85% yield) as blue solid. The product had the following characteristics:

[0121] Mp: >400° C.; .sup.1H NMR (300 MHz, THF-d.sub.8) δ 9.27 (d, J=8.5 Hz, 2H), 9.02 (d, J=7.7 Hz, 2H), 8.36 (d, J=8.4 Hz, 2H), 8.10 (d, J=8.3 Hz, 2H), 7.97 (t, J=9.1 Hz, 4H), 7.89 (d, J=9.3 Hz, 2H), 6.87 (s, 4H), 4.16 (t, J=6.2 Hz, 4H), 4.05 (t, J=6.2 Hz, 8H), 1.97-1.79 (m, 15H), 1.69-1.61 (m, 4H), 1.61-1.15 (m, 119H), 0.97-0.80 (m, 18H); .sup.13C NMR (75 MHz, THF-d.sub.8) δ 139.1, 137.5, 134.9, 132.7, 132.5, 131.3, 130.3, 130.1, 128.9, 127.9, 127.5, 127.3, 126.5, 125.5, 125.1, 124.7, 124.3, 123.6, 123.4, 122.1, 121.4, 111.0, 74.0, 70.0, 33.2, 33.1, 31.9, 31.1, 31.1, 31.1, 31.0, 30.9, 30.9, 30.8, 30.7, 30.7, 30.6, 27.6, 27.4, 23.9, 23.9, 14.9, 14.9; HRMS (MALDI-TOF): m/z Calcd for C.sub.122H.sub.16806: 1729.2841 [M].sup.+, found: 1729.2822 (error=−1.1 ppm).

[0122] Thus, it was shown that the product had the formula:

##STR00041##

[0123] The maximum absorption wavelength in toluene (10.sup.−5 mol/L) was 609 nm (molar extinction coefficient ε=3.85×10.sup.5 M.sup.−1 cm.sup.−1) with a high fluorescence quantum yield of 0.89.

EXAMPLE 7

Synthesis of 6,14-diphenyldibenzo[hi,st]ovalene [DBOV-Ph]

[0124] 6,14-diphenyldibenzo[hi,st]ovalene was prepared analogous to example 2 by using phenylmagnesium bromide solution instead of 2,6-dimethylphenylmagnesium bromide solution. It was evaluated that this compound has a narrow absorption spectrum as well as a narrow emission spectrum, which are both shown in FIG. 3c (Abs: absorbance; PI: PL-emission).

EXAMPLE 8

Synthesis of N-hexyl fumaric acid imide group substituted 6,14-dimesityldibenzo[hi,st]ovalene)

[0125] 6,14-dimesityldibenzo[hi,st]ovalene was reacted with N-hexyl fumaric acid imide in diphenylether for 2 days at 275° C. The analysis showed that the product had the chemical formula C.sub.76H.sub.58N.sub.2O.sub.4 with a measured mass of 1062.4324 g/mol. Thus, the formula was confirmed to be:

##STR00042##

[0126] It was evaluated that this compound has a narrow absorption spectrum as well as a narrow emission spectrum, which are both shown in FIG. 3d.

EXAMPLE 9

Toxicity of 3,11-bis(3,4,5-tris(2,5,8,11-tetraoxatridecan-13-yloxy)phenyl)-6,14-dimesityldibenzo[hi,st]ovalene)

[0127] 3,4,5-Tris(hexaethylene glycol) substituted 6,14-dimesityldibenzo[hi,st]ovalene was prepared analogous to example 4 and evaluated concerning its toxicity. It was found that the compound is non-toxic up to a concentration of 5 μM, which is a sufficiently high concentration in terms of bioimaging. The compound has a good solubility in DMSO.

EXAMPLE 10

Spectral Properties of Other Polycyclic Aromatic Hydrocarbons

[0128] The absorption and emission spectra of the following four polycyclic aromatic hydrocarbons have been recorded, which are shown in FIGS. 4a to 4d.

##STR00043## ##STR00044##

[0129] The absorption and emission spectra of all these compounds have been evaluated and were sufficiently narrow for both, SMLM, STED, MINFLUX and SIMFLUX microscopy. The respective spectra are shown in FIGS. 4a to 4d (Abs: absorbance; PI: PL-emission).

EXAMPLE 11

Evaluating the Suitability for SMLM/Performing SMLM Measurement

[0130] Different measurements were performed with 6,14-dimesityldibenzo[hi,st]ovalene being embedded in a polystyrene matrix and others with 6,14-dimesityldibenzo[hi,st]ovalene in air. A Leica GSD microscopy was used for the microscopy with an imaging laser wavelength: of 532 nm.

[0131] (Sample Preparation: Embedding with Polystyrene)

[0132] 6,14-dimesityldibenzo[hi,st]ovalene powder was dispersed in toluene at room temperature. The diluted solution having a concentration of e.g. 10.sup.−8 mol/L, 10.sup.−9 mol/L, 10.sup.−1° mol/L or 10.sup.−11 mol/L, respectively, was mixed with the same amount of toluene solution (0.08 mg/mL) of polystyrene.

[0133] A glass coverslip (#1.5) was cleaned by oxygen-plasma cleaner (250 W, 5 minutes). Approximately 1 μl of the so obtained solution was spin-coated on the oxygen-plasma-treated glass coverslip for 60 s, namely for 20 s at 2,000 rpm and for 40 s at 4,000 rpm.

[0134] The sample was dried by heating to 90° C. for 1 h on a hot plate.

[0135] The prepared glass coverslip was taped to a slide and put on the microscope for imaging test.

[0136] (Sample Preparation: In Air Environment)

[0137] 6,14-dimesityldibenzo[hi,st]ovalene powder was dispersed in toluene at room temperature to a concentration of 10.sup.−5 mol/L and was then further diluted to the needed concentration of e.g. 10.sup.−8 mol/L, 10 mol/L, 10.sup.−1° mol/L or 10.sup.−11 mol/L, respectively.

[0138] A glass coverslip (#1.5) was cleaned by oxygen-plasma cleaner (250 W, 5 minutes).

[0139] About 10 μl solution were put on top of the cleaned coverslip and then covered by a glass slide. It was waited until the whole toluene has volatilized completely and then the coverslip was taped to the slide. Afterwards, the prepared sample was put on the microscope for imaging testing.

[0140] (Sample Preparation: For Nano- and Micro-Structure Imaging Measurements)

[0141] The measured nano- and micro-structure sample was based on the gridded cover glass purchased from Ibidi GmbH in Martinsried, Germany. The nano structure was produced on this cover glass by Focus Ion Beam (FIB) or induced by mild “etching” method—treated with sodium hydroxide solution 4 M at room temperature for 24 h. For the sample preparation, 5 μl 10 mol/L solution were put on top of the micro/nano-structure sample. After a few minutes, when all of the toluene was volatilized completely, the prepared sample was put on the microscope for imaging testing.

[0142] (Blinking Behavior of 6,14-Dimesityldibenzo[Hi,St]Ovalene Embedded in Polystyrene or in Air)

[0143] SMLM were performed with 6,14-dimesityldibenzo[hi,st]ovalene embedded in polystyrene or in air. 30 000 frames were recorded with an exposure time of 30 ms each within 30 min. FIG. 5a shows a widefield image of the sample and FIG. 5b shows a part of the duty cycle diagram. It was thus confirmed that 6,14-dimesityldibenzo[hi,st]ovalene is well suited for SMLM.

[0144] (Surface Detection by SMLM)

[0145] A gridded cover glass, whose surface provides 400 squarish recesses, wherein each recess has a cross-section of 50 μm×50 μm and a depth of 5 μm, wherein some of the recesses contain numbers or letters, respectively, as shown in FIG. 6a. The sample was prepared as described above for the “Sample Preparation: For nano- and micro-structure imaging measurements”. 30,000 frames were recorded with an exposure time of 30 ms each. FIGS. 6a and 6b show SMLM images of a section of the sample showing a surface imperfection in field with the number 10. As is can be seen in FIGS. 6a and 6b the resolution of the SMLM images is so good that not only the surface structure is reproduced in detail, but so excellent that even small surface imperfections in form of a crack has been detected.

EXAMPLE 12

Evaluating the Suitability for STED/Performing STED Measurement

[0146] Different measurements were performed with 6,14-dimesityldibenzo[hi,st]ovalene being embedded in a polystyrene matrix and with 6,14-dimesityldibenzo[hi,st]ovalene in air. A Leica SP8 microscopy was used for the microscopy with an excitation laser wavelength of 561 nm and depletion (STED) laser wavelength of 775 nm.

[0147] (Stability of 6,14-dimesityldibenzo[hi,st]ovalene Embedded in Polystyrene)

[0148] The samples of 6,14-dimesityldibenzo[hi,st]ovalene being embedded in a polystyrene matrix were prepared as in example 11 except that the diluted solution had a concentration of 10.sup.−7 mol/L before being mixed with the toluene solution of polystyrene. The prepared glass coverslips were taped to a slide and kept in dark at room temperature until use.

[0149] A 15-months old sample of 6,14-dimesityldibenzo[hi,st]ovalene embedded in polystyrene as well as a freshly prepared sample of 6,14-dimesityldibenzo[hi,st]ovalene embedded in polystyrene were analyzed by STED. Both samples showed similar imaging results.

[0150] (Surface Detection by STED)

[0151] 6,14-dimesityldibenzo[hi,st]ovalene powder was dispersed in toluene at room temperature to a concentration of 10.sup.−5 mol/L and was then further diluted to the needed concentration of e.g. 10.sup.−7 mol/L. A gridded glass coverslip (#1.5) purchased from Ibidi GmbH in Martinsried, Germany which have microscopic/nano-structures was cleaned by oxygen-plasma cleaner (250 W, 5 minutes). 8 μl 10.sup.−7 mol/L solution were put on top of the micro/nano-structure sample. After a few minutes, when all of the toluene was volatilized completely, the prepared sample was put on the microscope for 3D STED imaging.

[0152] The STED was performed with the following microscopy settings of the Leica SP8 microscope:

[0153] Excitation light: 561 nm 1% power; STED light: 775 nm 80% power; Format: 2048×2048; Size: 38.7×38.7 um; Scanning speed: 400 Hz; pixel size: 18.94 nm×18.94 nm; Zoom factor: 3; Pixel Dwell time: 300 ns; Frame rate: 0.0121 s; Z stack: 8.29 μm/54 steps; Detector: HyD2; STED delay time: 0; Line average: 8; Total imaging time: about 74 minutes.

[0154] The obtained results are shown in FIGS. 7a to 7f. The depth of the nanostructures shown in FIGS. 7a, 7b, 7e and 7f was larger than 2 μm which was too deep and could not be imaged by AFM microscope.

[0155] The results reveal that 6,14-dimesityldibenzo[hi,st]ovalene is very stable and still very bright even with high STED beam power for 3D STED imaging of more than one hour and that 6,14-dimesityldibenzo[hi,st]ovalene has a significant STED effect both embedded with polystyrene as well as in air environments.

EXAMPLE 13

Evaluating the Compound Synthesized in Example 4 for Cell Imaging

[0156] 3, 11-bis(3,4,5-tris(2,5,8, 11-tetraoxatridecan-13-yloxy)phenyl)-6, 14-dimesityldibenzo[hi,st]ovalene (subsequently abbreviated as DBOV-Mes-OTEG) synthesized in example 4 was evaluated concerning its properties.

[0157] More specifically, the cytotoxicity of DBOV-Mes-OTEG was tested in living cells. The results are shown in FIG. 8. The test showed that the water soluble DBOV-Mes-OTEG does not show any significant toxicity to cells in a concentration of 1 μM. Moreover, the uptake of DBOV-Mes-OTEG into living cells was investigated by imaging 21 hours with low laser intensity at the spinning disk confocal microscopy (Visitron). The test revealed that DBOV-Mes-OTEG was taken into the cytoplasm at the beginning of the incubation and that its location was after 21 hours incubation in the nucleus and nuclear membrane.

[0158] After the aforementioned live cell imaging, the samples were fixed and imaged in phosphate-buffered saline (PBS) without special imaging buffer. The test revealed that DBOV-Mes-OTEG was able to be imaged in the blinking mode under continuous exposure with high laser intensity 10 kW/cm.sup.2 for 90 min. Furthermore, no significant decrease of blinking signals of DBOV-Mes-OTEG were detected. In addition, blinking signals could also be imaged with low laser intensity of 240 W/cm.sup.2 and this reveals the possibility of living cell imaging with DBOV-Mes-OTEG.

EXAMPLE 14

Comparison of the Blinking Properties of Different Compounds

[0159] The blinking properties of the following compounds have been measured and compared with each other:

##STR00045## ##STR00046##

[0160] DBOV-Mes, C.sub.60, C78 and C96 are compounds to be used in accordance with the present invention, whereas Alexa-647 is a commercially available organic dye distributed from ThermoFisher Scientific under the tradename Alexa Fluor® 647 dye, which is the gold standard for SMLM with excellent blinking properties, namely high photon numbers and low on-off duty cycles.

[0161] The blinking properties of DBOV-Mes and C60 were evaluated in phosphate buffered saline (DPBS), in air and in polystyrene, whereas the blinking properties of C78 and C96 were measured in a polystyrene film and the blinking properties of Alexa-647 were evaluated in the presence of an enzymatic oxygen scavenging system (glucose oxidase with catalase (GLOX)) and a primary thiol (MEA, 10 mM). More specifically, the duty cycle, the number of photons per switching event and the blinking time of the respective samples were measured.

[0162] The duty cycle is the fraction of time a molecule resides in its fluorescent on-state and was calculated according to the method described by Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M. and Zhuang, X in “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging”, Nat. Methods, 8, page 1027 (2011).

[0163] The number of photons per switching event and the blinking time of the respective samples were determined as follows: Fluorescence intensity traces were extracted by first generating a maximum intensity projection of the recorded frames. Fluorescence signals in this projection were localized using the Thunderstorm-plugin in Fiji as described by Ovesný, M., Křižek, P., Borkovec, J., Švindrych, Z. and Hagen, G. M. ThunderSTORM in “a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging”, Bioinformatics, 30, pages 2389 to 2390 (2014) and by Schindelin, J. et al. in “Fiji: an open-source platform for biological-image analysis”, Nat. Methods, 9, page 676 (2012). Then the intensity trace for each localization throughout all frames of the raw data was calculated as the total background corrected intensity in a 7×7 ROI around the localized coordinates. The local background for every localization in every frame was calculated within a 17×17 ROI. Pixel values bigger than 5 times the standard deviation within this ROI were excluded from background calculation as they were considered as fluorescence signal. Calculated total intensities within the ROls were then plotted for every frame. The calculation of photons per blinking event was done by localization of the compounds in every frame of the recorded imaging data. Localizations were then filtered according to the expected width of the signals. Localizations appearing in consecutive frames were then merged. To account for low photon yields that might lead to missed detections, we allowed one dark frame between two detections for merging. As spatial constraint we used a maximum distance of 80 nm, a rather large radius was chosen to allow localizations with low photon counts to be still properly merged. The histogram of photon counts was then generated and fitted by a monoexponential function. Reported mean values are derived from the fit. The mean blinking duration was extracted from the same localization data. The mean value was derived from a single exponential fit.

[0164] The obtained results are shown in table 1.

TABLE-US-00001 TABLE 1 Dye Alexa- 647 DBOV-Mes C60 C78 C96 Environment DPBS Buffer DPBS Air PS buffer Air PS PS PS Detected photons 3.438 4.918 5.570 4.902 3.673 4.960 4.960 5.740 5.020 per switching event Duty cycle (×10.sup.−4) 2.1 1.3 4.7 8 5.3 1.2 3.2 2.7 1.7 Blinking time (ms) 65 87 108 54 75 79 96 83 94

[0165] The results show that the compounds to be used in accordance with the present invention exhibit (in comparison to Alexa 647, the current gold standard small molecule dye) ideal properties for SMLM due to their environmentally-independent blinking behaviour, large photon numbers, good stability and well-defined excitation and emission spectra.