Rapid Fluorescence Switching by Using a Fast Photochromic [2.2

Subscriber access provided by University of Pennsylvania Libraries

Article

Rapid Fluorescence Switching by Using a Fast Photochromic [2.2]Paracyclophane-Bridged Imidazole Dimer Katsuya Mutoh, Michel Sliwa, and Jiro Abe J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/jp309917s • Publication Date (Web): 15 Feb 2013 Downloaded from http://pubs.acs.org on February 19, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry C is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Rapid Fluorescence Switching by Using a Fast Photochromic [2.2]Paracyclophane-Bridged Imidazole Dimer Katsuya Mutoh,†Michel Sliwa,§ and Jiro Abe*, †, ‡ †

Department of Chemistry, School of Science and Engineering, Aoyama Gakuin University, 5-10-1

Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5258, Japan ‡

CREST, Japan Science and Technology Agency (JST), 7 Gobancho, Chiyoda-ku, Tokyo 102-0076,

Japan. §

Laboratoire de Spectrochimie Infrarouge et Raman (UMR 8516 CNRS), Université de Lille 1 Sciences

et Technologies, Bât. C5, 59655, Villeneuve d’Ascq Cedex, France * E-mail: [email protected] RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 32

ABSTRACT Recently, we have developed a series of fast photochromic imidazole dimers with a [2.2]paracyclophane (PC) moiety that bridge diphenylimidazole units, and succeeded the acceleration of the thermal decoloration rate. The colorless [2.2]paracyclophane ([2.2]PC)-bridged imidazole dimers show a photoinduced homolytic bond cleavage of the C−N bond between the imidazole rings to give a pair of colored imidazolyl radicals upon UV light irradiation, followed by the radical−radical coupling reaction to form the initial C−N bond between the imidazole rings. The decoloration reaction to give the initial imidazole dimer proceeds only thermally. The high quantum yield close to unity of the photochromic reaction and the large extinction coefficient of the radical achieves both high optical density at photostationary state and rapid switching speed. The application to rapid fluorescence switching has been investigated to develop a new type of photochromic fluorescence switching molecule applicable to super-resolution microscopy. The widespread absorption of the colored radical lying between 500 nm and 900 nm enables the efficient quenching of the excited electronic state of the fluorophores by FRET from the fluorophores to the radical moiety. We successfully developed a [2.2]paracyclophane-bridged imidazole dimer possessing a fluorescein moiety as a fluorescence unit. This photochromic dye shows fast photochromism to give a pair of imidazolyl radicals that quench the fluorescence from the fluorescent unit by the FRET mechanism. The fluorescence intensity can be switched rapidly with the fast photochromism.

KEYWORDS

photochromism

·

fluorescence

switching

·

super

resolution

microscopy

·

hexaarylbiimidazole · imidazole dimer

1. Introduction Fluorescence microscopy is one of the essential tools for real time imaging of the organelles in vivo.1 The development of the microscope and discovery of the fluorescent probes like GFP enables not only the real-time imaging of the living cell but also the investigation of the interaction between an enzyme


2

Page 3 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and a substrate. However, for microscopy technique, the considerable weakness is a lower limit of its spatial resolution known as the diffraction limit (~200 nm) due to the wavelike character of light. Stimulated-emission depletion (STED) microscopy and photoactivated localization microscopy (PALM) / stochastic optical reconstruction microscopy (STORM) called super-resolution microscopy are the representative examples breaking the diffraction limit.2 The light switching microscopy (RESOLFT type microscopy) is also classified into the super-resolution microscopy, which follows the principle of STED microscopy.3 This method relies on the reversible transformation between the fluorescent on and off states upon photoexcitation. Photochromic molecules that change their structure reversibly with irradiation of light have a potential to switch the fluorescent ability.4 Raymo et al. demonstrated the rapid fluorescence “ON” and “OFF” switching by using a fast photochromic oxazine derivative with a fluorescent BODIPY unit. The oxazine dyad changes its structure to the 3H-indolium cation upon UV light irradiation and it shows fast thermal back reaction to the original state within 10 µs. Since the colored 3H-indolium cation absorbs in the range of wavelengths of light overlapping the fluorescnce spectrum of the BODIPY moiety, the fluorescence intensity of the oxazine dyad could be switched with photochromism, followed by Förster resonance energy transfer (FRET). However, the fluorescence intensity of the oxazine dyad reduces less than 10 % with the photochromic reaction, and the improvement of the contrast ratio of the fluorescence intensities between the ON and OFF states has been desired. Photochromic [2.2]paracyclophane ([2.2]PC)-bridged imidazole dimers that we have recently developed show a photoinduced homolytic bond cleavage reaction to give a pair of colored imidazolyl radicals upon UV light irradiation and the radicals thermally go back to the initial imidazole dimer by the radical−radical coupling reaction.5 The high quantum yield close to unity of the photochromic reaction and the large extinction coefficient of the radical achieves both high optical density at photostationary state and rapid switching speed. The excited electronic state of a radical is normally deactivated by a nonradiative process. Hence, the [2.2]PC-bridged imidazole dimer potentially has a rapid fluorescence switching ability and possibility to achieve high contrast ratio. In addition, the widespread absorption of the radical lying between 500 nm ACS Paragon Plus Environment

3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 32

and 900 nm enables the efficient quenching of the excited electronic state of fluorophores by FRET from the fluorophores to the radical moiety, leading to the multicolor super-resolution microscopy. Moreover, the significant temperature dependence of the reaction rate for the thermal back reaction from the colored radical to the colorless imidazole dimer enables the temperature mapping in a cell by the observation of the fluorescence recovery rate.6 Therefore, we have designed and synthesized a derivative of novel [2.2]PC-bridged imidazole dimer 1 possessing a fluorescein moiety as a fluorescent unit to demonstrate the fluorescence modulation with the rapid photochromic switching between the imidazole dimer, ON state, and the radical, OFF state, as a new fluorescent probe (Scheme 1).

2. Experimental Section 2.1 Synthesis All reactions were monitored by thin-layer chromatography carried out on 0.2 mm E. Merck silica gel plates (60F-254). Column chromatography was performed on silica gel (Silica Gel 60N (spherical, neutral), 40-50 µm, Kanto Chemical Co., Inc.). 1H-NMR spectra were recorded at 400 MHz on a Bruker AVANCE III 400 NanoBay. DMSO-d6 and CD3OD were used as deuterated solvent. MASS spectra (ESI-TOF-MS) were measured by using a Bruker micrOTOF II-AGA1. All glassware were washed with distilled water and dried. Unless otherwise noted, all reagents and reaction solvents except acetic acid were purchased from TCI, Wako Co. Ltd., Aldrich Chemical Company, Inc, and ACROS Oraganics, and were used without further purification. Acetic acid was purified by adding 5 wt % KMnO4, boiling under reflux for 2 h and then fractionally distilling to remove acetaldehyde. Pseudogem-[4-formyl-13-(4,5-diphenyl-1H-imidazol-2-yl)][2.2]paracyclophane5b (2) and compound 37a were prepared according to a literature procedure. pseudogem-(4,5-diphenyl-1H-imidazol-2-yl)-4-(3’,6’-dihydroxyspiro(isobenzofuran-1(3H),9’(9H)xanthen)-3-one)-5-phenyl-1H-imidazol-2-yl)[2.2]paracyclophane (4). pseudogem-[4-formyl-13-(4,5-diphenyl-1H-imidazol-2-yl)][2.2]paracyclophane (50 mg, 0.110 mmol), compound 3 (60 mg, 0.109 mmol) and ammonium acetate (120 mg, 1.557 mmol) were stirred at 110 ˚C ACS Paragon Plus Environment

4

Page 5 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


in acetic acid (2 mL) for 72 h. The reaction was quenched with water and the reaction mixture was extracted with ethyl acetate. The organic layer was washed with water and brine, dried over Na2SO4, filtered, and evaporated. The product was separated by preparative thin layer chromatography (PTLC) twice, using MeCN:CH2Cl2 = 10:11 and then EtOH:MeCN:CH2Cl2 = 2:2:25, as eluents respectively to give a red solid as a mixture of two structural isomers (11 mg, 11 %). 1H-NMR (400 MHz, DMSO-d6) δ: 11.90 (s, 1H, one structural isomer), 11.82 (s, 1H, one structural isomer), 11.70 (m, 2H, two structural isomers), 8.03 (m, 1H, one structural isomer), 7.57 (s, 1H, one structural isomer), 7.39-6.87 (m, 37H, two structural isomers), 6.86-6.41 (m, 20H, two structural isomers), 6.38 (s, 1H, one structural isomer), 4.74-4.58 (m, 4H, two structural isomers), 3.22-2.72 (m, 12H, two structural isomers); m/z (HR–ESI– TOF–MS): calcd. 899.3228 (C60H43N4O5); found 899.3203 [M+H]+. pseudogem-FPI-DPI[2.2]PC (1). All manipulations were carried out with the exclusion of light. Under nitrogen, to a solution of 4 (7 mg, 7.8 µmol) in benzene:EtOH = 4:1 (5 mL) was added PbO2 (19 mg, 79.5 µmol). The reaction mixture was vigorously stirred for 3 h at room temperature. The solution was filtered with celite and the filtrate was evaporated. Then the crude product was separated by PTLC, using EtOH:MeCN:CH2Cl2 = 3:3:50 as eluents to give a red solid (5 mg, 64 %). 1H-NMR (400 MHz, CD3OD, one of the structural isomer) δ: 7.92 (s, 1H), 7.65 (d, J = 4.0 Hz, 1H), 7.49-7.44 (m, 2H), 7.38-7.35 (m, 4H), 7.21-6.87 (m, 15H), 6.64 (s, 2H), 6.57-6.44 (m, 5H), 4.45-4.38 (m, 1H), 3.62-2.86 (m, 7H); m/z (HR–ESI–TOF–MS): calcd. 897.3071 (C60H41N4O5); found 897.3056 [M+H]+. 2.2 Laser Flash Photolysis Measurement of the thermal back reaction rate of the radical The laser flash photolysis experiments were carried out with a TSP-1000 time-resolved spectrophotometer (Unisoku). A 10 Hz Q-switched Nd:YAG (Continuum Minilite II) laser with the third harmonic at 355 nm (ca. 8 mJ per 5 ns pulse) was employed for the excitation light. The probe beam from a halogen lamp (OSRAM HLX64623) was guided with an optical fiber scope to be arranged in an orientation perpendicular to the exciting laser beam. The probe beam was monitored with a ACS Paragon Plus Environment

5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 32

photomultiplier tube (Hamamatsu R2949) through a spectrometer (Unisoku MD200). Sample solutions were deaerated by argon bubbling prior to the laser flash photolysis experiments. Measurement of the fluorescence recovery rate Luminescence switching in the millisecond range was recorded using a laser flash photolysis apparatus. 490 nm excitation light were provided by a Hamamatsu Light Source (LC8) combined with a long pass filter and 10nm band pass filter center at 490 nm, coupled to fiber and a condenser to get a pseudo collimated light (diameter 6 mm, 5 mW). Excitation pulses to switch the fluorescence (355 nm, fwhm 4 ns, 10 mJ, 0.5 Hz) were provided by a 10 Hz Nd:YAG laser (Continuum Surelite II) at the opposite direction from the excitation source. The diameter of the excitation beam was about 8 mm to ensure that all the volume of collected emission was excited. The emitted light was collected at 90°, dispersed by a monochromator (Horiba Jobin-Yvon, iHR320) and analyzed with a photomultiplier (R1477-06, Hamamatsu) coupled to a digital oscilloscope (LeCroy 454, 500 MHz). Synchronization of excitation pulses and acquisition time was secured with a PCI-6602 8 Channel counter/timer (National Instruments). The experiment was controlled by a homemade software written in LabView environment. The recorded traces were averaged for several pulses. 1 mm (excitation, emission length) × 1 cm spectroscopic cell was used. Optical density (1 mm) of the solution at the excitation wavelength was below 0.1 to avoid any inner effect. The stability of the solution was checked after several excitation that fluorescence intensity keeps constant. 2.3 Fluorescence microscopy The fluorescence microscopy was carried out with an Olympus BX51 microscope. The high-pressure mercury vapor lamp through a 480/20 nm bandpass filter was employed for the fluorescence excitation light. The stimulus light inducing photochromism (365 nm and 405 nm, 200 ms/pulse) is performed with a Mosaic Digital Diaphragm System (Photonic Instruments, Inc.). The fluorescence of compound 1 was monitored with a Q IMAGING Scientific Digital CCD Camera (QI Click, exposure time; 15 ms) through a 546/12 nm band-pass filter.


6

Page 7 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3. Results and Discussion 3.1 Synthesis Compound 1 was synthesized according to Scheme 2. Compound 4 was oxidized with PbO2 to avoid the oxidation of hydroxyl groups of fluorescein although K3[Fe(CN)3] is used as the oxidant normally. The introduction of fluorescein was confirmed by 1H NMR spectroscopy and the molecular formula was determined by HR−ESI−TOF−MS. The purity of 1 was estimated to be 98 % by HPLC analysis. Although 1 has four kinds of structural isomers depending on the position of fluorescein, the measurements were carried out using the mixture of the isomers.5f 3.2 Photochromic properties The steady state UV-vis absorption spectrum of 1 is shown in Figure 1. The absorption band around 470 nm can be assigned to the absorption of the fluorescein moiety.7 The excitation of this absorption band results in the characteristic fluorescence of the fluorescein moiety at 520 nm. The fluorescence quantum yield obtained with 480 nm excitation was estimated to be about 0.39 using Rhodamine 6G as a standard. The value is lower compared with that of fluorescein8 (about 0.8) and the low fluorescence quantum yield of 1 can be explained by an increase of the non-emissive relaxation pathway (nature of the relaxation is discussed in the next section), however this value is enough high for single molecule fluorescence imaging. Compound 1 shows the photochromism with the color change from yellow to blue upon UV light irradiation. The transient vis-NIR absorption spectrum of 1 shows a narrow absorption band at 400 nm and a broad absorption band ranging from 500 nm to 900 nm attributable to the radical 1R that absorbs the fluorescence in the range which fluorescein emits. Therefore, the efficient fluorescence quenching by FRET from the fluorescein moiety to the radical moiety would be expected. All of the absorption bands of 1R decay with the same time constant (Figure 2a), indicating the presence of one conformation of the colored radical. The half-life of 1R is 4.4 ms at 25 °C in ethanol. Figure 2b shows the decay profiles of the transient absorbance at 400 nm of 1R, measured over the temperature range from 5 °C to 40 °C. The thermal bleaching process obeys first order kinetics and the activation parameters are estimated from the Eyring plots. The ∆H‡ and ∆S‡ values estimated from ACS Paragon Plus Environment

7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 32

standard least-squares analysis of the Eyring plots are 49.6 kJ mol−1 and −36.4 J K−1 mol−1, respectively. The free energy barrier (∆G‡ = ∆H‡ − T∆S‡) for the thermal back reaction is 60.5 kJ mol−1 at 25 °C. 3.3 Fluorescence switching property Figure 3a shows the fluorescence recovery rate (green line) after 355 nm laser pulse excitation, under 490 nm irradiation at 20 °C. The fluorescence recovery rate is consistent with the thermal dimerization reaction rate of the radical (blue line). Moreover the photo-stability of the fluorescence switching was demonstrated after several 355 nm excitation (Figure 3b). The nature of the fluorescence quenching should be discussed here with more details because not only a FRET mechanism but also an electron quenching process from fluorescein to the radical can be considered. Therefore, the mechanism of the fluorescence quenching was investigated by comparing the switching efficiency in polar and apolar solvent since the efficiency based on the electron quenching process would be affected by the polarity of solvent. While 1 is only soluble in polar solvent, a derivative possessing PEG chains has been prepared to increase the solubility in apolar solvent (Supporting information). As shown in Figure S13, the fluorescence switching ratio is independent (40%) of the solvent and it is the critical evidence of the quenching based mainly on FRET by the radical. The absolute value of switching efficiency is an important parameter for the application and is asscociated to two different parameters: the photochromic reaction yield and FRET efficiency. The ultrafast photodissociation dynamics of pseudogembisDPI[2.2]PC was demonstrated and thus the quantum yield of the photochromic reaction of pseudogem-bisDPI[2.2]PC can be considered to be almost unity. However the photochromic reaction yield for pseudogem-FPI-DPI[2.2]PC would not be unity and the contribution of the electronic excitation state of the fluorescein moiety to the dissociative state of pseudogem-bisDPI[2.2]PC should be considered to determine the photochromic quantum yield because the excitation of the fluorescein moiety can also lead to the radical species (Figure S14) even if the [2.2]PC-bridged imidazole dimer moiety does not absorb the excitation light. The photochromic reaction of HABI has been reported to be efficiently sensitized with the visible light photosensitizing dyes such as aryl ketones and p(dialkylamino)aryl aldehydes.9 Krongauz et al. reported that ο-Cl-HABI cleaves into radicals upon ACS Paragon Plus Environment

8

Page 9 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


visible light irradiation in the presence of the photosensitizing dye, 2,5-bis[(2,3,6,7-tetrahydro1H,5Hbenzo[ij]quinolizin-1-yl)methylene]cyclopentanone (JAW).10 Previously, we also reported the photosensitized photochromism with pyrenyl units, Py-HABI.11 Because the difference of the energy level between the locally excited state of pyrenyl groups and the dissociative state of HABI is small, there are two possible deactivation processes of the excited electronic state, emission from the locally excited state of pyrenyl groups and the thermally excitation to the dissociative state of HABI, resulting in the cleavage of the C–N bond between the two imidazole rings. Altogether a maximum value of about 0.41 for the photochromic reaction yield of pseudogem-FPI-DPI[2.2]PC can be estimated for pure fluorescein excitation at 480 nm, which is identical with the ratio of the missing fluorescence by comparing the fluorescence quantum yield for 1 (0.39) and classical fluorescein (0.8)8. By assuming that the fluorescence quantum yield of classical fluorescein on excitation with 355 nm is almost same as that on excitation with 480 nm, the maximum photochromic reaction yield of pseudogem-FPI-DPI[2.2]PC for the excitation at 355 nm can be similarly estimated to be about 0.54 with the fluorescence quantum yield (0.26) which was determined by 355 nm excitation using sulfate quinine as a standard. However the extra vibrational relaxation was omitted in the above discussion and the new vibrational relaxation pathway should be considered to discuss about the photochromic quantum yield in detail because the relaxed biradical of pseudogem-bisDPI[2.2]PC is formed in a few picoseconds with a quantum yield about 1 whereas fluorescein fluorescence lifetime is about 4 ns.7b The FRET efficiency is even a more complex question as the cross section of the radical is not known and the ultrafast experiment is in this case needed to characterize it. 3.4 Fluorescence imaging microscopy As a preliminary step toward the super-resolution microscopy, the fluorescence imaging switching by using the fast photochromism of 1 was also demonstrated under the fluorescence microscopy. Though the contrast ratio could be enhanced by increasing the laser power, the fluorescence recovery (the halflife is 4.4 ms at 25 °C) is too fast to detect by a common CCD camera. Therefore, the fluorescence switching was investigated at −180 °C to decrease the reaction rate of the dimerization of the radical. ACS Paragon Plus Environment

9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 32

The high-pressure mercury vapor lamp is used as the fluorescence excitation light through a 480 nm bandpass filter and the fluorescence of 1 was monitored at 546 nm. The photochromism of 1 was induced by light stimulus at 365 nm and 405 nm of the high-pressure mercury vapor lamp guided with a Mosaic Digital Diaphragm System (Photonic Instruments, Inc.) even if the maximum absorbance of 1 is about 300 nm. Indeed the transmittance of the objective lens at wavelength below 360 nm is very low. The poly(ethyl acrylate) (PEA) film of 1 was prepared to remove the effect of the diffusion of molecules and convection of solvent. However, the photochromic properties of the film cannot be consistent with those in solutions because the reaction rate of photochromism is generally influenced by the environment around the photochrome.12 The irradiation of the stimulus pulse light (200 ms/pulse) inducing the photochromic reaction under the continuous excitation with 480 nm fluorescence excitation light leads to the efficient quenching of the fluorescence and the intensity of the fluorescence recovers rapidly within 30 seconds at −180 °C (Figure 4). The contrast of the fluorescence intensity is sufficient to detect because the population of the radical at photostationary state is high at −180 °C. 4. Conclusions We demonstrated the fluorescence switching based on the fast photochromism of a newly designed [2.2]PC-bridged imidazole dimer. Indeed, the fluorescence intensity of this photochromic dye can be switched by the photoconversion from the imidazole dimer to the radical. It was demonstrated that the radical moiety quenches the excited electronic state of the fluorescent moiety by FRET and the intensity recovers quickly within the thermal back reaction of the radical. The quantification of switching efficiency and the photochromic reaction yield is not easy and is under progress which involved ultrafast time resolved technique. The quenching rate is not high enough to apply to classical light switching fluorescence imaging microscopy because the population of the radical is low at room temperature due to the fast thermal back reaction. However the back reaction of a few milliseconds combined with the photo-switching achieved even by the excitation of the fluorophore makes these system as a great potential in high resolution fluorescence microscopy such as dSTORM13 or SOFI, 14 only one laser source can be used to probe and switch the fluorophore in millisecond dark state although ACS Paragon Plus Environment

10

Page 11 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


two laser sources are needed in PALM experiments. Hence, the more rational molecular design will be required to increase the photochromic quantum yield on the excitation at around 360 nm of the photochromic dye for classical fluorescence imaging microscopy. Moreover, the introduction of a fluorescence unit which has a high fatigue resistance like perylene will be demanded to achieve high resolution fluorescence imaging. These new systems have a great potential as a fast switchable fluorescent probe for light switching microscopy and high resolution localization microscopy, and will be expected for the future development.

ASSOCIATED CONTENT Supporting Information Available:

1

H NMR spectra, HR−ESI−TOF−MS spectra, HPLC

chromatogram, UV−vis absorption spectra, fluorescence spectra, fluorescence excitation spectra, kinetics for the thermal back reaction in ethanol, decay profiles for the thermal back reaction in phosphate buffer, experimental setup and the movie for the fluorescence microscopy. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. ACKNOWLEDGMENT This work was supported partly by the Core Research for Evolutional Science and Technology (CREST) program of the Japan Science and Technology Agency (JST), a Grant-in-Aid for Scientific Research (A) (22245025) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and NAIST Advanced Research Partnership Project. This work was also supported by ACS Paragon Plus Environment

11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 32

bilateral project, a Sakura Project between the French Ministry of Foreign and European Affairs and the Japan Society for the Promotion of Science (JSPS), a CNRS-JSPS joint research project, and the CNRS International Research Group on PHoto-switchablE orgaNIC molecular systems & deviceS (IRG PHENICS).


12

Page 13 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1) (a) Miyawaki, A. Innovations in the imaging of brain functions using fluorescent proteins. Neuron 2005, 48, 189–199. (b) Helmchen, F.; Denk, W. New Developments in Multiphoton Microscopy. Curr. Opin. Neurobiol. 2002, 12, 593–601. (c) Sakaki, E.; Kojima, H.; Nishimatsu, H.; Urano, Y.; Kikuchi, K.; Hirata, Y.; Nagano, T. Highly Sensitive Near-Infrared Fluorescent Probes for Nitric Oxide and Their Application to Isolated Organs. J. Am. Chem. Soc. 2005, 127, 3684–3685. (2) (a) Hell, S. W.; Wichmann, J. Breaking the Diffraction Resolution Limit by Stimulated Emission: Stimulated-Emission-Depletion Fluorescence Microscopy. Opt. Lett. 1994, 19, 780–782. (b) Klar, T. A.; Jakobs, S.; Dyba, M.; Egner, A.; Hell, S. W. Fluorescence Microscopy with Diffraction Resolution Barrier Broken by Stimulated Emission. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8206–8210. (c) Hess, S. T.; Girirajan, T. P. K.; Mason, M. D. Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy. Biophys. J. 2006, 91, 4258–4272. (d) Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.; Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; Lippincott-Schwartz, J.; Hess, H. F. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 2006, 313, 1642–1645. (e) Rust, M. J.; Bates, M.; Zhuang, X. Sub-Diffraction-Limit Imaging by Stochastic Optical Reconstruction Microscopy (STORM). Nature Meth. 2006, 3, 793–795. (f) Fernández-Suárez, M.; Ting, A. Y. Fluorescent Probes for Super-Resolution Imaging in Living Cells. Nat. Rev. Mol. Cell Biol. 2008, 9, 929–943. (3) (a) Dedecker, P.; Hotta, J.; Flors, C.; Sliwa, M.; Uji-i, H.; Roeffaers, M. B. J.; Ando, R.; Mizuno, H.; Miyawaki, A.; Hofkens, J. Subdiffraction Imaging Through the Selective Donut-Mode Depletion of Thermally Stable Photoswitchable Fluorophores: Numerical Analysis and Application to the Fluorescent Protein Dronpa. J. Am. Chem. Soc. 2007, 129, 16132–16141. (b) Brakemann, T.; Stiel, A. C.; Weber, G.; Andresen, M.; Testa, I.; Grotjohann, T.; Leutenegger, M.; Plessmann, U.; Urlaub, H.; Eggeling, C.; Wahl, M. C.; Hell, S. W.; Jakobs, S. A Reversibly Photoswitchable GFP-like Protein with Fluorescence Excitation Decoupled from Switching. Nature Biotechnol. 2011, 29, 942–947.


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 32

(4) (a) Deniz, E.; Tomasulo, M.; Cusido, J.; Sortino, S.; Raymo, F. M. Fast and Stable Photochromic Oxazines for Fluorescence Switching. Langmuir 2011, 27, 11773–11783. (b) Deniz, E.; Tomasulo, M.; Cusido, J.; Yildiz, I.; Petriella, M.; Bossi, M. L.; Sortino, S.; Raymo, F. M. Photoactivatable Fluorophores for Super-Resolution Imaging Based on Oxazine Auxochromes. J. Phys. Chem. C 2012, 116, 6058–6068. (c) Uno, K.; Niikura, H.; Morimoto, M.; Ishibashi, Y.; Miyasaka, H.; Irie, M. In Situ Preparation of Highly Fluorescent Dyes upon Photoirradiation. J. Am. Chem. Soc. 2011, 133, 13558– 13564. (d) Fukaminato, T.; Tanaka, M.; Doi, T.; Tamaoki, N.; Katayama, T.; Mallick, A.; Ishibashi, Y.; Miyasaka, H.; Irie, M. Fluorescence Photoswitching of a Diarylethene-Perylenebisimide Dyad Based on Intramolecular Electron Transfer. Photochem. Photobiol. Sci. 2010, 9, 181–187. (e) Beharry, A. A.; Wong, L.; Tropepe, V.; Woolley, G. A. Fluorescence Imaging of Azobenzene Photoswitching In Vivo. Angew. Chem. Int. Ed. 2011, 50, 1325–1327. (f) Osakada, Y.; Hanson, L.; Cui, B. Diarylethene Doped Biocompatible Polymer Dots for Fluorescence Switching. Chem. Commun. 2012, 48, 3285–3287. (g) May, F.; Peter, M.; Hütten, A.; Prodi, L.; Mattay, J. Synthesis and Characterization of Photoswitchable Fluorescent SiO2 Nanoparticles. Chem. Eur. J. 2012, 18, 814–821. (5) (a) Kishimoto, Y.; Abe, J. A Fast Photochromic Molecule That Colors Only under UV Light. J. Am. Chem. Soc. 2009, 131, 4227–4229. (b) Harada, Y.; Hatano, S.; Kimoto, A.; Abe, J. Remarkable Acceleration for Back-reaction of a Fast Photochromic Molecule. J. Phys. Chem. Lett. 2010, 1, 1112– 1115. (c) Kimoto, A.; Tokita, A.; Horino, T.; Oshima, T.; Abe, J. Fast Photochromic Polymers Carrying [2.2]Paracyclophane-Bridged Imidazole Dimer. Macromolecules 2010, 43, 3764–3769. (d) Mutoh, K.; Hatano, S.; Abe, J. An Efficient Strategy for Enhancing the Photosensitivity of Photochromic [2.2]Paracyclophane-Bridged Imidazole Dimers. J. Photopolym. Sci. Technol. 2010, 23, 301–306. (e) Takizawa, M.; Kimoto, A.; Abe, J. Photochromic Organogel Based on [2.2]Paracyclophane-Bridged Imidazole Dimer with Tetrapodal Urea Moieties. Dyes Pigm. 2011, 89, 254–259. (f) Mutoh, K.; Abe, J. Comprehensive

Understanding

of

Structure-Photosensitivity

Relationships

of

Photochromic

[2.2]Paracyclophane-Bridged Imidazole Dimers. J. Phys. Chem. A 2011, 115, 4650–4656. (g) Mutoh, K.; Abe, J. Photochromism of a Water-Soluble Vesicular [2.2]Paracyclophane-Bridged Imidazole ACS Paragon Plus Environment

14

Page 15 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Dimer. Chem. Commun. 2011, 47, 8868–8870. (h) Yamashita, H.; Abe, J. Photochromic Properties of [2.2]Paracyclophane-Bridged Imidazole Dimer with Increased Photosensitivity by Introducing Pyrenyl Moiety. J. Phys. Chem. A 2011, 115, 13332–13337. (i) Kawai, S.; Yamaguchi, T.; Kato, T.; Hatano, S.; Abe, J. Entropy-Controlled Thermal Back-Reaction of Photochromic [2.2]Paracyclophane-Bridged Imidazole Dimer. Dyes Pigm. 2012, 92, 872–876. (6) Tierney, H. L.; Murphy, C. J.; Jewell, A. D.; Baber, A. E.; Iski, E. V.; Khodaverdian, H. Y.; McGuire, A. F.; Klebanov, N.; Sykes, E. C. H. Experimental Demonstration of a Single-molecule Electric Motor. Nature Nanotech. 2011, 6, 625–629. (7) (a) Abo, M.; Urano, Y.; Hanaoka, K.; Terai, T.; Komatsu, T.; Nagano, T. Development of a Highly Sensitive Fluorescence Probe for Hydrogen Peroxide. J. Am. Chem. Soc. 2011, 133, 10629– 10637. (b) Alvarez-Pez, J. M.; Ballesteros, L.; Talavera, E.; Yguerabide, J. Fluorescein Excited-State Proton Exchange Reactions: Nanosecond Emission Kinetics and Correlation with Steady-State Fluorescence Intensity. J. Phys. Chem. A 2001, 105, 6320–6332. (c) Togashi, D. M.; Szczupak, B.; Ryder, A. G.; Calvet, A.; O’Loughlin, M. Investigating Tryptophan Quenching of Fluorescein Fluorescence under Protolytic Equilibrium. J. Phys. Chem. A 2009, 113, 2757–2767. (d) Ali, M.; Dutta, P.; Pandey, S. Effect of Ionic Liquid on Prototropic and Solvatochromic Behavior of Fluorescein. J. Phys. Chem B 2010, 114, 15042–15051. (8) Kellogg, R. E.; Bennett, R. G. Radiationless Intermolecular Energy Transfer. III. Determination of Phosphorescence Efficiencies. J. Chem. Phys. 1964. 41, 3042–3045. (9) Monroe, B. M.; Weed, G. C. Photoinitiators for Free-Radical-Initiated Photoimaging Systems. Chem. Rev. 1993, 93, 435-448. (10) (a) Qin, X. Z.; Liu, A.; Trifunac, A. D.; Krongauz, V. V. Photodissociation of Hexaarylbiimidazole. 1. Triplet-State Formation. J. Phys. Chem. 1991, 95, 5822-5826. (b) Liu, A.; Trifunac, A. D.; Krongauz, V. V. Photodissociation of Hexaarylbiimidazole. 2. Direct and Sensitized Dissociation. J. Phys. Chem. 1992, 96, 207-211. (c) Lin, Y.; Liu, A.; Trifunac, A. D.; Krongauz, V. V.


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 32

Investigation of Electron Transfer Between Hexaarylbiimidazole and Visible Sensitizer. Chem. Phys. Lett. 1992, 198, 200-206. (11) Miyasaka, H.; Satoh, Y.; Ishibashi, Y.; Ito, S.; Nagasawa, Y.; Taniguchi, S.; Chosrowjan, H.; Mataga, N.; Kato, D.; Kikuchi, A.; Abe, J. Ultrafast Photodissociation Dynamics of a Hexaarylbiimidazole Derivative with Pyrenyl Groups: Dispersive Reaction from Femtosecond to 10 ns Time Regions. J. Am. Chem. Soc. 2009, 131, 7256–7263. (12) (a) Evans, R. A.; Hanley, T. L.; Skidmore, M. A.; Davis, T. P.; Such, G. K.; Yee, L. H.; Ball, G. E.; Lewis, D. A. The Generic Enhancement of Photochromic Dye Switching Speeds in a Rigid Polymer Matrix. Nat. Mater. 2005, 4, 249–253. (b) Such, G. K.; Evans R. A.; Davis, T. P. Rapid Photochromic Switching in a Rigid Polymer Matrix Using Living Radical Polymerization. Macromolecules 2006, 39, 1391–1396. (c) Sriprom, W.; Néel, M.; Gabbutt, C. D.; Heron, B. M.; Perrier, S. Tuning the Color Switching of Naphthopyrans via the Control of Polymeric Architectures. J. Mater. Chem. 2007, 17, 1885–1893. (d) Ercole, F.; Malic, N.; Davis, T. P.; Evans, R. A. Optimizing the Photochromic Performance of Naphthopyrans in a Rigid Host Matrix Using Poly(dimethylsiloxane) Conjugation. J. Mater. Chem. 2009, 19, 5612–5623. (e) Norikane, Y.; Davis, R.; Tamaoki, N. Drastic Solvent Effect on Thermal Back Reaction of Spiroperimidine Photochromic Compounds. New J. Chem. 2009, 33, 1327– 1331. (f) Ercole, F.; Davis, T. P.; Evans, R. A. Comprehensive Modulation of Naphthopyran Photochromism in a Rigid Host Matrix by Applying Polymer Conjugation. Macromolecules 2009, 42, 1500–1511. (13) Heilemann, M.; van de Linde, S.; Schuttpelz, M.; Kasper, R.; Seefeldt, B.; Mukherjee, A.; Tinnefeld, P.; Sauer, M. Subdiffraction-Resolution Fluorescence Imaging with Conventional Fluorescent Probes. Angew. Chem. Int. Ed. 2008, 47, 6172–6176. (14) Dertinger, T.; Colyer, R.; Iyer, G.; Weiss, S.; Enderlein, J. Fast, Background-Free, 3D SuperResolution Optical Fluctuation Imaging (SOFI). Proc. Natl. Acad. Sci. U.S.A. 2009. 106, 22287–22292.


16

Page 17 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIGURE CAPTIONS. Figure 1. UV-vis absorption spectrum (red) and fluorescence spectrum (green) of 1 in ethanol (2 × 10−5 M), and transient vis−NIR absorption spectra (blue) of 1, recorded just after laser excitation in degassed ethanol (2 × 10−5 M; excitation wavelength, 355 nm; pulse width, 5 ns; power, 5 mJ/pulse). Figure 2. (a) Transient vis−NIR absorption spectra of 1 in degassed ethanol at 25 °C (2 × 10−5 M; 10 mm light path length). Each of the spectra was recorded at 3 ms intervals after excitation with a nanosecond laser pulse (excitation wavelength, 355 nm; pulse width, 5 ns; power, 5 mJ/pulse). (b) Decay profiles of the colored species generated from 1, monitored at 400 nm in degassed ethanol (2 × 10−5 M). Figure 3. (a) Time profile of the variation of absorbance at 400 nm and fluorescence at 525 nm (20 °C in ethanol, 490 nm excitation) after 355 nm excitation wavelength to switch the fluorescence (pulse width, 4 ns; power, 10 mJ/pulse). (b) Fluorescence switching cycle upon 355 nm irradiation. Figure 4. Fluorescence quenching and subsequent recovery images of a PEA film doped with 1 after excitation with 365 nm and 405 nm light (pulse width, 200 ms) at −180 °C.


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 32

SCHEME TITLES. Scheme 1. Photochromism and fluorescence switching behavior of 1. Scheme 2. Synthesis of 1.


18

Page 19 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. UV−vis absorption spectrum (red) and fluorescence spectrum (green) of 1 in ethanol (2 × 10−5 M), and transient vis−NIR absorption spectrum recorded just after laser excitation (blue) of 1 in degassed ethanol (2 × 10−5 M; excitation wavelength, 355 nm; pulse width, 5 ns; power, 5 mJ/pulse).


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 32

Figure 2. (a) Transient vis−NIR absorption spectra of 1 in degassed ethanol at 25 °C (2 × 10−5 M; 10 mm light path length). Each of the spectra was recorded at 3 ms intervals after excitation with a nanosecond laser pulse (excitation wavelength, 355 nm; pulse width, 5 ns; power, 5 mJ/pulse). (b) Decay profiles of the colored species generated from 1, monitored at 400 nm in degassed ethanol (2 × 10−5 M).


20

Page 21 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. (a) Time profile of the variation of absorbance at 400 nm and fluorescence at 525 nm (20 °C in ethanol, 490 nm excitation) after 355 nm excitation wavelength to switch the fluorescence (pulse width, 4 ns; power, 10 mJ/pulse). (b) Fluorescence switching cycle upon 355 nm irradiation.


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 32

Figure 4. Fluorescence quenching and subsequent recovery images of a PEA film doped with 1 after excitation with 365 nm and 405 nm light (pulse width, 200 ms) at −180 °C.


22

Page 23 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. Photochromism and fluorescence switching behavior of 1.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 32

Scheme 2. Synthesis of 1.


24

Page 25 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Content (Word Style “SN_Synopsis_TOC”).


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

UV−vis absorption spectrum (red) and fluorescence spectrum (green) of 1 in ethanol (2 × 10−5 M), and transient vis−NIR absorption spectrum recorded just after laser excitation (blue) of 1 in degassed ethanol (2 × 10−5 M; excitation wavelength, 355 nm; pulse width, 5 ns; power, 5 mJ/pulse). 57x42mm (300 x 300 DPI)


Page 26 of 32

Page 27 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(a) Transient vis−NIR absorption spectra of 1 in degassed ethanol at 25 °C (2 × 10−5 M; 10 mm light path length). Each of the spectra was recorded at 3 ms intervals after excitation with a nanosecond laser pulse (excitation wavelength, 355 nm; pulse width, 5 ns; power, 5 mJ/pulse). (b) Decay profiles of the colored species generated from 1, monitored at 400 nm in degassed ethanol (2 × 10−5 M). 113x179mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(a) Time profile of the variation of absorbance at 400 nm and fluorescence at 525 nm (20 °C in ethanol, 490 nm excitation) after 355 nm excitation wavelength to switch the fluorescence (pulse width, 4 ns; power, 10 mJ/pulse). (b) Fluorescence switching cycle upon 355 nm irradiation. 115x171mm (300 x 300 DPI)


Page 28 of 32

Page 29 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fluorescence quenching and subsequent recovery images of a PEA film doped with 1 after excitation with 365 nm and 405 nm light (pulse width, 200 ms) at −180 °C. 171x27mm (200 x 200 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Photochromism and fluorescence switching behavior of 1. 59x41mm (600 x 600 DPI)


Page 30 of 32

Page 31 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Synthesis of 1. 64x30mm (600 x 600 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

71x31mm (300 x 300 DPI)


Page 32 of 32

Rapid Fluorescence Switching by Using a Fast Photochromic [2.2

Recommend Documents