Article pubs.acs.org/JPCA
Emissive Photoconversion Products of an Amino-triangulenium Dye Zhiyu Liao,† Sidsel Ammitzbøll Bogh, Marco Santella, Christian Rein, Thomas Just Sørensen, Bo W. Laursen,* and Tom Vosch* Nano-Science Center and Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark S Supporting Information *
ABSTRACT: Upon prolonged exposure to intense blue light, the tris(diethylamino)-trioxatriangulenium (A3-TOTA+) fluorophore can undergo a photochemical reaction to form either a blue-shifted or a red-shifted fluorescent photoproduct. The formation of the latter depends on the amount of oxygen present during the photoconversion. The A3-TOTA+ fluorophore is structurally similar to rhodamine, with peripheral amino groups on a cationic aromatic system. The photoconversion products were identified by UV−vis absorption and steady-state and timeresolved fluorescence spectroscopy, and further characterized by HPLC, LC-MS, and 1H NMR. Two reaction pathways were identified: a dealkylation reaction and an oxidation leading to formation of one or more amide groups on the peripheral donor groups. The photoconversion is controlled by the experimental conditions, in particular the presence of oxygen and water, and the choice of solvent. The results highlight the need to characterize the formation of fluorescent photoproducts of commonly used fluorescent probes, since these could give rise to false positives in multicolor/multilabel imaging, colocalization studies, and FRET based assays. Finally, an improved understanding of the photochemical reaction leading to bleaching of fluorescent dyes can lead to the creation of specific probes for fluorescence based monitoring of chemical reactions.
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INTRODUCTION As the field of single molecule spectroscopy and fluorescence nanoscopy (e.g., localization microscopy, STED) keeps expanding, it is becoming clear that understanding the photophysical properties and excited state pathways plays an important role in tailoring fluorophores for the abovementioned applications.1−4 In these applications, the fluorophores are usually exposed to far more excitation cycles compared with conventional cuvette based bulk measurements, which can result in photobleaching. Besides photobleaching, photoconversion into other fluorescent species is something that must be taken into consideration.5,6 If these photoproducts are photostable and fluorescent in another spectral region than the original dye, this could give problems in multicolor− multilabel imaging, colocalization studies, and FRET assays. Rhodamine fluorophores are a class of dyes that have been used extensively for single molecule spectroscopy and fluorescence nanoscopy.7−9 The dealkylation of rhodamine B (Figure 1A) and the formation of blue-shifted photoproducts have been studied previously but required the addition of a substrate like CdS or TiO2.10,11 Watanabe et al. noted that rhodamine B without CdS is more photostable and attributed this to the low rate of intersystem crossing in water and ethanol.10 Also for Texas Red (Figure 1C), a blue shift in the emission was observed by Johnson et al. upon photobleaching, but no explanation for it was given.6 For rhodamine 6G (Figure 1B), the formation of a radical has been proposed to play a role © 2016 American Chemical Society
Figure 1. Molecular structures of (A) rhodamine B, (B) rhodamine 6G, (C) Texas red, (D) A3-TOTA+.
in the photobleaching, although here no information was given on the final photoproducts.12−14 Received: March 27, 2016 Revised: May 5, 2016 Published: May 5, 2016 3554
DOI: 10.1021/acs.jpca.6b03134 J. Phys. Chem. A 2016, 120, 3554−3561
The Journal of Physical Chemistry A
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Article
RESULTS AND DISCUSSION Steady-State UV−vis Absorption and Fluorescence. Figure 3A,B (black curves) shows the absorption and emission
The photoinduced formation of radicals in rhodamine derivatives has also been used as a reversible blinking mechanism in subdiffraction limited localization microscopy applications.9 In this paper, we show that tris(diethylamino)trioxatriangulenium (A3-TOTA+, Figure 1D) upon excitation can form fluorescent photoproducts that are spectrally shifted compared with the original fluorophore. We show that the photoconversion of A3-TOTA+ can follow two parallel pathways leading to a dealkylation or oxidation to form amide groups, most likely via the same photogenerated intermediate (Figure 2). The latter amide product has to the
Figure 2. Photoconversion of tris(diethylamino)-trioxatriangulenium A3-TOTA+. The process takes place via a photogenerated species that can undergo dealkylation to form a blue-shifted species upon full dealkylation. Partial dealkylation does not significantly change the spectral characteristics of the dye (minimal blue shift). Oxidation of the photogenerated species leads to formation of an amide donor group and a spectral red-shift.
Figure 3. (A) Evolution of absorption spectra of tris(diethylamino)trioxatriangulenium A3-TOTA+ in MeCN as a function of irradiation time. Spectra from black to red correspond to irradiation times from 0 to 5 h, with steps of 1 h. (B) Normalized emission spectra of A3TOTA+ in MeCN excited at 467 nm (solid line) and 507 nm (dashed line), before (black curve) and after 5 h irradiation (red curve). For the 507 nm excitation data, the black curve is normalized by the same value as the red curve.
best of our knowledge not been reported before for rhodamine type dyes.15 Similar reaction pathways were reported for the oxidation of simpler amine compounds.15−17 The A3-TOTA+ fluorophore18−20 is structurally and electronically a 3-fold symmetric, extended rhodamine,21−25 which makes our findings directly relevant for this important class of fluorophores. The D3h symmetry of the A3-TOTA+ system results in a degeneracy of the first electronic transition. However, any perturbation of the dye that breaks this symmetry will result in a splitting of the first absorption band into two. In this way, the A3-TOTA+ dye becomes a highly sensitive probe for even relative small changes to the chromophore or its surroundings.19,20 While this report will lead to a better understanding of what causes “photobleaching” and how one could try to find schemes to lower the probability of this process occurring, it also demonstrates that spectrally shifted fluorophores can be efficiently formed by irradiation, a result that highlights the possibility of interference from photoproducts in multicolor fluorescence experiments.6 Additionally, our results could also open up possibilities to use this knowledge to design fluorophores for the detection of specific chemicals (e.g., oxygen or water) or to follow chemical reactions at the single molecule level.5,26−28
spectra of A3-TOTA+ in MeCN. A3-TOTA+ has an absorption maximum at 472 nm in acetonitrile (MeCN) and a fluorescence maximum at 507 nm.19,29 Under illumination using a 488 nm laser beam, the absorption of the sample at 472 nm decreased as a function of irradiation time (black to red curve, see Figure 3A) and a red-shifted absorption peak around 520 nm appeared, together with new blue-shifted peaks (around 400 and 425 nm). Cursory inspection of the data suggests isosbestic points at 260, 288, 318, 427, and 496 nm, indicating the presence of two spectrally different species. Figure 3B shows the emission spectrum broadening as a function of irradiation time in MeCN, when excited at 467 nm (black curve before and red curve after 5 h laser irradiation). When exciting at 507 nm, before laser irradiation (black dashed curve) we only see the tail of the original A3-TOTA+ emission, while after 5 h of laser irradiation, a clear red-shifted emission spectrum with a maximum at 545 nm is observed (red dashed curve). Figure 4 shows excitation spectra of A3-TOTA+ in MeCN after 5 h laser irradiation. The excitation spectrum, monitoring the emission at 575 nm, clearly shows the new spectral features that can also be seen in the absorption spectrum at 400, 425, and 520 nm. The excitation spectrum is a clear indication of the absorption spectrum of the red-shifted photoproduct. The excitation spectrum monitored at an emission wavelength of 500 nm is a bit broader on the blue side of the main absorption band after bleaching, which could 3555
DOI: 10.1021/acs.jpca.6b03134 J. Phys. Chem. A 2016, 120, 3554−3561
Article
The Journal of Physical Chemistry A
Figure 4. Normalized excitation spectra of A3-TOTA+, before (black curve) and after (red curves) bleaching. Solvent, bleaching time, and probed emission wavelength are indicated on the figure.
hint to the presence of partially dealkylated photoproducts, which we expect to show only a minimal blue shift.10 Optical Characterization of the Red Photoproduct. The fluorescence excitation spectrum of the newly formed red species clearly indicates that the symmetry of the A3-TOTA+ has been broken, giving rise to two distinct bands corresponding to absorption into S1 and S2 on each side of the original A3-TOTA+ absorption (See Figure 4).19,20,29−31 Time-correlated single photon counting experiments were performed to investigate the red-shifted emissive species further. The decay curves are shown in Figure 5. For the sample before irradiation, the decay curve can be fitted with a monoexponential decay function with a decay time of 2.14 ns (see Figure 5A). After 5 h of irradiation, the decay curves at the same excitation and detection wavelength could be fitted with a monoexponential decay function with a similar value of 2.16 ns, indicating the partly dealkylated components must have a similar decay time (see Figure S1). To more efficiently excite the newly formed red-shifted product, we used a red-shifted excitation laser and detection wavelength (see Figure 5B). This decay curve can be fitted with a biexponential decay with values of 2.16 ns (15% amplitude) and 1.22 ns (85% amplitude). The dominant component of 1.22 ns can be attributed to the redshifted product. The Effect of Solvent, Concentration, Water, and Oxygen on the Photoconversion Rate. We repeated the A3-TOTA+ bleaching experiments in MeCN several times as a function of A3-TOTA+ concentration. Figure 6A shows the effect of the A3-TOTA+ starting concentration on the bleaching rate. The drop in the absorbance is 0.19, 0.26, and 0.31 starting from 1.15, 0.88, and 0.55, respectively. The relative drop after 5 h is at 16%, 30%, and 56% much larger for the low starting concentration compared with the higher ones. This could be due to experimental conditions (no stirring and increased light intensity gradient in the sample at higher concentrations) or a self-quenching mechanism for the reactive intermediate with another A3-TOTA+ molecule. Further experiments are needed to elucidate this. Next, we performed the bleaching experiments in a mix with another solvent (at a 80:20 ratio volume percent solvent/ MeCN). We noticed a dramatically increased photostability when we added methanol (See Figure 6C). In contrast to this observation, photoconversion was more significant when CCl4 was added (See Figure 6D). This indicates that hydrogen bonding stabilizes the A3-TOTA+ fluorophore. Even anhydrous
Figure 5. Time-resolved single photon counting measurements. (A) Fluorescence decay recorded from tris(diethylamino)-trioxatriangulenium A3-TOTA+ in MeCN before bleaching, 467 nm excitation and detection at 510 nm. (B) Fluorescence decay recorded from A3TOTA+ in MeCN after 5 h irradiation, 507 nm excitation and detection at 535 nm. In the fitting of curve B, one decay component was fixed to 2.16 ns. The χ2 values for the fits in panels A and B were 0.987 and 1.076, respectively.
MeCN still contains a low amount of water (
