Super long green persistent luminescence from X-ray excited β-NaYF 4 : Tb 3 +

hwzhang@iue.ac.cn (Hongwu Zhang). Abstract: Here, we have discovered a X-ray excited long afterglow phosphor β-NaYF4: Tb3+. After the irradiation of X-ray, the green emission can persist for more than 240 h. After 36 h, the afterglow intensity arrived at 0.69 mcd•m-2, which can clearly be observed by naked eyes. Even after 84 h, the afterglow emission brightness still reached 0.087 mcd•m-2. Also, combined with the results of thermoluminescence and photoluminescence, the super long afterglow emission of βNaYF4: Tb3+ can be ascribed to the tunneling model associated with F centers. More importantly, the super long green afterglow emission of β-NaYF4: Tb3+ has been successfully used as in vivo light source to activate g-C3N4 for photodynamic therapy(PDT)and bacteria destruction. Furthermore, super long persistent luminescence of β-NaYF4: Tb3+ could be repeatedly charged by X-ray for many circulations, which indicates that the phosphors have high photo stability under repeated cycles of alternating X-ray irradiation.

X-ray activated ZnS: Cu, Co afterglow nanoparticles-TBrRh123 conjugates for cancer cell destruction in vitro 38 . Chen and coworkers exploited LiGa5O8: Cr X-ray excited afterglow nanoparticles for imaging-guided X-ray induced photodynamic therapy of deep-seated tumors 39 . However, The usual afterglow material is charged by mercury lamp, and the excitation light is absorbed by valence electrons.
When X-ray charging is used, the X-ray absorption is from the inner layer electron. Due to the different energy storage mechanism of the two afterglow materials, the better afterglow materials excited by mercury lamps are usually not ideal after X-ray excitation, which results in that the intensity of the developed X-PLNPs decayed quickly and is not strong enough to stimulate the photosensitizer. This meant that these phosphors cannot be regarded as efficient in vivo exposure light source to realize PDT.
Furthermore, until now, as a novel afterglow phenomenon, the mechanism of X-ray excited long persistent luminescence is still unclear, which have limited the further development of novel higher efficiency X-PLNPs. Thus, exploring their luminescent mechanism and then development of novel higher efficiency X-PLNPs are indispensable to the design of PDT antibiotics in deep tissue.
In present work, we have successfully fabricated X-ray excited super long time green phosphors β-NaYF4: Tb 3+ , which not only can be quickly charged by X-ray and then emit green luminescence for several weeks after the removal of X-ray source but also can be excited by 980 nm laser to release the remaining energy efficiently. And, β-NaYF4: Tb 3+ is regarded as excellent model to explore the

Results
The XRD patterns and the electron micrographs of the as-prepared samples were shown in Fig. 1a-b. All the diffraction peaks of the samples were in perfect match with the standard powder peak positions of β-NaYF4: Tb 3+ hexagonal phase (JCPDF No.  and no other phase and apparent shift can be found, indicating the as-prepared samples were pure β-NaYF4. The TEM images showed that the samples consisted of uniform micro-rods with a diameter of ∼1 um. And, the hexagonal prisms morphology was further confirmed that the as-prepare samples were hexagonal β-NaYF4. The X-ray excited emission spectrum (black) showed typical narrow emission peaks of Tb 3+ which all show the Aband emission at 543 nm, as well as additional emission bands at 350-500 nm and 570-630 nm. All these peaks can be ascribed to the multiples transitions from f 8 (Supplementary Fig. 1). The more important was that the sample possessed excellent X-ray induced afterglow properties, which afterglow emissions (red line, Fig. 1c) were similar to the photoluminescence and also can be ascribed to the transitions of Tb 3+ . At the same time, we can observed the strong green afterglow emission from the Hebei University logo covered by the phosphors after irradiating by X-ray irradiation for 10 min in Fig. 1d, indicating the strong afterglow emission of β-NaYF4: Tb 3+ .
In order to further investigate the X-ray induced long persistence luminescent properties of β-NaYF4: Tb 3+ , the super long persistent luminescence decay curve of the ceramic discs monitored at 543 nm after irradiation by an X-ray irradiator for 10 min had been obtained as shown in Fig. 2a. The afterglow intensity decreased quickly in the first several hours and then decays slowly. Even after 36 h, the afterglow intensity still arrived at 0.69 mcd•m -2 , which can be can be clearly observed by naked eyes.
Even after 84 h, the afterglow emission brightness still reached 0.087 mcd•m -2 (Fig. 2c). In addition, the inset of Fig. 2a gave afterglow spectra acquired at different times during the 20 days afterglow process.
The profiles of the afterglow spectra did not change with decay time, indicating that the persistent luminescence originated from the Tb 3+ emitting centres and existed in the whole photoluminescence emission band. Even compared with background, the afterglow spectrum of β-NaYF4: Tb 3+ can be clearly measured after 20 days. The long-lasting afterglow of the phosphors was further confirmed by a digital SLR camera. Fig. 2b showed the change in afterglow brightness over time for four discs after exposure to a 30 mA 50 KV X-ray irradiator for different time. The green afterglow light can be rapidly charged and even 1 min X-ray irradiation can induce more than ten days of persistent visible emission.
At the same time, the enhancement of X-ray irradiation time can obviously prolong the afterglow time and increase the afterglow intensity. Among the 10-15 min irradiation was the most suitable for X-ray induced long persistence luminescence of β-NaYF4: Tb 3+ , which was consistent with the afterglow curves induced different irradiation time (Supplementary Fig. 2), considering the reduction of X-ray dosage makes sense here we chose X-ray irradiation for 10 min. Furthermore, when the afterglow emissions decayed undetectable after 240 h, the 100℃ heat-treatment can induce the recovery of the strong green emission (Fig. 2b-7) which became even brighter than the ones that underwent 5 min of decay ( Fig. 2b-1). This can be ascribed to the rapid and massive release of the residual trapped electrons located in traps by the heat. More important, this intensive release process lasted for more than 10 min until the traps are completely emptied. The heat-assisted intense visible emission indicates that the energy released during the 240 h RT decay process was just part of the energy collected and stored by the materials during excitation and thus that the actual decay time should be much longer than 240 h. All these results have indicated that β-NaYF4: Tb 3+ possessed super-long green afterglow emission under the irradiation of X-ray.
Due to its X-ray-excited super long green persistent luminescence, β-NaYF4: Tb 3+ can be potentially regarded as in vivo light source to activate photosensitizer to conduct PDT in deep tissue.
Owing to strong absorption of blue-green light, g-C3N4 (Supplementary Fig. 3), a famous photo-catalyst, was chosen as PDT agent combined with β-NaYF4: Tb 3+ to treat bacterial infection in deep implant. It is well known that PAO1 is a common gram-negative rod-shaped bacterium that is susceptible to cause diseases in organisms 40 and used here as processing object (Supplementary Fig. 4 and Fig. 5). Experimental data indicated that after irradiated for 2 min by X-ray under 30 mA 50 kVp, treated with the composite of β-NaYF4: Tb 3+ -g-C3N4, the viabilities of PAO1 cells rapidly decreased to 67.14%, while the X-ray irradiation hardly induced the harm to the growth of PAO1 in the control group ( Fig. 5a). More importantly, it could be observed that after remove of X-ray source, during 5 h, the viabilities of PAO1 cells could decrease largely to about 30.92% ( Fig. 5f and Fig. 5g), which indicated that the X-ray excited afterglow emission of β-NaYF4: Tb 3+ could continuously provide light energy to activate g-C3N4, which thus largely prolonged the photodynamic treating time and obviously decrease the X-ray irradiation dose. These results suggest that X-ray excited long afterglow green emission of β-NaYF4: Tb 3+ would develop a novel PDT method with more safety and efficient for future medical therapy.

Discussion
It is well known that thermally stimulated luminescence ( TSL ) can only explore shallow traps due to the high temperature can cause the thermal quenching of the luminescent center and photo stimulated luminescence (PSL) can explore deeper traps [41][42][43][44][45] . Thus, here, the TSL and PSL methods were utilized to explore the mechanism of X-ray induced super long afterglow emission of β-NaYF4: Tb 3+ . No TSL spectrum from the sample was detected after being excited by UV lamp, however, a strong TSL emission at 107℃ occurs from the 4f 8 → 7fJ transition of Tb 3+ after X-ray excitation as shown in Fig. 3a.
Since β-NaYF4: Tb 3+ was a broad bandgap material (7.3 eV), UV light was not enough sufficient to charge defects but the X-ray irradiation can provide high energy to realize the charge of traps. Fig. 6 showed a clear linear relationship between the reciprocal of the square of the persistent luminescence intensity (I -1 ) at 543 nm and time (t) in the latter stage of the decay of the afterglow which confirmed that a tunneling process occurs 46-48 . The persistent luminescence at -196℃ more confirmed the tunneling process (Supplementary Fig. 7). Then the trap depth was estimated to be 0.764 eV via the equation of E=2kTm 2 /δT 49 . In order to explore the nature of the deeper traps, we further stimulated the sample locally by 450 nm, 532 nm, 635 nm, 808 nm and 980 nm fiber lasers respectively after X-ray charging. The afterglow intensity of the laser-irradiated portion was enhanced after a short period of irradiation, and as the irradiation time increased, the afterglow fluorescence rapidly weakened and eventually disappeared completely (Fig. 3b). The part that was blackened by laser irradiation will not show the afterglow emission even if it was heated (Supplementary Video 1). However, if the shallow trap electrons were completely released by high-temperature heating before the laser irradiation, the afterglow illumination can be seen again in the portion excited by the laser after the short time irradiation was stopped (Supplementary Video 2). All of these phenomena lead us to believe that under laser excitation, part of the electrons of the deep traps will be transferred to the shallow traps, and the other part will act to illuminate with the center of the luminescence. In a short time, the former was dominant and the afterglow emission was enhanced; when the excitation time was increased, the latter was dominant and the afterglow emission was weakened.

Supplementary
The color center phenomena of halide alkali metal was studied in depth in the 1920s and 1970s, which provided a theoretical basis for the fluoride afterglow mechanism [50][51][52][53][54] . Hence, we proposed a tunneling model associated with F centers (Fig. 4a). The simple process of storing and releasing energy is: the electrons of VB (electrons in F 2p orbital) are excited to the CB (free state) after the sample is excited via X-ray, they will drop to the bottom of the CB (Y 4d orbital) after loss of kinetic energy ( Fig.   4a① and Fig. 4bⅰ). Meanwhile, portion of the Fions are excited by X-ray to escape, resulting in F vacancies, while the electrons at the bottom of the CB will be captured by F-vacancies to form F-centers (the electron of Y 4d orbital shifts into F vacancies (Fig. 4a② and Fig. 4bⅲ)). Tb 3+ ion is easy to lose an electron to a stable structure with eight electrons in its outermost shell (equivalent to capturing a hole from the VB and the electron will fill the F 2p orbital (Fig. 4a③ and Fig. 4bⅱ)) to form Tb 4+ 55, 56 . At this point, the F center forms tight bound exciton (Frenker exciton) with Tb 4+ ion 57, 58 , the electron in F center and the hole bound by Tb 3+ are directly recombined through tunneling process (Fig. 4c3 and Fig.   4bⅳ), the ground state electron of Tb 3+ absorb energy and are activated to a high energy state and then followed by luminescence of the 4f → 4f transition.
Although the recharge and release process from above can explain the afterglow phenomenon, it cannot explain the extra long afterglow properties of this material. Based on the light excitation and thermal excitation data, we realize that there are deep traps and shallow traps, and electrons can pass between them. In fact, R. W. Pohl has already studied the electron transfer between deep traps and shallow traps in fluoride, and pointed out that the deep trap is F centers , and the shallow trap is from F ' centers 50 . Hence, the simple electron transfer process of F centers and F ' centers with tunneling process was shown in Fig. 4c. The electron in the F center nearest of the luminescent center is easily recombined with the luminescent center through Auger effect to leave the F vacancy (Fig. 4c1). The electron of the F ' center enter the F vacancy through tunneling energy level under thermal excitation to form a new F center (Fig. 4c2) and then recombine with the luminescence center through Auger effect (Fig. 4c3). The energy required for this process is low, which is the initial process of afterglow decay at RT. The electron in F center recombine with the F vacancy adjacent to the luminescent center through tunneling effect to form a new F center to achieve the energy transfer between the F centers (Fig. 4c5), and causing the luminescent center to recombine with its nearest neighbor F center (Fig. 4c6). The energy required for this process is high, which is the slow decay process of the afterglow at RT. Although the above model can explain the long afterglow phenomenon, the actual situation is much more complicated.
The color center includes not only F centers, but also R centers, M centers, etc 52 . How they interact with each other requires more research.
In summary, We have developed β-NaYF4: Tb 3+ persistent phosphors that can be effectively activated by X-ray and exhibit super long lasting visible afterglow. After the irradiation of X-ray, the green emission can persist for more than 240 h. After 36 h, the afterglow intensity arrived at 0.69 mcd•m -2 , which can clearly be observed by naked eyes. Even after 84 h, the afterglow emission brightness still reached 0.087 mcd•m -2 . Furthermore, due to the excellent overlap between the afterglow emissions of β-NaYF4: Tb 3+ and the absorption of g-C3N4, β-NaYF4: Tb 3+ has been explored as irradiation source to activate the g-C3N4 to conduct PDT. The obtained results showed that the afterglow emission of β-NaYF4: Tb 3+ can inhibit the viabilities of PAO1 cells largely to about 30.92% during 5 h after ceasing the X-ray irradiation. Furthermore, super long green light of β-NaYF4: Tb 3+ could be repeatedly charged by a X-ray activation for many circulations, which indicated that the phosphors had high photo stability under repeated cycles of alternating X-ray irradiation. All the results suggested that X-ray excited long afterglow green emission of β-NaYF4: Tb 3+ would develop a novel PDT method with more safety and efficient for future medical therapy. β-NaYF4: 10%Tb 3+ (Supplementary Fig. 8) microparticles (MPs) were produced through a conventional Hydrothermal method, Briefly, according to the designed composition certain amount of NaNO3, Y(NO3)•6H2O and Tb(NO3)3•6H2O were weighed and dissloved in deionized water with a molar ratio of 1.0:0.9:0.1. An aqueous solution containing EDTA-2Na (5 mmol) was then added to the above solution and stirred slowly for 30 min. Also 40 mmol of NH4F were dissolved in the deionized water Using ultrasound and added into the mixture and then adjusted the PH to 5 with dropping dilute HNO3. After vigorous stirring for another 30 min, The final mixture was transferred into a stainless Teflon-lined autoclave, sealed and maintained at 180℃ for 20 h. As the system was spontaneously cooled to room temperature, the reaction products were collected by centrifugation and washed in ethanol for three times, then dried at 80℃ for 24 h, finally ground to obtain fine β-NaYF4: 10%Tb 3+ phosphors.

Synthesis of β-
The obtained powder (3 g) was pressed into discshaped samples respectively with diameters of 10 mm using a uniaxial hydraulic press at a pressure of 15 MPa.

Synthetic of g-C3N4:
The photocatalyst of g-C3N4 was prepared by directly heating melamine in the semiclosed system to prevent sublimation of melamine. 10 g of melamine powder was put into an alumina crucible with a cover, then heated to 500℃ in a muffle furnace for 2 h at a heating rate of 20℃/min; the further deammonation treatment was performed at 520℃ for 2 h.
Bacterial culture: All glass apparatuses used in the experiments were autoclaved at 121℃ for 20 min to ensure sterility. pseudomonas (P.) aeruginosa PAO1 was grown in the beef extract peptone medium incubator at 35℃.Three days later, the sealing film on the culture dish with cultivate good bacteria was stripped, and the appropriate size quartz plate was fixed on the mouth of the Petri dish. The initial PAO1 population in this batch medium was about 106 colony-forming units (cfu)/mL. Characterization. The structural characterization of the synthesized phosphors were supported by X-ray diffraction (XRD) with Bruker D8 advance X-ray diffractometer (Bruker Optics, Ettlingen, Germany)

Bacterial inactivation by X-PDT
using CuKα radiation at a scanning step of 0.02 o . The morphology of the prepared samples was obtained using a Nova Nano SEM200 scanning electron microscope (FEI, Inc.). X-ray excited emission spectra, afterglow emission spectra and afterglow decay curves were recorded using an Andor SR-500i spectrometer (Andor Technology Co. Belfast, UK) equipped with a Hamamatsu R928 photomultiplier.
The thermoluminescence (TSL) spectra of samples were measured using a FJ-427A1TL dosimeter  pattern of β-NaYF4, the position of XRD peaks of the β-NaYF4 crystalline are shown at the below. b, The electron microscopy image of the synthesized phosphors recorded at high magnification corresponding to its phases. c, X-ray excited luminescence spectra (black) and afterglow spectra (red) of the phosphors. d, A paint of the Hebei University logo prepared by mixing raw phosphors with anhydrous ethanol. The logo is photographed after being irradiated by Xray irradiation for 10 min. Strong green luminescence is emitted from the material. The paint is taken by a digital SLR camera. The imaging parameter is: auto/ISO 200/0.5 sec.   Group1 of PAO1 irradiated by X-ray. b, Group2 of PAO1 with β-NaYF4: Tb 3+ irradiated by X-ray. c, Group3 of PAO1 with g-C3N4. d, Group4 of PAO1 with g-C3N4 irradiated by X-ray. e, Group5 of PAO1 with β-NaYF4: Tb 3+ and g-C3N4 irradiated by X-ray but took the β-NaYF4: Tb 3+ away immediately after ceasing the X-ray irradiation. f, Group6 of PAO1 with β-NaYF4: Tb 3+ and g-C3N4 irradiated by X-ray. X-ray Exposures: upon 30 mA, 50 kVp for 2 min. Images of all groups were obtained after being placed in a darkroom for 5 h. g, Comparison of PAO1 cells viability in six groups.