S. K. Singh
Department of Physics, Indian Institute of Technology (Banaras Hindu University), Varansi-221005, India. E-mail: sunilcfsl@gmail.com; sunilks.app@itbhu.ac.in; Fax: +91 542 2369889; Tel: +91-8574027822
First published on 15th October 2014
The introductory aspect of this review starts with a prologue on bioimaging in general and optical imaging in particular, and finally focuses on the most recently explored red and near infrared (NIR) emitting persistent luminescence nanoparticles (PLNPs) for bioimaging applications. Accordingly, a pre-requisite towards a better understanding of the subject makes it vital to talk about persistent luminescence, and the developments in red and NIR emitting persistent phosphors. In this context, different synthesis techniques to design nanoparticles and chemically modified (surface modification) nanostructures have also been summarized. Finally, the use of these nanostructures as bioimaging and targeting probes, both for in vitro and in vivo studies, in diverse frameworks, has been reviewed in detail. The significant findings suggest that, Mn2+ and/or Cr3+ doped nanostructures, particularly gallogermanates, are able to give an intense red-near infrared persistent emission with a longer afterglow time for more than 2 weeks and are suitable for bio-imaging applications. The review also talks about the remaining challenges, new dimensions and future course of research in this field.
Optical imaging, also commonly referred as to fluorescence imaging, is one of the fastest growing fields and is expected to be an alternative for other well established imaging techniques.3–21 The technique basically requires luminescent bio-markers/tracers and advanced detection techniques, such as microscopic instruments, for most applications. Thus, the current research interest in this field is focused on the development of efficient optical detectors/sensors as well as to discover exotic luminescent materials as fluorescence probes. As typical bioimaging (or biological labelling) materials, fluorescent markers, such as organic dyes,3–5 quantum dots (QDs),6–11 plosmonic nanomaterials,12–14 fluorescent proteins,15,16 near infrared (NIR) fluorescent molecules,17–19 have been used successfully; however, most of them usually suffer from a high photo-bleaching rate (photochemical destruction of a fluorophore under light exposure), a high background light (due to intrinsic fluorescence by bio-entities itself, e.g. body fluids and proteins are well known to give emission under UV light exposure, which results in a poor signal-to-noise ratio), broad emission features (due to molecular/band to band transitions), short luminescence lifetimes (typically of the order of nano/sub-micron seconds), and poor biocompatibility (organic dyes and many of the QDs are readily toxic), which have made their use challenging until recently.20,21 In principle, these bio-markers give emission by the usual fluorescence process, in which a high energy excitation source (e.g. UV radiation) is used to excite a fluorophore, and the emission thereby is observed in the visible region, which is used for the visualization of different bio-entities.
Recently, a colloidal solution of lanthanide doped nano-phosphors has been identified as a potential luminescent entity for imaging applications because of the unique excitation and emission features associated with it.22–27 The doped lanthanide ions bear the property of frequency upconversion (UC), which allows the use of NIR (usually 980 nm) light as an excitation source. Herein, UC describes a nonlinear optical process, in which low energy photons are used to generate high-energy photons, usually IR radiation is used to generate visible and UV light.28–35 The IR to visible UC method has been extensively applied for bioimaging applications in recent decades due to its inherent merits. Remarkably, NIR light is not absorbed by biological samples, increases penetration depth in tissues, and avoids the problem of auto-fluorescence and a light scattering background.36–38 The complete NIR region is considered in two main parts as far as the imaging application is concerned, i.e. NIR I (650–900 nm) and NIR II (1000–1450 nm). The choice between these two regions for imaging application is case dependent as both of them have their own features of merit. This could be understood in terms of the variation in absorption and scattering by bio-entities/body fluids in these two regions. The absorption coefficient of water, haemoglobin and scattering coefficient for skin, adipose tissue and mucous tissue in the NIR region have been studied well. Fig. 1 shows that the absorption coefficient for both haemoglobin and water is reasonably good for imaging applications. The absorption coefficient of water increases for the higher wavelength side (NIR II region), but at the same time the scattering coefficient, for different types of tissues, is reduced for the NIR region II.37,38 Furthermore, looking into more detail, lanthanide ion doped inorganic nanoparticles (NPs) could maintain a photoluminescence characterized by a high photochemical stability, sharp emission bands (∼10 nm, f–f transitions in lanthanide ions which remain shielded by the outer s and p orbitals), long luminescence lifetimes (usually of the order of μs or ms), lower photo-bleaching potentials, and low toxicity (LD50 value for lanthanide doped oxides is ∼1000 times higher compared to QDs) as well.39 However, despite their apparent advantages, such anti-Stokes probes rely strictly on higher-order coherent excitation, e.g. laser sources, which is a fundamental limitation with such materials. Lasers are costly and can excite in a limited area (due to a small beam diameter, typically of the order of only a few mm), and, at the same time, the NIR laser may also create a heating effect (significantly the temperature may rise up to the physiological order) at high input laser power.
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Fig. 1 Absorption coefficient of haemoglobin and water in the 400–1000 nm range. Reprinted by permission from Macmillan Publishers Ltd: [Nature Biotechnology] (ref. 36), copyright (2001). |
Persistent luminescent materials, which particularly emit in the red and NIR region, also known as long-lasting/afterglow materials (both lanthanide and transition metal ions are known for persistent emission), could be utilized as an option to stay away from these issues with extra preferences such as nano-scale thermometry and delayed detection.40 In persistent phosphors, two kinds of active centres remain involved: traps and emitters. Trap levels originate due to lattice defects, impurities, or codopants in the material just below (sub eV to few eV) the conduction bands. On excitation with suitable energy (such as by electron beam, X rays, and UV light), electron–hole pairs are generated and the excited electrons from the conduction band are captured in the trap levels. Traps usually do not emit radiation, but they store the excitation energy for a longer time (thus primarily act as reservoir). The process is called charging of the material. This energy can then be liberated by thermal, optical or other physical stimulations, resulting in stimulated emissions from the active centers (process is called discharge). Thus, emitters are the centers that emit in the region of interest after getting suitable excitation energy via stimulation of the free charge from the trap level (sometimes also called persistent energy transfer process). The key feature of such persistent phosphor is the possibility of tuning the location of the trap band (by controlling the lattice defects, doping ions, and impurities in the host), thus obtaining flexibility for selecting the stimulation energy source. A representative scheme to understand the general idea about the different processes involved in persistent luminescence is sketched in Fig. 2.41
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Fig. 2 Energy band model depicting anti-Stokes emission under incoherent excitation. During an optical charging process, the gap between valence and conduction band is decreased by generating an electron–hole pair. The trapped electron mitigates into a satellite trapping band, from where it can be extracted by optical pumping. Emission of a secondary photon with a lower wavelength compared to that occurring through the recombination of the electron–hole pair. Reproduced from ref. 41. |
The supremacy of the imaging process with such persistent luminescence materials could be acknowledged with three distinctive features.41 The first and the most paramount one is the persistent luminescence itself: intense, long-lasting persistent light (up to several hundred hours) emanating by the material extensively, much after the removal of the excitation (usually UV, X-rays, electron beam) source. This allows imaging without external excitation, which completely removes the likelihood of auto-fluorescence/background noise, and thus a significant improvement in the signal-to-noise ratio and sensitivity is attained. Secondly, the emission in red-NIR region (particularly in 650–1000 nm range) lies in the tissue transparency window,42,43 in which light attenuation is largely due to scattering rather than absorption, which further increases the detection depth. The third one is the possibility of photo-stimulation, to rejuvenate the persistent luminescence after a long period, through an incoherent light source, e.g. by light emitting diodes (LEDs)/flash lamps, and even by sunlight, which makes imaging for longer times effective, easier, cost effective and safe. Recently, in last few years, this imaging technique has been quite popular and significant progress has been achieved in such a short span of time, and still great efforts are in progress. Nevertheless, a review on this subject is still not available. A brief review on NIR persistent luminescence based nanostructures with a special reference to bioimaging thereof may be quite useful not only to follow the new progress in this field, but also to construct and design new probes for imaging applications.
This review is an attempt to present an overview of persistent luminescence, particularly covering red and NIR persistent materials, and their applications for bioimaging. The main focus is on NIR persistent luminescence in transition metal (Mn and Cr) doped nano-structures. Different synthesis techniques to design nanocrystalline particles and chemically modified (surface modification) nanostructures of such materials for imaging purposes are summarized. A detail of the structure, effect of doping of different ions on the structure, origin of defect levels (trap levels) and different mechanisms to tune the depth of the trap levels in such materials has also been included in the study so as to build up an understanding of persistent luminescence. Furthermore, different optical parameters, e.g. thermal stimulation, photo-stimulation by coherent/incoherent/sunlight sources, persistent energy transfer from the trap level to the active ion, persistent luminescence stability and reproducibility, effect of power/temperature/charging time, on persistent luminescence have also been considered. Finally, the use of these materials as bioimaging probes, both for in vitro and in vivo studies in different system, along with acute and chronic toxicity studies have been reviewed in detail. In the final remarks, the review talks about the remaining challenges, and future direction of the research in this field.
Persistent luminescence was at first not all that appealing for researchers working in the area of luminescent materials. Infact, it was viewed as an undesired property for luminescent materials, as the presence of lattice defects usually quenches the luminescence property considerably, and its utilization was likewise exceptionally constrained. Some typical sulfide based materials with a persistent luminescence time on the order of few hours with a weak intensity were in use, but at the cost of the potential risk of being environmental hazards.46,47 The application of lanthanide ions for persistent luminescence revolutionized this field. In last few years, up to this point, more than 200 combinations of host materials and activator ions have been depicted, of which around 20% or thereabouts are singularly focused around divalent europium (Eu2+) and co-dopant ions.44 Generally, most of the lanthanide ions have been used to get persistent luminescence, essentially in different colours. Recently, Tadashi et al. explored the persistent luminescence of most of the lanthanide ions in a Ca2SnO4 host.45 Fig. 3 shows a beautiful digital image of both the emission and afterglow by different lanthanide ions. A large number of potential applications of persistent luminescence, e.g. traffic signs, emergency signage, watches, clocks, textile printing, forensic applications such as security ink, latent fingerprint detection, and photovoltaic applications, to increase the conversion efficiency of silicon solar cells, and certainly for bioimaging/tagging, have been successfully demonstrated and few of them have been also commercialized to date.48–53 The use of lanthanide ions for persistent luminescence is considered among one of the latest applications of lanthanide ions following its application for lasers, fibres, IR to visible frequency UC, advance lighting and display devices, and imaging.54,55
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Fig. 3 Phosphorescence observation for La, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu doped samples, (a) excited at 254 nm (photoluminescence), (b) after 5 seconds afterglow (emission). Reproduced from ref. 45 with permission from The Royal Society of Chemistry. |
The original concept for the development of lanthanide doped persistent luminescent materials was centred on Eu2+ ions in different host matrices, including glass (silicate and borate) and ceramics (aluminates, aluminosilicates). The Eu2+ doped alkaline earth (Ca, Sr) aluminates remain, however, the most important persistent phosphors, especially because the afterglow is greatly enhanced by co-doping with some trivalent lanthanide ions, particularly Dy3+ and Nd3+ ions.56 It was proposed that in such phosphors, the reduction of Eu2+ to Eu3+ and oxidation of Nd3+/Dy3+ to Nd4+/Dy4+ occurs in the persistent luminescence processes, which was not completely acceptable due to many reasons.57–59 However, among them, CaAl2O4:Eu2+, Nd3+ and SrAl2O4:Eu2+, Dy3+ have already been in use commercially for many of the applications listed above. Smet's group summarizes the origin, developments and advancements in the field of lanthanide doped persistent luminescence phosphors for the blue, red, green and white light colours in two review articles.40,60
In addition to this, a recent review by Tanabe's group gives a short description about red and NIR emitting phosphors, which was planned to basically emphasize the state-of-the-art development of such persistent phosphors only.61 The review comments on the evaluation method of the red to NIR persistent luminescence intensity. They summarize that the generally adopted evaluation method for persistent phosphors is luminance (in units of cd m−2), which is not suitable for red to NIR persistent phosphors particularly aimed at the application of in vivo imaging. Instead, the use of radiance (in units of W sr−1 m−2), which is not related to the human eye sensitivity, is more suitable and can be adopted. Furthermore, the review lists a brief detail of the host and activator ions for red-NIR emission and the mechanism involved in persistent luminescence; however, it completely overlooks the bioimaging part, which is the major attraction of the research and development for NIR persistent phosphors nowadays.
CaS and SrS are phosphors that have a great variety of emission centres and perform at a wide range of colours. However, such phosphors are extremely sensitive to moisture and thus chemically unstable, and to make them stable, surface modification becomes essential. To remove this lacuna, oxysulfide phosphors were tried and developed at the next step.75–80 In this line, Y2O2S:Eu3+ is very well known as an afterglow phosphor, which emits in the orange-red region, close to 621 nm. Other co-dopant such as Ti and Mg are also used to basically control the defect/trapping centres. Sm3+ has also been used as an activator to obtain orange-red emission in oxy-sulphide hosts. Several other phosphors in different hosts, e.g. silicates,81–90 germanates,91,92 nitrides,93 stannates,94 phosphates,95–102 aluminates,103 titannates,104,105 metal oxides,106,107 fluorides,108 and others,109 have also been developed for getting long afterglow emission in the red region. Table 1 gives a comprehensive detail of the host, dopant ions, colour/wavelength of the afterglow emission, persistent time, and remarks, if any of red emitting persistent phosphors.64–109
Host (References) | Dopant ion(s) | λemission (nm) | Persistent time | Remarks |
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CaS (ref. 64) | Eu2+, Tm3+/Bi3+ | 650 | 1 h | Effect of Na+ co-doping |
CaS (ref. 65) | Eu2+ | 655/670 | — | Effect of Cl− co-doping |
CaS (ref. 66) | Bi3+ | 650 | 1 h | Effect of Cl− co-doping |
CaS (ref. 67) | Ce3+, Pr3+ | Green/red | 1 h | Effect of Li+ co-doping |
Ca1−xSrxS (ref. 68) | Eu2+ | 650 | 1 h | White light emission |
SrS (ref. 69) | Eu2+, Dy3+ | 616 | — | Nano-size particle |
CaS (ref. 70) | Eu2+, Pr3+ | — | — | Effect of Li+ co-doping |
Ca1−xSrxS (ref. 71) | Eu2+, Pr3+ | 650 | — | Effect of Li+ co-doping |
SrS (ref. 72) | Pr3+ | Blue/green/red | — | — |
SrS (ref. 73) | Eu2+ | 615 | — | Lanthanide (3+) co-doping |
CaS (ref. 74) | Eu2+, Dy3+ | 650 | — | Nano, photostimulated |
Y2O2S (ref. 75) | Eu3+, Mg2+, Ti4+ | 611 | 1 h | Nanorods |
Y2O2S (ref. 76) | Eu3+, Mg2+, Ti4+ | 611 | 1 h | Solid state reaction |
Y2O2S (ref. 77) | Ti4+/Ti2+ | 565 | 5 h | High temp sintering |
Y2O2S (ref. 78) | Sm3+ | 610 | 1 h | — |
Y2O2S (ref. 79) | Mg2+, Ti4+ | 594, broad | — | High temperature |
Gd2O2S (ref. 80) | Er3+/Ti4+ | 555, 675 | 1.2 h | — |
MgSiO3 (ref. 81 and 82) | Mn2+, Eu2+, Dy3+ | 660 | 4 h | High temp sintering |
CdSiO3 (ref. 83) | Mn, Ni, Cr | 550–720 | <1 h | Solid state reaction |
CaMgSi2O6 (ref. 84 and 85) | Mn | 550–800 | <1 h | Sol–gel route |
CaMgSi2O6 (ref. 86) | Mn2+, Dy3+ | 550–800 | <1 h | Nanoparticle synthesis |
SrMg(SiO3)2 (ref. 87) | Mn2+, Dy3+ | 500–800 | <1 h | Solid state reaction |
Sr3MgSi2O8 (ref. 88) | Eu2+, Mn2+, Dy3+ | 670 | 2 h | Solid state reaction |
BaMg2Si2O7 (ref. 89) | Mn2+, Ce3+ | 620/670 | 2 h | Solid phase reaction |
Ca3MgSi2O8 (ref. 90) | Eu2+, Dy3+ | Green/red | 5 h | — |
MgGeO3 (ref. 91 and 92) | Yb3+/Bi2O3 | 600–720 | — | — |
M2Si5N8 (ref. 93) | Eu (M = Ca, Sr, Ba) | 580–620 | — | — |
Ca2SnO4 (ref. 94) | Sm3+ | Orange-red | — | — |
β-Zn3(PO4)2 (ref. 95) | Mn2+ | 616 | 2 h | Role of excess Zn2+ |
β-Zn3(PO4)2 (ref. 96 and 97) | Mn2+, Al3+, Ga3+ | 570–700 | — | Solid state reaction |
β-Zn3(PO4)2 (ref. 98) | Sm3+, Mn2+ | 616 | 2 h | Solid state reaction |
YPO4 (ref. 99) | Pr3+, Ln3+ | Red | — | High temperature |
Ca3(PO4)2 (ref. 100 and 101) | Mn2+/Tb/Dy | 660 | — | Biocompatible host |
Ca9Ln(PO4)7 (ref. 102) | Lanthanides, Mn | Red | — | — |
CaO–Al2O3 (ref. 103) | Tb3+ | Green/red | — | Glass |
CaTiO3 (ref. 104) | Pr3+ | 612 | 0.1 h | — |
CaTiO3 (ref. 104) | Al3+ | 612 | 0.2 h | — |
Ca2Zn4Ti16O38 (ref. 105) | Pr3+ | 614, 644 | — | Dual phosphorescence |
MO (ref. 106) | Eu (M = Ca, Sr, Ba) | Orange-red | 2 h | Solid state reaction |
Lu2O3 (ref. 107) | Pr, Hf | 610, 632 | — | Thermal/photostimulation |
CaF2 (ref. 108) | Tm/Dy/Mn | Miscellaneous | — | — |
Ca2Si5N8 (ref. 109) | Eu2+, Tm3+ | 610 | — | Bioimaging application |
The work by Lecointre et al. presents a new concept to design red persistent phosphors in yttrium phosphates.99 They codoped the material with Ce3+, Ln3+ and Pr3+, and Ln3+ (Ln = Nd, Er, Ho and Dy) to realize the red persistent luminescence. The study was explored on the basis of TSL measurements and it was found that similar to Ce3+, Pr3+ was also able to trap holes at high temperatures while being the radiative recombination center, whereas the Ln3+ codopants played the role of the electron trap. In a similar work, Bessiere et al. proposed Ca3(PO4)2, a highly biocompatible compound, for colour tunable red long lasting luminescence.101 Mn2+ was selected as the dopant to serve as the active luminescent centre, displaying its 4T1 (4G)/6A1 (6S) broad emission at 660 nm and substituting the small octahedral Ca site of the host. Furthermore, codoping with trivalent lanthanide ions (Dy3+, Tb3+) enhances the Mn2+ optical luminescence (X-ray excited), while delayed luminescence originating from carrier trapping is suppressed. Ca3(PO4)2:Mn2+, Dy3+ showed the most intense long lasting luminescence. Lian et al. developed a Ca and Zn based titanate phosphor and realized dual persistence luminescence originating at 614 and 644 nm, induced by two separate kinds of doping defects. Furthermore, they suggest the application of this material in energy conversion.105
All of the listed compositions of red phosphors in Table 1 were prepared basically to realize red-persistent luminescence; however, there were very few specific applications for persistent luminescence based bioimaging. In this context, Maldiney et al. synthesized Ca2Si5N8(CSN) simply by mixing stoichiometric amounts of the required compounds, and then sintered the material at 1300 °C under a reducing atmosphere (90% N2, 10% H2).109 Furthermore, the powder material (pressed to form a small target with a diameter of 8 mm) was subjected to a pulsed laser ablation technique to extract the NPs in solution. Significantly, small NPs with diameters in the range of 3-–5 nm were obtained, but the low quantity in solution prevents further use of the material for bioimaging. However, they further used a wet grinding technique to extract the NPs, which gave enough NPs to realise the use of this material as an optical probe for small animal in vivo imaging. This technique successfully translates the present bulk nitridosilicate powder to obtain a narrow size distribution of negatively charged hydroxyl-terminated CSN NPs with a mean diameter close to 200 nm; however, the yield is only 10%. Surface functionalization of the NPs was performed by using poly-ethylene glycol (PEG) grafting so as to obtain a better distribution of NPs after the systemic injection, thus delaying their uptake by the reticulo-endothelial system (RES).
Except such few rare examples, it was hard to find bioimaging applications by red emitting persistent phosphors. Indeed, few of them, particularly those emitting in the red region (≥650 nm) could be used for imaging applications in principle. Nevertheless, most of the developed phosphors have been synthesized by high temperature synthesis techniques, e.g. solid state reaction methods or by high temperature solution combustion synthesis, followed by high temperature post-annealing, which ultimately results in the development of particle sizes in the sub-micron to micron range and are not usable for bioimaging applications. Thus, new synthesis strategies for the evolution of ultrafine particles, effective surface functionalization strategies along with the bio-compatibility issues need more attention to make them useful for bioimaging applications.
A wide variety of luminescence centres have been developed and used as NIR luminescent probes for imaging applications. Recently, a review by Wang et al. gives a comprehensive detail of different types of nanomaterials that can be excited/can emit in the NIR I (650-–900 nm) and NIR II (1000-–1450 nm) regions.112 A wide range of materials was reviewed, e.g. lanthanide based NPs (both UC and downconversion based), carbon based nanostructures (e.g. carbon dots, carbon nanotubes, and graphene), quantum dots (e.g. CdS, CdTe, CdSe, ZnSe, InAs, PbS, and Ag2S) and noble metal nanomaterials (silver and gold metal nanoclusters), for fabrication and applications in bioimaging. The review, of course, also puts forward the shortcomings, challenges and opportunities associated with them. However, when it comes to NIR persistent luminescence based materials such comprehensive article is very rare or completely missing. This is due to the fact that, the number of known activators for NIR persistent luminescence is relatively low and less explored. Contrary to the wide availability of research articles related to IR emission in lanthanide doped materials, only few works related to lanthanide doped (only) materials possessing NIR persistent luminescence is reported to date. The majority of research for NIR persistent luminescence is based on transition metals, mainly either based on Mn2+ and/or on Cr3+ as one of the dopant ions. In addition to these two main classes of activators, scarcely any other ions have been found to exhibit persistent luminescence in the NIR region, particularly in the interest of bioimaging applications. Therefore, the available articles/materials based on these activators are further reviewed in three different subsections as follows.
Recently, Jumpei et al. reported NIR persistent luminescence due to the Nd3+(4F3/2 → 4IJ/2) ion in a CaAl2O4 host co-doped with Eu2+ ion.115 They prepared samples with compositions Ca0.995Eu0.005Al2O4, Ca0.99Nd0.01Al2O4 and Ca0.985Eu0.005Nd0.01Al2O4 by a conventional solid state reaction method and further studied optical properties such as reflectance, photoluminescence, persistent luminescence and afterglow decay curves. The decay curves for the persistent luminescence of Eu2+ and Nd3+, after 10 min of excitation by UV (330 nm) light, is shown in Fig. 4(a). The decay profiles of Eu2+ and Nd3+ are quite similar. Authors explain the persistent luminescence in terms of the energy transfer from the Eu2+ to Nd3+ ions. The Eu2+ ion is photo-oxidized into Eu3+ or (Eu2+ + h+) and the electron can be trapped by some defects derived from Nd3+ codoping. The process of de-trapping takes place via heat and recombination with the photo-oxidized Eu3+ ions. The persistent luminescence intensity ratio for Nd3+ to Eu2+ remains almost constant with a variation in time. This concludes that, persistent luminescence from both the ions originates from a common electron trap and electron transfer process. Furthermore, this also supports the energy transfer from Eu3+ to Nd3+. Teng et al. have also reported the NIR persistent emission of the Nd3+ ion in a strontium aluminate host.116
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Fig. 4 (a) Afterglow curves for the persistent luminescence of Eu2+ and Nd3+ in CaAl2O4:Eu2+–Nd3+ after 10 min of excitation by 330 nm light. (b) Phosphorescence decay curves of SrAl2O4:1.0%Eu2+, 1.5%Dy3+, and 2%Er3+ phosphor for 525 and 1530 nm emissions. The upper right inset is the proposed charge trapping and energy transfer processes among Eu, Dy, and Er ions. The bottom left inset is the phosphorescence decay curves of SrAl2O4:1.0%Eu2+, 2%Er3+ phosphor for 525 and 1530 nm emissions. (a) Reprinted with permission from [ref. 115]. Copyright [2013], Optical Society of America. (b) Reprinted with permission from [ref. 117]. Copyright [2009], AIP Publishing LLC. |
Another example of NIR persistent luminescence in lanthanide, Er3+, has been reported by Yu et al. in a SrAl2O4 host codoped with Eu2+ and Dy3+ ions.117 Detailed structural, excitation, emission and decay time characteristics have been explored. Herein, again an energy transfer mechanism has been identified behind the persistent NIR emission. The phosphors were synthesized through the combustion method with the composition SrAl2O4:1%Eu2+, 1.5%Dy3+, x%Er3+, where x = 0.5-–2.5, and particle size of the phosphor lies in the range of 17-–27 nm. The excitation spectrum depicts that, the peak at 518 nm arises due to the 4I15/2 → 2H11/2 transitions of the Er3+ ions totally overlapping with the 525 nm green emission bands of the Eu2+ ions, which suggests a fair possibility of an efficient energy transfer from the Eu2+ ions to the Er3+ ions. Fig. 4(b) shows the afterglow decay curves of the SrAl2O4:1.0%Eu2+, 1.5%Dy3+, and 2%Er3+ phosphor for 525 nm green emission and 1530 nm NIR emission. The charge trapping and ET process is shown in the inset to Fig. 4(b). In comparison to the green afterglow (more than 10 h), the NIR decay time is much shorter (only about 10 minutes). This big difference in afterglow time is because of the fact that, the Er3+ ions not only act as efficient NIR emitters but also function as effective charge trap centres. The authors discussed in detail the different processes involved in trapping and de-trapping. Except for these few specific examples, it is hard to find lanthanide based NIR persistent phosphors. Thus, it is summarized that, although lanthanide doped NIR persistent luminescent materials are available, they are very small in number and at the same time the persistent time is quite low (only upto few minutes), which limits their applications in bioimaging.
Tanabe and Sugano estimated the energy levels of Mn2+ by considering the interaction between the d electrons and different types of the point charge/crystal field environment, e.g. linear, square planar, tetrahedral and octahedral.118–120 The energy levels of a free Mn ion includes terms such as 6S, 4G, 4D, 4P, and 4F. The levels of the free ions are demonstrated as 2S+1L, where S presents the total spin quantum number and L is the total orbital angular momentum. Many of these levels split into two or more levels and the degeneracy of these levels is given by 2L + 1. These crystal field modified degenerate energy levels are represented by 2S+1X, where X usually corresponds to A for no degeneracy, E for a two-fold degeneracy and T for a three-fold degeneracy. The red/NIR emission in Mn2+ is observed due to the lowest energy multiplet of the first excited state 4T1 (G) to the ground state 6A1 (S). Usually, red persistent emission is observed in most of the hosts by Mn2+ ions (refer to Table 1), while NIR persistent luminescence has been realized in a few hosts only. Table 2 gives a comprehensive detail of the host, dopant ions, wavelength of the afterglow emission, and remarks, if any of the NIR emitting persistent phosphors activated with the Mn2+ ion.40,123–129
HostReferences | Co-dopants | λemission | Remarks |
---|---|---|---|
Ca0.2Zn0.9Mg0.9Si2O6 (ref. 40) | Eu2+, Dy3+ | 699 nm | Persistent time more than 24 h, surface functionalization with PEG and amino groups, imaging and tumour targeting applications |
Ca0.2Zn0.9Mg0.9Si2O6 (ref. 123) | Eu2+, Dy3+ | 699 nm | Surface functionalization with methoxy-PEG of different molecular weight, cancer imaging |
Ca0.2Zn0.9Mg0.9Si2O6 (ref. 124) | Eu2+, Dy3+ | 699 nm | Biotinylated PEG-PLNPs, protein binding and avidin expressing glioma cells targeting application |
Ca0.2Zn0.9Mg0.9Si2O6 (ref. 125) | Eu2+, Dy3+ | 699 nm | Functionalization with Rak-2, prostate cancer cells imaging application |
CaMgSi2O6 (ref. 126) | Eu2+, Ln3+ (Ln = Dy, Pr, Ce, Nd) | 685 nm | Eu, Pr, Mn co-doping yield best results bioimaging application |
SiO2/CaMgSi2O6 (ref. 127) | Eu2+, Pr3+ | 660 nm | Biocompatible, imaging application |
Zn2P2O7 (ref. 128) | Tm3+ | 690 nm | Blue (Tm only), red (Mn only) and NIR (Tm, Mn both) persistent luminescence |
MAlO3 (ref. 129) | Mn4+/Ge4+ (M = La, Gd) | 730 nm | Bioimaging in pork tissue |
The credit for the first use of Mn2+ doped persistent nanophosphors for in vivo imaging applications goes to Daniel Scherman's group.40 The material was co-doped with lanthanide ions (Eu2+ and Dy3+). The group selected a MgSiO3 host for the purpose, which was already reported for red persistent luminescence arising due to Mn2+.81 The group accomplished two new things: firstly, they altered the composition of the host lattice, by introducing Ca and Zn, to optimize the emission of Mn2+ in the IR region, and secondly, they synthesized this phosphor (Ca0.2Zn0.9Mg0.9Si2O6) via a sol–gel synthesis technique, followed by a selective sedimentation process so as to reduce the particle size usable for imaging applications. In this way, they were able to obtain particles with diameters ranging from 50 to 100 nm. The X-ray diffraction (XRD) pattern and transmission electron microscopy (TEM) image of the sample is shown in Fig. 5(a) and (b), respectively.
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Fig. 5 Physical characteristics of long afterglow Ca0.2Zn0.9Mg0.9Si2O6 nanoparticles, (a) XRD pattern showing clinoenstatite-like structure, (b) transmission electron microscopy images (scale bar: 200 nm), (c) excitation spectrum, (d) long afterglow emission spectrum, (e) time dependence of the luminescence intensity of the nanoparticles (NPs). NPs (10 mg) were placed in 96-well plates under direct exposure to a 6 W UV lamp for 5 min. The luminous intensity was quantified straightforward by using an intensified charge-coupled device (ICCD) camera (Photon Imager; Biospace). Data analysis was performed by signal integration for 5 s. The luminous decay data were fit by a power law function for time 100 s. (f) Schematic representation of NP surface modification, (i) amino-NPs, (ii) carboxyl-NPs, (iii) PEG-NPs, and (g) optical imaging of mouse with 1 mg tail vein injections of amino-NPs. Reproduced with permission from ref. 40, Copyright (2007) National Academy of Sciences, U.S.A. |
The authors explained that the persistent luminescence was due to Mn2+ (activator ion). Trap centres are created due to Dy3+ doping and lanthanide ions act as the primary acceptor of energy, which is thermally released to Mn2+ ions (through tunnelling and PET processes). The change in composition changes the symmetry and crystal field strength at the Mn2+ site, which is responsible for emission from red to NIR corresponding to the well-known 4T1(4G) excited state to the 6A1(6S) fundamental state. The excitation (for λems = 690 nm) and emission (for λexc = 340 nm) spectrum are shown in Fig. 5(c) and (d), respectively. The broad nature of the excitation band also support an energy transfer process, as this could not be solely due to the transitions of the Mn2+ ion. The decay curve of luminous intensity shows that the persistent luminescence is detectable for about 24 h, when kept in the dark. The decay kinetics (shown in Fig. 5(e)) were found to be close to a power law I ≈ I0xt−n (n = 0.96, R2 = 0.996) after the first 100 s.
In the next step, the persistent NPs were surface functionalized to make them amiable for imaging applications. Three different surface modifications, namely, amino, carboxyl and PEG, were tried. Amino-NPs were synthesized by a reaction with 3-aminopropyltriethoxysilane, carboxyl-NPs were obtained from a reaction of amino-NPs with diglycolic anhydride, and PEG-NPs were achieved by a peptidic coupling of amino-NPs with PEG5000COOH. A schematic representation of the surface modifications of NPs through different capping agents is shown in Fig. 5(f). These surface functionalized particles were developed with different surface charges. The authors have showed different aspects of imaging with these surface modified NPs. In the first step, simple visualization of persistent luminescence in mice is observed after subcutaneous (SC) and intramuscular (IM) injection of the NPs. Even the lowest dose administered, 20 ng, produces a detectable signal with a satisfactory signal-to-noise ratio superior to 5 during SC injection; however, a comparatively higher dose, 20 μL at 10 mg mL−1, is required for a satisfactory result during IM injection. In the second step, optical imaging of a mouse with 1 mg tail vein injections of differently charged NPs were carried out. Fig. 5(g) shows the images with amino-NPs. The results depict that, the bio-distribution of NPs depends on the surface charge. Note that differently charged NPs possess markedly altered bio-distributions. Furthermore, the use of anionic liposomes before NP injection has been found to be a powerful method to improve the targeting of specific organs, and PEG-NPs (after preinjection of anionic liposomes) have been used to visualize tumours in a mouse successfully.
In the continuation of their work towards the enhancement of the imaging process in many aspects, in their next work, the group modified the surface coating of the persistent NPs with methoxy-PEG of varying molecular weights (5, 10, and 20 kDa) and also varied the particle size of the persistent phosphor.123 The earlier used PEG-NPs were mainly absorbed by RES organs, mostly the liver and spleen. Nevertheless, the use of methoxy-PEG extends the persistent luminescence nanoparticles (PLNP) circulation in mice and concludes that, the blood retention capability of PLNPs is highly dependent on the particle core diameter as well as on the surface coating. However, they further point out that, increasing the molecular weight of the PEG moiety, from 5 to 20 kDa, had no significant effect on the combined uptake in major RES organs such as the liver and spleen. Small and stealth PEGylated PLNPs were shown to circulate for a much longer period, postponing the uptake from the RES organ only, which shows its promise for future applications in targeting and cancer imaging.123
In another novel application of the same PLNPs, the group successfully reported the first use of biotinylated PLNPs to target avidin-expressing glioma cells.124 In this process, PEGylated PLNPs were functionalized by biotin to form biotin-PEG-PLNPs and the system further was used to target biotin-binding proteins, e.g. streptavidin and neutravidin. Firstly, biotinylated PLNPs are allowed to bind with streptavidin coated on a plate. It is marked that, the presence of a PEG spacer is critical to allow the efficient binding to streptavidin coated on a plate. Secondly, the interaction with free neutravidin in solution was confirmed by fluorescence microscopy. Finally, an in vitro binding study on BT4C cells expressing lodavin fusion protein, bearing the extracellular avidin moiety, was performed and was used to target the malignant glioma cells through a specific biotin–avidin interaction. In a similar work for targeting strategies, the group reports the design and functionalization of PLNPs with small targeting molecules.125 The first example they take is the functionalization of PLNPs with biotin in order to evaluate their binding affinity on a streptavidin coated plate, which is also somewhat explained in their earlier work .124 The second and novel example they target is the functionalization of PLNPs with Rak-2 molecule, a well-known molecule for its affinity toward PC-3 cells. The Rak-2 functionalized PLNPs show high stability in a complete DMEM (Dubecco's modified Eagle's medium) culture medium, and the preliminary results reported by the group show the immobilization of PLNP-PEG-biotin on a streptavidin plate and binding of PLNP-PEG-Rak-2 on prostate cancer cells.
Thus, in this way, the group exploited a few important bioimaging applications (both in vivo and in vitro) of the Mn2+ doped persistent phosphor; however, at the same time, they realized that improvement in the persistent emission intensity/persistent time is essential for long-term monitoring of in vivo probe accumulation, which needs more work towards the development of new PLNPs with improved optical characteristics. The authors came up with a new idea that, controlling the electron trap depth could help to enhance the optical properties of PLNPs. In this context, they synthesized several Mn2+ doped diopside NPs, either codoped with trivalent lanthanide ions, e.g. CaMgSi2O6:Mn2+, Ln3+ (Ln = Dy, Pr, Ce, Nd) abbreviated as CMSO:Ln, which can be excited with X-rays only, or tridoped with divalent europium and trivalent lanthanide ions, e.g. CaMgSi2O6:Mn2+, Eu2+, Ln3+, to enable UV excitation, abbreviated as CMSO.126 The main aim was to tune the trap depth by co-doping with Ln3+ ions and to find a particular lanthanide ion for an optimized IR emission suitable for bioimaging applications. A schematic energy level diagram of Mn2+ and Ln3+ in CMSO is shown in Fig. 6(a). The relative positions of the electron trap levels originate due to the different lanthanide ion co-doping with respect to the conduction band edge is expanded as shown in the inset of the energy level diagram in Fig. 6(a). The trap depth due to Pr, lying below Ce3+ and above Nd3+, has been identified as the optimal electron trap in the diopside host, which led to an improved nanomaterial, excitable with UV light and displaying the most intense afterglow in the NIR region. Furthermore, they used the novel and optimized composition (CaMgSi2O6:Eu2+, Mn2+, Pr3+) for in vivo imaging via intravenous injections of such PLNPs in mice for sensitive detection through living tissues. They also compared this material with the earlier reported phosphor composition abbreviated here as CZMSO, i.e. Ca0.2Zn0.9Mg0.9Si2O6 co-doped with Eu2+, Dy3+, Mn2+, during the imaging application, shown in Fig. 6(b and c), respectively. They report improved imaging with the CaMgSi2O6:Eu2+, Mn2+, Pr3+ composition.
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Fig. 6 (a) Schematic energy level diagram of Mn2+ and Ln3+ in a CaMgSi2O6 (CMSO) host. The main hole traps are Mn2+ ions in the Mg2+ site, whereas electrons are trapped by oxygen vacancies (VO) and Ln3+ ions. Thermally stimulated luminescence (TSL) and persistent luminescence occur by thermally activated electron release and capture by Mn3+ ions, giving the Mn2+ emission. The inset shows the relative positions of electron trap levels with respect to the conduction band edge. In vivo imaging of (b) Ca0.2Zn0.9Mg0.9Si2O6 (CZMSO):Eu, Dy; and (c) CMSO:Eu, Pr PLNPs under the photon-counting system. The signal was recorded for 15 min following systemic injection of the probes (100 μg, excited 5 min under a 6 W UV lamp at 254 nm before injection). Luminescence intensity is expressed in false color units (1 unit = 2800 photons per s cm2 steradians). For both compositions (b and c), the left picture represents the luminescence signal from PLNP, and the right picture represents the photograph of the imaged mouse. Reprinted (adapted) with permission from ref. 126. Copyright (2011) American Chemical Society. |
In a similar work, Li et al. synthesized a SiO2/CaMgSi2O6:Eu0.01, Pr0.02, Mn0.10 persistent phosphor, which emitted its persistent emission maximum at 660 nm.127 The article covers the synthesis and characterization (structural-phase, morphology, optical) of the NPs, surface functionalization, cell viability (through cytotoxicity) test, and in vivo imaging (on mouse). The NPs were obtained with control over morphology and size. Hydrophilic modification was carried out further, which showed that the nanoprobes exhibit good biocompatibility. The cell viability test shows that, NPs are metabolized from the lymph circulation and transferred from the abdomen to the bladder. The afterglow emission of the as-prepared nano-probes was successfully used to track the real time bio-distribution in vivo with a reasonably good signal-to-noise ratio.
Different from a silicate host, Pang et al. recently reported NIR persistent emission due to Mn2+ ions in a zinc pyrophosphate, Zn2(0.97−x)P2O7:0.06Tm3+, 2xMn2+ (0 ≤ x ≤ 0.05), host.128 A series of new phosphors were synthesized by using the typical high temperature solid-state reaction method. Both single doped and co-doped samples crystallize into mono phase, α-Zn2P2O7. The doping of Mn2+ and/or Tm3+ does not alter the phase, only a slight variation of the cell volume is seen, and this may be due to the differences of ionic radius and valence between Zn2+ and the doping ions (Mn2+, Tm3+), which confirms the substitution by the doping ions into the host lattice. Furthermore, detailed photoluminescence, persistent luminescence and decay characteristics have been studied. The material doped with Mn2+ ions gives an intense wide emission band from 610 nm to 780 nm with a maximum emission intensity at 690 nm (λexc = 412 nm), which is ascribed to the forbidden d–d transition of Mn2+ from the 4T1 to 6A1 energy states. Co-doping of Tm3+ further causes a splitting of the broad band at around 670 nm and at 694 nm.
The material shows excellent persistent luminescence in the blue (if Tm3+ is doped only, ZPOT), red (if Mn2+ is doped only, ZPOM) and NIR region (both Mn2+ and Tm3+ are co-doped, ZPOT:2xM). The long lasting phosphorescence (LLP) emission spectra of the samples ZPOT:2xM (0 ≤ x ≤ 0.05) is shown in Fig. 7(A). Characteristic blue LLP emissions of Tm3+ peaking at 368 nm (1D2 → 3H6), 454 nm (1D2 → 3H4) and 482 nm (1G4 → 3H6), respectively is observed in Tm3+ singly doped materials. However, in the ZPOT:2xM samples, the intensity of the blue LLP emissions of Tm3+ decreases gradually with the increasing concentration of Mn2+ and finally disappears when the value of x exceeded 0.005. This results in the intensity enhancement of the red emission of Mn2+, which attains a maximum value at x = 0.01. In addition, a longer wavelength shift of the emission maximum of Mn2+, from about 625 nm to 690 nm, is also observed, which is explained due to the two possible distinct Zn sites in the structure of α-Zn2P2O7. Thus, the change of the emission intensity ratio of the 625 nm to 690 nm band leads to a colour shift of Mn2+ LLP from red to NIR. Fig. 7(B) describes the LLP decay curves monitored for the emissions of Mn2+ at 690 nm. The LLP decay curve of Mn2+ exhibits a third exponential decay process, obeying the decay function:
I = A1![]() ![]() ![]() |
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Fig. 7 (A) The LLP emission spectra of ZPOT:2xM (0 ≤ x ≤ 0.05) measured at 5 min after ceasing the 254 nm excitation source, (B) LLP decay curves (black circle) and the plots of inverse of LLP intensity (I−1) versus time (t) (blue line) monitored at 690 nm for ZPOT:0.02M, (C) persistent luminescence properties of sample LAM21, afterglow intensity monitored at 731 nm as a function of time. The phosphor was pre-irradiated by a xenon lamp for 10 min, (D) long persistent luminescence tissues imaging of pork tissue (a) pre-injection image, and (b) 1, (c) 10, (d) 30, (e) 120, and (f) 720 min post-injection fluorescence images. The sample GAM23 was pre-excited at 325 nm for 10 min before the injection. Injection of sample (1 g) mixed in normal saline (10 mL); injection depth was 3 mm under the epidermis. (a and b) Reproduced from ref. 128, (c and d) reproduced from ref. 129 with permission from The Royal Society of Chemistry. |
Similarly, Li et al. studied the NIR luminescence of Mn4+ in La and Gd based aluminates and proposed a holistic design concept for NIR long persistent phosphors.129 The samples were synthesized using typical solid state reaction methods and characterized for phase, microstructure, photoluminescence, persistent luminescence, decay curves, and defect properties using TL and ESR studies. Mn4+-doped MAlO3 (M = La, Gd) persistent phosphors gives an emission maximum around 730 nm. Different concentrations of Mn4+ and Ge4+ were used and their effect on persistent emission was studied. The co-doping of Ge4+ tailors the defect level trap depths, and thereby a considerable improvement of persistent time over 20 h was attained for the sample La1Al0.99O2.985:0.1%Mn4+, 0.9%Ge4+ (LAM21) and Gd1Al0.99O2.985:0.1%Mn4+, 0.9%Ge4+ (GAM23). The NIR long persistent luminescence of both GAM23 and LAM21 (Fig. 7(C)) phosphors were detectable by a supersensitive camera even up to 24 h, after pre-irradiation of the samples by 325 nm radiation of a Xe flash lamp for 10 min. The materials were used for imaging applications also. Fig. 7(D) shows the deep tissue images of pork tissues injected with the GAM23 phosphor. For imaging, phosphors were first irradiated with 325 nm light of a Xe flash lamp for 10 min and then injected in tissues. The authors claim that the phosphors give a very high resolution even after 2 h post-injection, due to the absence of auto-fluorescence and a higher signal-to-noise ratio. This allows tissue bioimaging to be monitored for more than 12 h. A few other works on Mn doped NIR persistent phosphor materials may also be found; however, as far as the bioimaging application is concerned, the abovementioned works are the representative works in the area and are the signposts for researchers working in this area to plan and design further developments. In the next step towards the development of efficient NIR persistent phosphors for bioimaging applications, in last few years, Cr3+ doped persistent materials have been widely explored. The works are summarized in the next section.
HostReferences | λEmission (nm) | Time (h) | Remarks |
---|---|---|---|
ZnGa2O4 (ref. 130, 131 and 136) | 650-–730 | >1 | Solid state reaction, variation of Cr3+ concentration |
ZnGa2O4 (ref. 132, 133 and 135) | 650-–730 | >1 | Zn deficiency improves persistent luminescence |
ZnGa2O4 (ref. 134) | 650-–730 | >1 | Low temperature sintering, imaging of vascularization, tumours and grafted cells |
Zn3Ga2Ge2O10 (ref. 137) | 650-–1000 | >360 | Solid state reaction, sunlight activated, all weather material |
Zn3Ga2Ge2O10 (ref. 41) | 630-–750 | >360 | Photostimulation through incoherent light, imaging of pork tissue, cytotoxicity |
Zn3Ga2Sn1O8 (ref. 50) | 650-–800 | >300 | Imaging in goldfish |
Zn2.94Ga1.96Ge2O10 (ref. 138) | 650-–800 | >360 | Pr3+ co-dopant, Zn deficiency, surface functionalization with PEG and RGDyK |
Zn1+xGa2−2x(Ge/Sn)xO4 (ref. 139) | 640-–780 | >360 | Replacement of Ga by Sn/Ge in ZnGa2O4 |
Zn(Ga1−xAlx)2O4 (ref. 140) | 640-–780 | — | Replacement of Ga by Al in ZnGa2O4, co-doping of Bi |
LiGa5O8 (ref. 141) | 640-–780 | >1000 | In vivo imaging, visible light stimulation |
LiGa5O8 (ref. 142) | 640-–780 | — | Surface functionalization with PEG-OCH3, in vivo imaging |
LiGa5O8 (ref. 143) | 640-–780 | — | Embedded in glass matrix |
Ga2O3 (ref. 144) | 640-–780 | >1 | Nanowire assemblies |
Ga2O3 (ref. 145) | 600-–800 | <1 | Co-doping with Zn2+, photocatalytic activity |
Mg/SrGa2O4 (ref. 146–148) | 630-–780 | — | New phosphors |
Gd3Ga5O12 (ref. 149 and 150) | 630-–800 | — | — |
Y3Al2Ga3O10 (ref. 151) | 660-–750 | — | — |
La3Ga5GeO14 (ref. 152 and 153) | 600-–1400 | — | Excitation dependent emission |
Sr4Al14O25 (ref. 154) | 680-–850 | <1 | Eu2+ and Dy3+ as co-dopants |
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Fig. 8 (a) Cubic spinel structure of ZnGa2O4(ZGO); (b) normalized PL spectra of ZGO:Cr and LLP emission spectrum at RT, 10 s after the end of a 290 nm excitation; (c) LLP decays measured after a 15 min laser excitation at 550 nm (11 mJ) and 290 nm (3 mJ); (d) schematic energy level diagram showing typical electron trapping processes. CB and VB refer to the conduction band and the valence band of the host; GS and ES are the ground state and excited state of the luminescent center. Arrows (i), (ii), and (iii) show three different excitation ways for electrons moving to the trap center through the CB. The quantum tunnelling (QT) effect is another possible path for electron trapping. (a–c) Reprinted (adapted) with permission from ref. 132. Copyright (2014) American Chemical Society. (d) Reproduced from ref. 133 with permission from The Royal Society of Chemistry. |
The paper additionally remarks on to how one can effectively determine the relative locations of the VB and CB of the host, the GS and ES of the luminescent centre, and the position of trap levels by making use of different characterization techniques, which is the most extreme prerequisite to propose the right mechanism involved in the persistent luminescence process. The energy of the topmost level of the VB may be characterized as zero by embracing the idea of a host referred binding energy (HRBE) scheme. Furthermore, the absorption spectra of an un-doped sample (host) could be used to determine the band-gap energy, which would give information about the position of the CB bottom. Moreover, photoconductivity excitation (PCE) spectra, persistent luminescence excitation spectra (PersLE), or photoluminescence excitation (PLE) spectra measurements can be used to evaluate the energy of the electronic transition from the GS to the bottom of the CB. Similarly, for transition metal doped samples, the absorption spectra or PLE spectra can confirm the 3d–3d intra-transitions from the GS to various ES. At last, the depth of the defect/trap levels (for stimulation: thermal activation energy, or photostimulation) can be attained through thermoluminescence (TL) glow curve measurements.133
NPs of the zinc gallate phosphor (ZGO) were prepared using a hydrothermal method followed by low-temperature sintering in air. The hydroxylation of these NPs was performed by basic wet grinding of the powder (ZGO-OH). Furthermore, surface functionalization was performed according to their earlier protocol in case of Mn2+ doped silicates with a small modification to obtain ZGO-NH2 and ZGO-PEG, as shown in the scheme in Fig. 9(a). Fig. 9(b) shows the TEM image of the NPs with a core diameter of 20-–60 nm. As compared to the ZGO-NH2, PEG grafting increases the hydrodynamic diameter (80 nm) and causes an opposite shift in the zeta potential (−6.70 mV) (see Fig. 9(c and d)); however, the optical characteristics remains same for both types of particles. Remarkably, surface functionalization with PEG helps the colloidal solution of NPs to remain in blood circulation for a longer time.
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Fig. 9 The surface functionalization and characterization of red-excitable PLNPs. (a) A schematic representation of ZGO-OH surface functionalization; (b) a transmission electron micrograph of ZGO-OH nanoparticles. Scale bar, 100 nm; (c) the hydrodynamic diameter measured by dynamic light scattering in 5% glucose before and after PEG coverage; (d) the evolution of zeta potential as a function of surface coverage. Reprinted by permission from Macmillan Publishers Ltd: [Nature material] (ref. 134), copyright (2014). |
The PEG functionalized particles were able to circulate in the blood circulation route for more than 2 h, which favours tumour targeting in vivo, and the group successfully demonstrated the first proof of in vivo tumour passive targeting. Initially, during the first 2 h, the bio-distribution of ZGO-PEG was similar for both tumour-bearing and healthy mice, and tumours were not visible; however, after 4 h, tumours were clearly visible, and the NPs needed activation by an orange/red source (photo-stimulation) after such a long period. The luminescence from the regions of interest (ROIs) was compared with the global luminescence signal from the entire animal, which shows only a little variation in intensity from tumours (subcutaneous CT26) due to the permeability and retention effect; however, a significant enhancement in intensity by the liver (from 18% to about 50%) was observed during the 6 h period, which confirms the major accumulation of stealth NPs within the liver (Kuffer cells). The results are summarized in Fig. 10(a–e). Heavy accumulation of the particles in the liver after 6 h strained the authors to further examine the cellular and systemic toxicity of these ZGO-based PLNPs to answer the biocompatibility issue of the used NPs. Importantly, there was no signature of inflammation or any change in morphology during histopathology even after 24 h of the injection of NPs, which, although preliminary, confirms that ZGO-PEG NPs are free from acute toxicity in healthy mice.
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Fig. 10 The biodistribution of stealth ZGO-PEG nanoparticles in tumour-bearing mice (n = 3), (a) optical image of a tumour-bearing mouse; (b) persistent luminescence image of a tumour-bearing mouse, 2 h after the injection of ZGO-PEG nanoparticles excited by an ultraviolet lamp for 2 min (acquisition time: 10 min); (c) persistent luminescence image of a tumour-bearing mouse immediately after LED illumination, 4 h after the injection of ZGO-PEG nanoparticles excited by an ultraviolet lamp for 2 min (acquisition time: 3 min); (d) schematic ROI drawn for semi-quantization analysis: L-liver, T-tumour; (e) semi-quantization of the accumulation kinetics in the main labelled tissues expressed as a percentage of the total luminescence detected from the whole animal. Persistent luminescence intensity is expressed in false colour units (1 unit D 2800 photons s cm2 sr) in all images. Reprinted by permission from Macmillan Publishers Ltd: [Nature material] (ref. 134), copyright (2014). |
Finally, the authors investigated the use of surface functionalized ZGO NPs (ZGO-OH, ZGO-NH2 and ZGO-PEG) to track injected cells (RAW264.7 macrophages) in vivo. In this context, first they performed a preliminary investigation regarding suitable NPs, internalization of NPs in the cell, and of course the cell viability. The ZGO-NH2 NPs underwent superior phagocytosis with a clearly detectable persistent luminescence, after orange/red excitation (photo-stimulation). In addition to this, incubation with ZGO-NH2 did not show a significant effect on cell viability, and the results are summarized in Fig. 11(a–c). Efficient internalization was clearly verified through confocal microscopy (Fig. 11(d–f)) and TEM measurements. After confirming these, the bio-distribution of free ZGO-NH2 was compared with the bio-distribution of RAW 264.7 cells incubated with ZGO-NH2NPs. As pointed our earlier, free NPs are accumulated mostly in liver and spleen (RES organ); however, labelled cells leads to a strong luminescence signal in the lungs due to the rapid sequestration of phagocytes in the lung capillary bed (shown in Fig. 11(g and h)). This different behaviour was also further verified by an ex vivo persistent luminescence acquisition 24 h after intravenous injection (shown in Fig. 11(i)).
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Fig. 11 Cellular tracking with persistent luminescence after LED excitation. (a) Phagocytosis efficiency expressed as persistent luminescence signal retained by RAW macrophages that incorporated differently charged ZGO nanoparticles; (b) optical and persistent luminescence image of tagged RAW cells suspended in culture medium (3 min acquisition time); (c) in vitro cellular toxicity of ZGO-NH2 nanoparticles towards RAW cells (MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, assay). Nanoparticles were incubated with cells for 6 h; (d) confocal microscopy image of TOPRO staining (blue) on tagged RAW cells; (e) confocal microscopy image of FITC-grafted ZGO nanoparticles (green), taken up by RAW cells; (f) merged confocal microscopy image of RAW cells stained with both TOPRO and FITC-grafted PLNPs; (g) biodistribution of ZGO-NH2 nanoparticles in healthy mouse, 15 min after systemic injection (acquisition time: 1 min); (h) biodistribution of RAW cells tagged with ZGO-NH2 nanoparticles in healthy mouse, 15 min after systemic injection (acquisition time: 1 min); (i) ex vivo biodistribution of free ZGO-NH2 and RAW macrophages tagged with ZGO-NH2 nanoparticles 24 h after intravenous injection, Li-liver, S-spleen, K-kidneys, H-heart, Lu-lungs. Persistent luminescence intensity is expressed in false colour units (1 unit D 2800 photons s cm2 sr) for all images. Reprinted by permission from Macmillan Publishers Ltd: [Nature material] (ref. 134), copyright (2014). |
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Fig. 12 NIR afterglow decay of Zn3Ga2Ge2O10:0.5%Cr3+ discs irradiated by a 4 W 365 nm ultraviolet lamp. (a) Afterglow intensity (I) monitored at 713 nm as a function of time (t). The sample was irradiated for 5 min. The bottom inset shows the same data plotted as I−1 versus t. The upper inset shows four afterglow spectra recorded at 0 h, 6 h, 12 h and 24 h after the stoppage of the irradiation; (b–h) NIR images of four phosphor discs taken at different afterglow times (5 min to 360 h) after irradiation by a 365 nm lamp for 10 s to 5 min. The discs were placed on a hot plate surface for imaging. Imaging parameters: (b–d) auto/ISO 200/0.3–4 s; (e) manual/ISO 200/30 s; (f–h) manual/ISO 400/30 s. (i) NIR image of the four 360 h-decayed discs when heated at ∼400 °C on the hot plate. Imaging parameters: auto/ISO 200/0.3 s. Reprinted by permission from Macmillan Publishers Ltd: [Nature material] (ref. 137), copyright (2012). |
Li et al. used Cr3+ doped zinc gallogermanates for the imaging of pork tissue with incoherent excitation.41 The group initially verified well established facts such as the existence of defect states through ESR measurements. The ESR peak appears at a g-value of 1.9996 (3374.9 G), which persists for an extended period >6 h. Moreover, through PCE measurements at different temperatures (in the range of 20-–300 K), they confirmed the possibility of the energy charging of this material by UV-visible light (250-–450 nm). Detailed excitation, emission and decay characteristics were presented. Moreover, the group demonstrated the stability and reproducibility of the antiStoke luminescence of the material through continuous irradiation with a LED and through cyclic operation over 17 individual on/off cycles of 10 min. Finally, for imaging applications the NPs were dispersed in ordinary saline (100 mg mL−1) and injected into tissue at various injection depths (0.1-–1 cm). Charging was performed ex situ (Xenon short-arc lamp) as well as in situ (X-ray activated) and photostimulation was performed by an IR LED (980 nm and 940 nm). The probe signal was clearly detectable in both the cases. Activation was possible for a large area (6 cm2) as well as for a deep penetration depth (1 cm). A good cellular viability (>95%), in the presence of the injected NPs with a concentration of 100 mg cm−2, and thus a low toxicity of the employed probe was clearly visible through the cytotoxicity studies. In a similar work, the substitution by Sn was used (Zn3Ga2Sn1O8:0.5Cr3+), which gives an almost equally good persistence time of over 300 h.50 Along with all the essential studies, such as optical properties, trap level analysis, photostability, persistent luminescence characteristics, bioimaging and cytotoxicity studies have also been carried out. Note that bioimaging was carried out on a goldfish. NIR persistent luminescence could be clearly imaged for about 2 h, which on photostimulation, through NIR LEDs, recover the initial intensity of the persistent emission.
The work of Abdukayum et al. also reports bioimaging with zinc gallogermanates doped with Cr3+.138 The novelty includes the following major changes, (i) co-doping of a lanthanide Pr3+ ion and creating a suitable Zn deficiency in the zinc gallogermanate (ZGGO) host, (ii) bioconjugation of PEG modified NPs with c(RGDyK) peptide, and (iii) extensive toxicity study covering in vitro (on 3T3 normal cell lines and U87MG cancer cell lines by cell counting assay) and in vivo (monitoring histological changes in several susceptible organs, including the heart, liver, spleen, lung, and kidney) as well as acute (7 days) and chronic (30 days) toxicity. The first change allowed them to obtain a suitable host composition (Zn3Ga1.96Ge2O10:Cr0.01Pr0.03), which could give intense persistent luminescence for more than 15 days. Herein, Pr3+ adjusts the trap density and trap, while control over the zinc content facilitates persistent energy transfer between the host emission and Cr3+ ions, which altogether results to prolong the afterglow. Secondly, PEGylated NPs were obtained, as already reported in other works, to increase the blood circulation time. Furthermore, PEGylated NPs were conjugated with a RGD peptide-c(RGDyK) to perform a tumour-targeted imaging application. The complete surface functionalization steps are shown in Fig. 13(A).
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Fig. 13 (A) Schematic illustration for the surface modification of the LPLNPs; (B) in vivo NIR luminescence images of U87MG tumour-bearing mice (white circles locate the tumour site): (a and b) and normal mice, (c and d) after intravenous injection of PEG-LPLNPs (a and c) and RGD-LPLNPs (b and d) (0.4 mg, 10 min irradiation with a 254 nm UV lamp before injection). (e) Representative ex vivo NIR luminescence images of isolated organs and tumour from a U87MG tumour-bearing mouse at 6 h post-intravenous injection of RGD-LPLNPs (0.4 mg): (1) heart, (2) lung, (3) liver, (4) spleen, (5) kidney, (6) stomach, (7) pancreas, (8) intestine, (9) bladder, (10) bone, (11) tumour. (f) Semi-quantification of RGD-LPLNPs in the isolated organs and tumour of the mice, error bars represent one standard deviation of triplicate measurements from individual animals. Reprinted (adapted) with permission from ref. 138. Copyright (2013) American Chemical Society. |
PEG functionalized NPs, administered into a normal mouse through a subcutaneous injection, show excellent persistent luminescence and allows in vivo bioimaging to be monitored (with SNR > 5) for more than 15 h. NIR stimulation by a 980 nm laser light recovers persistent luminescence after 5 days and is further retained even after 11 days, which shows the suitability of the material for long-term in vivo imaging via repeated stimulation with NIR light. Similar to the earlier report for biodistribution, the material is mainly accumulated in the RES organs, persistent luminescence through which was still visible for about 450 min (7.5 h) after injection. Furthermore, tumour targeting was carried out both by PEG-LPLNPs and RGD-LPLNPs (for a comparative study) by injecting the material into U87MG tumour-bearing mice and normal mice. Both materials were able to mark the tumour site (see Fig. 13(B, a–d)), but the luminescence signal from the RGD-LPLNPs was visible for a longer time in the tumour site. This was due to the high affinity of the RGD-LPLNPs to inter in αvβ3 on the tumour vasculature. Ex vivo imaging of the different organs, including the tumour was also performed, and the persistent luminescence from the tumour was clearly visible, Fig. 13(B-e).
Thus, in summary, Ge/Sn/Al substitution in ZnGa2O4 significantly improves the persistent emission intensity and persistent time, as a result of which the imaging is better performed in terms of both long time imaging and targeting.
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Fig. 14 Images of PEI-LiGa5O8:Cr3+ nanoparticles labeled 4T1 cells injected into a nude mouse for a 10 day tracking using an IVIS imaging system. The PEI-LiGa5O8:Cr3+ nanoparticles labeled 4T1 cells (∼2.5 × 107 cells) were illuminated by a 4 W 254 nm UV lamp for 15 min, and then subcutaneously injected into the back of a nude mouse. (a) Image taken at 4 h after cell injection. To get the PSPL signal, the mouse was exposed to a white LED flashlight (Olight SR51, 900 lumens) for 15 s. (a1) and (a2), Images taken at 10 s and 5 min after the stimulation, respectively. The signals were attributed to PSPL. (b–e) The mouse was exposed daily to the LED flashlight for 15 s, and images were taken at 10 s and 5 min after the stimulation. All images were acquired in the bioluminescence mode with an exposure time of 2 min. The images were processed using Living Image software at binning of 4 and smooth of 535. The color scale bar represents the luminescence intensity in the unit of radiance, p per s per cm2 per sr. Reprinted by permission from Macmillan Publishers Ltd: [Scientific Reports] (ref. 141), copyright (2013). |
The process of chelation of PLNPs with Gd ions involves surface coating of NPs with APTES (3-amino propyltriethoxysilane, amino functional group), binding of diethylenetriamine pentaacetic acid (DTPA), through 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide hydrochloride (EDC-HCl), and N-hydroxysuccinimide (NHS)coupling reaction, to these amino groups followed by chelation with Gd(III). The persistent luminescent emission and decay characteristics of the aqueous solution of the as-synthesized Gd(III)-PLNPs were investigated compared with PLNPs. Although the persistent luminescence intensity and persistent time are both affected by surface modification with the gadolinium complexes, it was good enough for imaging applications; moreover, the NIR persistent luminescence (for the solution with a Gd(III)-PLNPs concentration of 1 mg mL−1) was detectable even after 24 h after stopping the excitation with SNR = 5.2. Furthermore, to evaluate the capability of this material for MRI, longitudinal proton relaxation times (T1) were determined and compared with a commercial Gd-DTPA complex, which was found better for the case of the synthesized Gd(III)-PLNPs material. This confirmed the potential of Gd(III)-PLNPs as an effective contrast agent for T1-weighted MRI.
The authors successfully demonstrated the use of this material for in vivo NIR luminescence imaging (Fig. 15(a)) as well as for in vivo T1-weighted MRI (Fig. 15(b)). For the first type, a Gd(III)-PLNPs solution (1 mg mL−1) was excited using a 254 nm UV lamp for 10 min and then injected (intravenous, 300 μL, PBS) into a mouse. Imaging was possible for more than 6 h (SNR = 5) with a major luminescence signal from the liver sites (Fig. 15(a)). This clearly demonstrates that the Gd(III)-PLNPs are capable of long-term in vivo imaging without in situ excitation. Furthermore, in vivo T1-weighted MRI of Kunming mice was performed before and after an intravenous injection of a Gd(III)-PLNPs solution (300 μL, 1 mg mL−1) on a 1.2 T MRI system. The spatial resolution was better with MRI. The liver of the preinjection mouse was not very clear; however, visibility was significantly pronounced after 15 min postinjection of Gd(III)-PLNPs (Fig. 15(b)). Thus, the successful demonstration of both the imaging technique probes the multimodality of the material.
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Fig. 15 (a) In vivo NIR luminescence images of a normal mouse after an intravenous injection of Gd(III)-PLNPs (0.3 mg, 10 min irradiation with a 254 nm UV lamp before injection but without irradiation during imaging); (b) in vivo T1-weighted MR images of the mouse before and after intravenous injection of Gd(III)-PLNPs (0.3 mg); the yellow arrow indicates the liver. Reprinted (adapted) with permission from ref. 51. Copyright (2014) American Chemical Society. |
In another work by Chen et al., calcium gallogermanate (Ca3Ga2Ge3O12) was synthesized and codoped with Cr3+ ion and Tm3+, Yb3+ ions.163 The authors successfully observed NIR persistent luminescence due to the Cr3+ ion (with a persistent time of about 2 h) and NIR to NIR upconversion emission due to the Tm3+, Yb3+ ion couple. Interestingly, co-doping of Tm3+ enhances the persistent luminescence. The authors suggest its potential application in imaging both through persistent luminescence as well as through UC, but admit that a long distance still remains to be traversed to achieve this goal. Thus, in summary, already some research work have been started to introduce multimodality in persistent phosphors; however, the results are preliminary and needs further attention to develop efficient materials, which could meet the pragmatic aspects of multimodal imaging applications.
Mn2+ doped silicates (Ca0.2Zn0.9Mg0.9Si2O3:Eu2+, Dy3+) were first identified as suitable NIR persistent phosphors for imaging applications. Trap centres in this material is created due to Dy3+ doping and lanthanide ions act as the primary acceptor of energy, which is thermally released to the Mn2+ ions, through tunnelling and persistent energy transfer processes. The change in composition brings out changes in the symmetry and crystal field strength around the Mn2+ site, which is responsible for emission from red to NIR, corresponding to the well-known 4T1(4G) excited state to the 6A1(6S) fundamental state. The biodistribution of these NPs, which depends significantly on surface charge, is improved by surface functionalization with poly ethylene glycol (PEG). The blood retention capability highly depends on the core diameter of the NPs as well as on the surface coating; thus, methoxy-PEG modified NPs were identified as more suitable to improve tumour targeting in mice. In addition, the functionalization of PEG-PLNPs with small molecules, such as biotin and Rak-2, make it useful to even target malignant glioma cells and prostate cancer cells, respectively. Thus, the material was good enough for different imaging and targeting applications; however, improvement in the emission intensity/persistent time was realised as essential for long-term and deep tissue monitoring of in vivo probe accumulation, which led to the development of Cr3+ doped nanostructures. In the next step, Cr3+ doped zinc based gallates and substituted structures attracted the major attention due to its excellent NIR persistent emission with a longer afterglow time of more than 2 weeks. Moreover, the persistent emission can be rejuvenated/recovered by photostimulation, especially by incoherent light sources (e.g. LEDs), which offers real-time long term imaging applications of these PLNPs. Although the concept evolved recently (first reported in 2011), the theory developed successfully addresses wide aspects related to the structure of the material, effect of the structure on persistent luminescence of Cr3+ ions, photostimulation, surface functionalization, imaging, and toxicity.
Important findings can be summarized as follows. The basic requirement in such materials is to generate suitable trap levels. The existence and localization of such defects states could be estimated through electron spin resonance (ESR) and thermoluminescent (TL) glow measurements. The peak temperature and full width at half maximum (FWHM) of the TL glow curves reflect the trap depth and the trap depth distribution. In the next step, the charging, emission and processes involved in between become important. Photocurrent excitation (PCE) measurements can be used to determine information about the energy charging range for a particular material, which basically lies in the UV-visible regime for gallogermanates. Routine excitation, emission and decay curve analysis can be applied for photoluminescence and persistent emission measurements. Persistence time is a technical term here, which refers to the duration for which the eye can see with the aid of a night-vision monocular in a dark room. Bioimaging involves surface functionalization, bio-distribution and targeting applications, which is somewhat similar to that of Mn2+ doped materials with better resolution, high signal-to-noise ratio, longer retention time in blood circulation and better targeting ability. Both acute and chronic toxicity in in vivo and in vitro studies shows that these particles are safe for bioimaging applications.
In conclusion, Cr3+ doped zinc gallogermanates NIR PLNPs have evolved as the most adaptable and easy to use optical nanoprobes. These PLNPs emit intense, long-lasting persistent light even after the removal of the excitation, which allows imaging without external excitation and completely removes the possibility of auto-fluorescence/background noise and thus improves the signal-to-noise ratio and sensitivity greatly. Furthermore, emission in the NIR region by Cr3+ lies in the tissue transparency window, which further increases the detection depth. Furthermore, the plausibility of photo-stimulation by incoherent light source, (LEDs)/flash lamps, makes long term imaging conceivable, which is simple, financially savvy and safe. Taking into account the recent improvements in NIR persistent phosphors, a guaranteed future for this material might be predicted especially for scientists and pharmacologists involved in numerous distinctive sorts of research related to cancer diagnosis, vascular biology and cell research.
The majority of the reported NIR PLNPs covers the NIR I region; therefore, it would be a future viewpoint to develop persistent phosphors for the NIR II region also and look for their response in imaging and targeting applications. Moreover, further investigations and endeavours to develop multimodal persistent phosphors, which may work both as optical as well as magnetic probes will certainly be exciting for the future course of research.
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