Red and near infrared persistent luminescence nano-probes for bioimaging and targeting applications

Abstract

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.

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S. K. Singh

Sunil K Singh, born in 1982, received his doctoral degree in Physics in 2011 from the Banaras Hindu University, India. Currently, he is a DST-INSPIRE faculty at the Indian Institute of Technology (Banaras Hindu University), Varanasi, India. His research interest includes the physics of multimodal luminescence (upconversion, downconversion/quantum-cutting, downshifting), including persistent luminescence in lanthanide and transition metal doped nanostructures. In terms of applications, the focus is on bio-imaging, to increase the conversion efficiency of photovoltaic cells, sensors, and for security purposes. He has authored/co-authored a book chapter, a review article and more than 30 peer-reviewed journal articles, and his current h-index is 11 (Sep. 2014).

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1. Overview

Bioimaging is related to methods that can non-invasively envisage structural and functional processes in biological materials with no/minimal affect on the life processes therein.¹ In recent decades, the demand for bioimaging tools has grown very rapidly, particularly for bio-medical research and medical applications. This has consequently resulted in the development of many advance techniques, including magnetic resonance imaging (MRI), micro-computed tomography (micro CT), positron emission tomography (PET), optical coherence tomography (OCT), electron tomography (ET), ultrasonic imaging, and X-ray imaging.^1,2 These excellent bioimaging techniques cover a wide observation range commencing with sub-cellular structures, cells, tissues up to entire multi-cellular organisms. Each technique has its own advantages and also limitations, and thus more than one technique may also be used for complete imaging applications. However, the high cost of several of these techniques, bio-compatibility issues with tracers/markers, and a number of technological barriers impair their widespread use for routine imaging in medical as well as for specialized research.

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.

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

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.

2. Persistent luminescence

A pre-requisite towards a better understanding of the subject makes it essential to discuss persistent luminescence, its development, present status of the field, and a literary review of the existing persistent phosphors. Persistent luminescence is a phenomenon whereby luminescence can last for hours (or even more, recently reported upto few days) after the stoppage of the excitation.^44–53 As mentioned earlier, it is generally accepted that, the presence of lattice defects causes the evolution of trap levels, which play a central role for the afterglow/persistent luminescence in some potentially efficient luminescent materials. However, even after a long history of work on persistent luminescence, the fundamental of each and every step involved in this phenomenon is still not unanimously accepted, and more than one explanations are available in published articles to explain certain processes. The review article by Brito et al. focuses on the mechanisms of persistent luminescence. In addition to the mechanism, this interesting article also comments on the diverse names utilized for persistent luminescence.⁴⁶ The article remarks that, some of the names are used as adjectives, e.g. long, persistent, long-duration and long-lasting, whereas a few others are used as nouns, e.g. afterglow, fluorescence, phosphorescence and luminescence. Not just these, but names have likewise been utilized even as a part of a set of two, or in greater gatherings such as long-persistent phosphorescence.⁴⁶ However, persistent luminescence is alluded to a special case of thermally stimulated luminescence at a given temperature (usually at room temperature), which ought to be different from phosphorescence due to the forbidden transition in the luminescence center.

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

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.

3. Red emitting persistent materials and bioimaging applications

After making an initial introduction and background of the subject in first two sections, this section put forwards a proper focus on red emitting persistent materials and their biological applications. Initially, most of the red emitting afterglow phosphors were sulphide based lanthanide doped phosphors such as CaS, SrS, and BaS or their combination such as Ca_1−xSr_xS doped with different combinations of dopants.^62–74 Most of these sulphide based phosphors contain Eu²⁺ as one activator ion along with other co-dopants such as Tm³⁺, Dy³⁺, Ce³⁺, Pr³⁺, and Bi³⁺. In addition to this, the effect of alkali ions (e.g. co-doping of Li⁺, Na⁺) and halides (Cl⁻) on luminescence/persistent luminescence has also been studied. A review article by Ghosh et al. throws light on the developments of alkaline earth sulphides (e.g. CaS, SrS and BaS) as luminescent and energy storage materials.⁶² This includes the development of a brief understanding of the subjects, particularly defect states in these sulphides, which is attained through studies of electron spin resonance (ESR), thermally stimulated luminescence (TSL) and fluorescence emission spectra at low and elevated temperatures. This review article also covers the evolution of new preparation methods for polycrystalline powders, thin film layers and single crystals. In a recent work on sulphide phosphors, Burbano et al. developed NPs of CaS codoped with Eu²⁺ and Dy³⁺, having a particle size of nearly 80 nm, and realized efficient red persistent luminescence by photo-stimulation (through 900 nm, 950 nm and 980 nm IR laser), and they suggested its potential application for optical information storage. The storage of energy by UV excitation can serve as the writing process and the release of the stored energy by photo-stimulation through near IR light excitation can serve as the reading process.⁷⁴

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

Table 1 Detail of host matrix, dopant ion(s), emission colour/wavelength, and duration of persistent luminescence of different types of red emitting persistent materials

Host (References)	Dopant ion(s)	λemission (nm)	Persistent time	Remarks
CaS (ref. 64)	Eu^2+, Tm^3+/Bi^3+	650	1 h	Effect of Na^+ co-doping
CaS (ref. 65)	Eu^2+	655/670	—	Effect of Cl^− co-doping
CaS (ref. 66)	Bi^3+	650	1 h	Effect of Cl^− co-doping
CaS (ref. 67)	Ce^3+, Pr^3+	Green/red	1 h	Effect of Li^+ co-doping
Ca1−xSrxS (ref. 68)	Eu^2+	650	1 h	White light emission
SrS (ref. 69)	Eu^2+, Dy^3+	616	—	Nano-size particle
CaS (ref. 70)	Eu^2+, Pr^3+	—	—	Effect of Li^+ co-doping
Ca1−xSrxS (ref. 71)	Eu^2+, Pr^3+	650	—	Effect of Li^+ co-doping
SrS (ref. 72)	Pr^3+	Blue/green/red	—	—
SrS (ref. 73)	Eu^2+	615	—	Lanthanide (3+) co-doping
CaS (ref. 74)	Eu^2+, Dy^3+	650	—	Nano, photostimulated
Y2O2S (ref. 75)	Eu^3+, Mg^2+, Ti^4+	611	1 h	Nanorods
Y2O2S (ref. 76)	Eu^3+, Mg^2+, Ti^4+	611	1 h	Solid state reaction
Y2O2S (ref. 77)	Ti^4+/Ti^2+	565	5 h	High temp sintering
Y2O2S (ref. 78)	Sm^3+	610	1 h	—
Y2O2S (ref. 79)	Mg^2+, Ti^4+	594, broad	—	High temperature
Gd2O2S (ref. 80)	Er^3+/Ti^4+	555, 675	1.2 h	—
MgSiO3 (ref. 81 and 82)	Mn^2+, Eu^2+, Dy^3+	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)	Mn^2+, Dy^3+	550–800	<1 h	Nanoparticle synthesis
SrMg(SiO3)2 (ref. 87)	Mn^2+, Dy^3+	500–800	<1 h	Solid state reaction
Sr3MgSi2O8 (ref. 88)	Eu^2+, Mn^2+, Dy^3+	670	2 h	Solid state reaction
BaMg2Si2O7 (ref. 89)	Mn^2+, Ce^3+	620/670	2 h	Solid phase reaction
Ca3MgSi2O8 (ref. 90)	Eu^2+, Dy^3+	Green/red	5 h	—
MgGeO3 (ref. 91 and 92)	Yb^3+/Bi2O3	600–720	—	—
M2Si5N8 (ref. 93)	Eu (M = Ca, Sr, Ba)	580–620	—	—
Ca2SnO4 (ref. 94)	Sm^3+	Orange-red	—	—
β-Zn3(PO4)2 (ref. 95)	Mn^2+	616	2 h	Role of excess Zn^2+
β-Zn3(PO4)2 (ref. 96 and 97)	Mn^2+, Al^3+, Ga^3+	570–700	—	Solid state reaction
β-Zn3(PO4)2 (ref. 98)	Sm^3+, Mn^2+	616	2 h	Solid state reaction
YPO4 (ref. 99)	Pr^3+, Ln^3+	Red	—	High temperature
Ca3(PO4)2 (ref. 100 and 101)	Mn^2+/Tb/Dy	660	—	Biocompatible host
Ca9Ln(PO4)7 (ref. 102)	Lanthanides, Mn	Red	—	—
CaO–Al2O3 (ref. 103)	Tb^3+	Green/red	—	Glass
CaTiO3 (ref. 104)	Pr^3+	612	0.1 h	—
CaTiO3 (ref. 104)	Al^3+	612	0.2 h	—
Ca2Zn4Ti16O38 (ref. 105)	Pr^3+	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)	Eu^2+, Tm^3+	610	—	Bioimaging application

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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.

4. Near infrared emitting persistent nanostructures and bioimaging applications

Bioimaging, undoubtedly, is best performed with red-NIR radiation (both as excitation and/or emission), and the reasons for this have already been discussed in previous sections. Although it is worthwhile here to mention that probe detection is governed mainly by the emission intensity of the luminescent probe, the optical path length through tissue and volumetric energy distribution, which are significantly related to the absorption and scattering properties of biological media and tissues.¹¹⁰ As a reference, the most favourable window for arteries is considered between 634 nm and 756 nm with the minimal absorption coefficient at 686 nm, whereas for veins the optimal NIR window lies between 664 nm to 932 nm with the minimal absorption coefficient at about 730 nm for imaging applications.¹¹¹

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.

4.1 Lanthanide (only) doped persistent materials and bioimaging

Lanthanides are well known for their ladder-like dense energy level structures through which they can generate emission ranging from the UV-visible to NIR regions. However, in a more specific sense, lanthanide NIR-emitting ions are usually limited to those ions, which mainly emit prominently in the NIR spectral regime (700-–2500 nm). Recent articles by Bünzli's group summarizes the most important NIR-emitting Ln(III) ions and their characteristic transitions in the NIR region.^113,114 The main lanthanides described are Pr, Nd, Sm, Dy, Ho, Er, Tm and Yb, which gives prominent NIR emission. However, when it comes to NIR persistent emission, most of the lanthanide ions, capable of generating NIR emissions, cannot be adopted. This is mainly due to the fact that the 5d state of lanthanides is too high to charge, and in addition to this, they usually posses prominent visible emissions. Thus, the key barrier that limits the development of NIR persistent phosphors using lanthanide ions is the lack of carrier traps with a suitable thermal release rate at room temperature. Recently, the concept of persistent energy transfer (PET) between two different emitting centres has been proposed to direct the design of visible/NIR persistent phosphors. In this concept, persistent emission at a shorter wavelength (say, UV/visible) by entity 1 is absorbed by entity 2, which later on may emit persistently at a longer wavelength (say, visible/NIR). Very few research articles are available that report NIR persistent luminescence from lanthanide ions, and if available, most of them are based on PET among different lanthanide ions.^115–117 However, bioimaging from any such NIR persistent phosphor is rarely reported.

Recently, Jumpei et al. reported NIR persistent luminescence due to the Nd³⁺(⁴F_3/2 → ⁴I_J/2) ion in a CaAl₂O₄ host co-doped with Eu²⁺ ion.¹¹⁵ They prepared samples with compositions Ca_0.995Eu_0.005Al₂O₄, Ca_0.99Nd_0.01Al₂O₄ and Ca_0.985Eu_0.005Nd_0.01Al₂O₄ 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

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 ⁴I_15/2 → ²H_11/2 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.

4.2 Mn²⁺ doped persistent materials and bioimaging

Mn²⁺ possess an incompletely filled d shell and its electronic configuration is d⁵ (transition metals are most stable when their d orbitals have either 5 or 10 electrons; therefore, Mn losses the 4s electrons first to form the structure Mn²⁺). Since the energy levels lie within the d shell, all transitions from the ground state to excited levels are spin and parity forbidden. Nevertheless, the selection rule is relaxed a little bit through a spin–spin interaction and a vibronic mechanism, in which the electronic transitions are coupled with vibrations of suitable symmetry.^118–122 Due to the involvement of the outermost d shells, unlike the lanthanide ions where the inner f–f transitions are involved, the transitions from transition metal ions, e.g. Mn²⁺, are affected considerably by the crystal/ligand field. The interaction between a central metal ion surrounded by anions in different possible geometries (i.e., spherical, linear, square planar, tetrahedral, or octahedral) is portrayed utilizing the crystal field theory (CFT). Complete spherical symmetry is not observed in real aspects usually. The geometry of the negatively charged point charges influences the energy level of the central metal ion. In the case of a transition metal like Mn²⁺ and Cr³⁺, the 3d orbital being the outermost shell is affected most. Specific geometry due to point charges produces a characteristic splitting pattern. The extent of the splitting in energies is represented by the splitting energy Δ. A subscript is used further to specify the geometry of the surrounding point charges such as t for tetrahedral and o for octahedral.

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

Table 2 Detail of host, co-dopant ion(s), emission wavelength, etc. of NIR persistent materials activated with Mn²⁺ ions

Host^References	Co-dopants	λemission	Remarks
Ca0.2Zn0.9Mg0.9Si2O6 (ref. 40)	Eu^2+, Dy^3+	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)	Eu^2+, Dy^3+	699 nm	Surface functionalization with methoxy-PEG of different molecular weight, cancer imaging
Ca0.2Zn0.9Mg0.9Si2O6 (ref. 124)	Eu^2+, Dy^3+	699 nm	Biotinylated PEG-PLNPs, protein binding and avidin expressing glioma cells targeting application
Ca0.2Zn0.9Mg0.9Si2O6 (ref. 125)	Eu^2+, Dy^3+	699 nm	Functionalization with Rak-2, prostate cancer cells imaging application
CaMgSi2O6 (ref. 126)	Eu^2+, Ln^3+ (Ln = Dy, Pr, Ce, Nd)	685 nm	Eu, Pr, Mn co-doping yield best results bioimaging application
SiO2/CaMgSi2O6 (ref. 127)	Eu^2+, Pr^3+	660 nm	Biocompatible, imaging application
Zn2P2O7 (ref. 128)	Tm^3+	690 nm	Blue (Tm only), red (Mn only) and NIR (Tm, Mn both) persistent luminescence
MAlO3 (ref. 129)	Mn^4+/Ge^4+ (M = La, Gd)	730 nm	Bioimaging in pork tissue

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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 (Ca_0.2Zn_0.9Mg_0.9Si₂O₆) 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.

Fig. 5 Physical characteristics of long afterglow Ca_0.2Zn_0.9Mg_0.9Si₂O₆ 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 λ_ems = 690 nm) and emission (for λ_exc = 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. Ca_0.2Zn_0.9Mg_0.9Si₂O₆ 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.

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 (V_O) 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) Ca_0.2Zn_0.9Mg_0.9Si₂O₆ (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:Eu_0.01, Pr_0.02, Mn_0.10 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, Zn_2(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 (λ_exc = 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 exp(−t/τ₁) + A₂ exp(−t/τ₂) + A₃ exp(−t/τ₃),

where I is phosphorescence intensity; A₁, A₂ and A₃ are constants; t is time; and τ₁, τ₂ and τ₃ are decay times for the exponential components, respectively. The obtained value for τ₁, τ₂ and τ₃ is 4.1 s, 25.1 s, and 135.2 s, respectively. Fig. 7(B) also shows the LLP decays as a function of the inverse of LLP intensity (I⁻¹) versus time (t), through which the authors explain the tunnelling process of electrons transferring from defects to the activator ions.

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 La₁Al_0.99O_2.985:0.1%Mn⁴⁺, 0.9%Ge⁴⁺ (LAM21) and Gd₁Al_0.99O_2.985:0.1%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.

4.3 Cr³⁺ doped persistent materials and bioimaging

The electronic configuration for Cr³⁺ is 1s²2s²2p⁶3s²3p⁶3d³. Note that the ionization of an atom expels an electron from the orbital with the highest principle quantum; thus, the Cr³⁺ ion is a d³ ion. These three electrons are distributed among the five 3d orbitals, depending upon the environment in which the ion exists. The ground state terms are as follows: ⁴F, ⁴P, ²G, ²H, ²P, and ²D. Similar to the case of Mn²⁺, many of these levels split into two or more levels, and their degeneracy is represented 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 NIR emission in Cr³⁺ is observed due to the spin-forbidden ²E → ⁴A₂ transition, or a broadband emission (650-–1600 nm) due to the spin-allowed ⁴T₂ → ⁴A₂ transition, which strongly depends on the crystal-field environment of the host lattices. Herein, the ⁴T₂ excited state strongly depends on the crystal/ligand field, e.g. in a tetrahedral symmetry, the ⁴T₂ excited state lies below the ²T₁ and ²E excited states and gives rise to a broad-band NIR emission via radiative transition to ground state ⁴A₂(F). Therefore, the NIR emission wavelength from Cr³⁺ can be controlled by modifying the ligand field strength. Further defect levels/traps plays important role to make the emission long persistence. Keeping this in mind, Cr³⁺ has been studied for NIR persistent luminescence in different crystal lattices, manly in ZnGa₂O₄ and substituted structures, to vary the crystal field strength. Table 3 lists the detail of different hosts, emission wavelengths, and duration of persistent luminescence (in h) of NIR persistent materials activated with Cr³⁺.^{41,50,130–154} On the basis of the available research articles, a detailed overview may be given in following sub-sections.

Table 3 Detail of host, emission wavelength, and duration of persistent luminescence (in h) of NIR persistent materials, etc. activated with Cr³⁺ ions

Host^References	λEmission (nm)	Time (h)	Remarks
ZnGa2O4 (ref. 130, 131 and 136)	650-–730	>1	Solid state reaction, variation of Cr^3+ 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	Pr^3+ 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 Zn^2+, 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	Eu^2+ and Dy^3+ as co-dopants

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4.3.1 Zinc-gallate (ZnGa₂O₄) nanostructures and bioimaging. Ga₂O₃ based wide band gap materials are considered to be one of the most applicable wide band gap materials, not only in its intrinsic phase but also in many other phases particularly with alkali, alkaline or with some transition metals such as lanthanides. For persistent luminescence purposes, Zn based gallates, e.g. ZnGa₂O₄, is of great interest nowadays as it exhibits different emission colours when doped with transition metal elements and is a chemically and thermally stable wide band gap semiconductor.^130–136 After the first report on NIR persistent luminescence in Cr³⁺-doped ZnGa₂O₄ by Bessière et al. in 2011, this material has been attracting the most attention, particularly for bioimaging applications.¹³⁰ The detailed study on this host is further subdivided as follows.
4.3.1.1 Structure of zinc gallate. As far as the structure is concerned, Ga₂O₃ has polymorphism in its crystal structure. The polymorph of Ga₂O₃ mainly includes α-Ga₂O₃ (R3m, a = 4.979 Å and c = 13.429 Å), β-Ga₂O₃ (C2/m, a = 12.23, b = 3.04, c = 5.80 Å), and γ-Ga₂O₃ (Fd3m, a = 8.22 Å). Of these phases, β-Ga₂O₃ is the only stable modification,^155–158 whereas zinc gallate, ZnGa₂O₄ (ZGO), is an AB₂O₄ compound with a spinel structure, in which Zn²⁺ and Ga³⁺ ions occupy tetrahedral A and octahedral B sites, respectively (Fig. 8(a)). Despite the fact that it is most generally considered as a typical spinel, it shows a slight reversal character; therefore, a few percent of Zn²⁺ and Ga³⁺ occupy B and A sites, respectively. Such defects in the host matrix are called antisite defects, and thus a Zn ion goes at a Ga site and a Ga ion goes at a Zn site (noted as ZnGa'and GaZn° in the Kröger–Vink notation, respectively) in an ideal normal spinel structure type. ZGO displays about 3% inversion, which indicates that 3% of Zn²⁺ involve Ga destinations, and correlatively the same measure of Ga³⁺ possess Zn sites.^132,159,160

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.

4.3.1.2 Photoluminescence excitation, emission and persistent luminescence. The photoluminescence excitation spectrum of Cr³⁺ doped samples (with its well known emission at around 700 nm) shows four important regions that all together cover the UV and visible spectral regime. The first broad excitation band is observed below 250 nm, due to the O²⁻–Ga³⁺ charge transfer band. The second excitation band arises due to the band-to-band transition at around 290 nm. The rest of the bands, in the visible region (425-–570 nm), appears due to the d–d transitions of the Cr³⁺ ion.^130–136 Bessière et al. studied emission by exciting these three distinct excitation bands, as seen in Fig. 8(b).¹³² They observed that the ²E(2G) → ⁴A₂(4F) transition of Cr³⁺ in an octahedral crystal field is composed of a large number of peaks. It has been explained due to two different sites for Cr³⁺ ions in this crystal structure: (i) Cr³⁺ in unperturbed octahedral sites (denoted as CrR), and (ii) Cr³⁺ ions with a neighbouring antisite defect (denoted as CrN2). The peak corresponding to zero phonon R lines (for CrR) is observed at around 688 nm, while the peak corresponding to zero phonon N2 lines (for CrN2) is observed at around 695 nm. Stokes and anti-Stokes phonon side bands (PSB) associated with zero phonon lines have also been reported. Sharp peaks at 708 and 715 nm (Stokes) and 663, 670, and 680 nm (anti-Stokes) have been reported for R lines. PSB for N2 lines are observed in the 650-–750 nm regime; however, it appears with a relatively weak intensity. These zero phonon lines and associated PSB give rise to a broad band from 650 nm to 750 nm composed of a large number of sharp peaks. Cr³⁺ ions with a neighbouring antisite defect (CrN2) play an important role for persistent luminescence. Fig. 8(b) shows the persistent luminescence spectrum of the Cr³⁺ doped zinc gallate after few seconds of stoppage of excitation at 290 nm for 15 minutes. The spectrum is dominated by an N2 line. The decay curves after 290 nm and 550 nm excitation are also shown in Fig. 8(c).
4.3.1.3 Mechanism involved in persistent luminescence. If one looks towards the mechanism involved for the persistent luminescence of Cr³⁺ in zinc gallate, several models have been proposed by different groups and it seems difficult enough to get a conclusive remark. Recently, Zhuang et al. discussed this issue in his paper very clearly.¹³³ They remarked that, there are two major controversial points in proposed models to date: (i) species of charge carriers (whether a negative electron or a positive hole), and (ii) path of the trapping process (whether through the conduction band or through the valence band or by a quantum tunnelling effect in the band-gap). They simplify it by an energy level diagram, shown in Fig. 8(d). According to this energy level, there are three ways by which electrons can be excited to the conduction band (CB): (i) from the ground state (GS) to the excited state (ES) situated above the bottom of the CB; (ii) directly from the GS to the CB; and (iii) from the GS to the ES situated below the bottom of the CB. In the third case, the electron can reach the CB with thermal energy, which is called a thermally assisted photoionization process. The population from the CB is further moved to the trap centre. This trapping mechanism has been mostly adopted by many authors. However, quantum tunnelling (QT), which is another possible path for electron trapping, cannot be completely ignored. These trap charges give rise to the persistent luminescence after the removal of the excitation source either through thermal excitation (possible even at room temperature) and/or by photo-stimulation with a suitable frequency of light.

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

4.3.1.4 Nanoparticles, surface functionalization and bioimaging. Even after the first report of persistent luminescence in Cr³⁺ doped ZnGa₂O₄, in 2011, the bioimaging application of this material only first appeared in 2014. This was due to the fact that, the substituted structure of zinc gallate, e.g. zinc gallogermanate, was found to be more efficient in terms of both persistent luminescence intensity and persistent time; thus, it drew more attention and was used successfully for imaging applications much earlier, as discussed in detail in Section 4.3.2. However, the group, which first reported the NIR persistent luminescence by Mn²⁺ doped phosphors for imaging applications, made the first use of zinc gallate phosphors for imaging applications, and found it suitable for the optical imaging of vascularization, tumours and grafted cells for the first time.¹³⁴

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.

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.

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)).

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).

4.3.2 Ge/Sn substituted zinc-gallate (ZnGa₂O₄) nanostructures and bioimaging. Cr³⁺ doped ZnGa₂O₄ was found to be an excellent NIR emitting material, particularly a probe for bioimaging applications. The persistent time was found to be a few hours only, but photostimulation by LEDs for a small period may recover the persistent emission and can complement the process for long period imaging applications also. However, at the same time, it strained researchers to improve the afterglow time so as to exploit their wide applications, and thus Ge/Sn/Al substituted zinc gallates were developed further.^{41,50,137–140} Pan et.al. (in 2012) came up with an innovative composition, which holds persistence time >360 h.¹³⁷ Herein, persistence time refers to the duration for which the eye can see with the aid of a night-vision monocular in a dark room. In their new composition, they substituted Ga for Ge and suggested the general chemical formula Zn_xGa_yGe_zO(x + (3y/2) + 2z):tCr³⁺, mR, where R is a co-dopant selected from a group consisting of alkaline earth ions, lanthanide ions and Li⁺ ions; x, y and z are integers from 1 to 5; t is 0.01–5 mol%; and m is 0–5 mol% of the Cr³⁺-doped zinc gallogermanates. Detailed excitation, emission, decay curve and thermoluminescence measurements were carried out to explore the different properties. Fig. 12 shows the persistent decay curves and persistent luminescence spectra (a) along with beautiful digital images of the persistent emission at different time intervals (b–h) for the Zn₃Ga₂Ge₂O₁₀:0.5%Cr³⁺ phosphor. Although the persistent emission is weak, it is clearly visible even upto 360 h, after which the initial intensity can be recovered by heating, i.e. by thermal stimulation, (Fig. 12(i)). The other beauty of this material is its activation by sunlight in different outdoor conditions such as different weather conditions (sunny, cloudy, overcast and rainy days), at different moments between sunrise and sunset and at various outdoor locations (for example, in open areas and in the shadows of trees and buildings). Moreover, the persistent luminescence was retained even in aqueous solutions, including tap water, salt (NaCl) water and NaCl/bleach/bicarbonate (NaHCO₃). The authors suggested different potential applications of this material such as in night surveillance and photovoltaics, including in vivo imaging. Bioimaging applications of this material were further realized by different researchers in subsequent years.^41,50,139

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 (Zn₃Ga_1.96Ge₂O₁₀:Cr_0.01Pr_0.03), 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).

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 α_vβ₃ 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.

4.3.3 Lithium gallate (LiGa₅O₈) nanostructures and bioimaging. Another material of interest, i.e. Cr³⁺-doped LiGa₅O₈, in this continuation, bearing excellent NIR persistent luminescence (persistent time more than 1000 h) under visible light or a NIR light stimulation, was reported by Liu et al.¹⁴¹ They pointed out different promising applications, namely, optical information storage and night-vision surveillance, including the in vivo bioimaging of this material. To demonstrate the potential of the material for in vivo bioimaging, NPs (particle diameter of the order of 50-–150 nm) were prepared by using the sol–gel method followed by high-temperature calcination, and further surface functionalization of these NPs was performed by using polyethylenimine (PEI). In the next step, these functionalized NPs were used to label 4T1 murine breast cancer cells. This system was charged with a 254 nm UV lamp and then injected subcutaneously into the back of a nude mouse. Persistent luminescence was monitored at different time intervals with and without photostimulation. Persistent luminescence was clearly visible upto 4 h after the injection (Fig. 14(a)), and then after it no longer remains visible. However, stimulation by a white LED flashlight recovers/rejuvenates the NIR persistent luminescence. Fig. 14(a1–e1) and (a2–e2) show the images of persistent luminescence after stimulating the sample by a white LED for 15 s, 10 s, and 5 min after the stimulation. It is obvious that the resulting persistent luminescence signals retain sufficient intensity for more than 5 min (Fig. 14a2–e2). Thus, by repeated stimulations, the authors were able to continue imaging, possible even up to 10 days after the injection. Some other authors have also worked on similar host compositions and found them suitable for bioimaging applications.^142,143 Abdukayum et al. compared this material with zinc gallogermanate codoped with Cr and Pr, and they found that the latter one is more suitable in many ways for imaging applications.¹³⁸

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).

4.3.4 Miscellaneous hosts. In spite of lithium gallate, zinc gallate and its substituted structures, several other host matrices doped with Cr³⁺ ions were also developed for realising NIR persistent luminescence. A few important materials, such as gallium oxide,^144,145 alkaline gallates,^146–148 lanthanide garnets,^149–151 lanthanum gallogermanates,^152,153 and aluminates,¹⁵⁴ give NIR persistent emission; however, as far as the imaging application from them is concerned, it is still not explored. Lu et al. reported NIR persistent emission from hydrothermally synthesized β-Ga₂O₃:Cr³⁺ nanowire assemblies and speculated its potential applications as identification taggants in security and optical probes in bioimaging.¹⁴⁴ On the other hand, Wang et al. showed the photocatalytic properties from a Ga₂O₃:Cr_0.01³⁺, Zn_0.005²⁺ persistent phosphor.¹⁴⁵ Magnesium and strontium gallates have been claimed as new phosphors for in vivo imaging, but not implemented thus far.^146–148 Similar is the case with other studies, which also focus on structural and optical characteristics, and on the basis of these results, speculate their possible application in in vivo imaging. It would be exciting to see a novel composition in this line with better performances than the existing materials, which are usable for bioimaging applications.

5. NIR persistent phosphors and multimodal imaging

Every specific imaging technique has its own particular peculiarity of benefits and certain constraints too; thus, more than one imaging strategy (also called multimodal imaging) is utilized sooner or later for more precise, complete, and dependable data on a diagnosis. In this context, Abdukayum et al. put forward the idea to develop NIR PLNPs for multimodal imaging, both exhibiting NIR fluorescence imaging and magnetic resonance imaging.⁵¹ The NIR fluorescence imaging is excellent for high sensitivity, while its limitation towards poor spatial resolution can be complemented with MRI.^161,162 To realise this concept, zinc gallogermanates (Zn_2.94Ga_1.96Ge₂O₁₀) doped with Cr³⁺ and Pr³⁺ was developed as NIR PLNPs and further it was chelated with Gd ions to obtain Gd(III)-PLNPs. The material was found safe under in vivo and in vitro toxicity tests as well.

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.

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.

6. Summary and outlook

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 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, its development, present status of the field, literary review of the existing red and NIR emitting persistent phosphors. The significant findings on NIR persistent luminescence and its application for bioimaging are based on transition metals, primarily either on Mn²⁺ and/or on Cr³⁺ as one of the dopant/co-dopant ion. Other than these two fundamental classes of activators, barely any other ions have been found to exhibit persistent luminescence in the NIR region, which is usable for bioimaging application.

Mn²⁺ doped silicates (Ca_0.2Zn_0.9Mg_0.9Si₂O₃: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.

Acknowledgements

The author gratefully acknowledges the financial support from Department of Science and Technology (DST), New Delhi, India, through its DST-INSPIRE Faculty award (IFA-12 PH-21) program.

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