Red and NIR light could have much deeper tissue penetration depth compared with UV and other visible light (e.g., blue and green light). Typically, UCL of UCNP is known to have multiple emission bands with various optical intensities after 980 nm excitation. For example, the emissions of NaYF₄:Er/Yb/Tm nanoparticles include UV (330–350 nm, 350–370 nm), blue (390–420 nm, 440–460 nm, 460–500 nm), green (510–530 nm, 530–570 nm), red (630–660 nm), and NIR (770–810 nm) bands [17]. Recently, there has been an increasing focus on the design and synthesis of UCNPs with tunable UC emission from UV to near-infrared (NIR) through various doping strategies or by introducing well-designed core@shell nanostructures [40, 43]. In particular, engineering of UCNPs with excellent NIR emission (λ = ~800 nm) or single-band red emission (λ = ~650 nm) could result in ideal optical biolabels for future deep tissue imaging and has become a new attractive topic currently [44].

The mostly used UCNP for achieving a NIR-to-NIR in vivo imaging is Tm³⁺ and Yb³⁺ co-doped β-NaYF₄, which, after 980 nm excitation, could give out strong NIR emission located at 800 nm, ideally for deep tissue imaging [8, 40]. Several interesting methods have been developed to improve the NIR emission intensity of NaYF₄:Tm/Yb nanoparticles.

Increasing the concentration of Tm³⁺ ions in the host lattice is one of the simplest techniques to achieve the goal. Wang et al. demonstrated that NaYF₄:Tm/Yb nanoparticles with Tm³⁺ doping fixed at 0.2 mol % could only have a weak emission at 800 nm, and increasing the Tm³⁺ concentration up to 2 mol % could result in a significant enhancement thanks to the enhanced ³H₄ level [45]. By further replacing all Y³⁺ in NaYF₄:Tm/Yb to Yb³⁺ ions, Chen et al. have developed ultrasmall-sized (7–10 nm) NaYbF₄:Tm (2 mol %) nanoparticles with 3.6-fold enhancement in total upconversion emission intensity in comparison with that of 25 nm-sized NaYF₄:Tm/Yb (2/20 mol %) [46]. Such enhancement has been suggested to arise from the increased absorptions induced by the Yb³⁺ ions and the increased efficiency of energy transfer between Yb³⁺ and Tm³⁺ ions. In a followed-up study, Liu et al. reported a lutetium (Lu)-based UCNP with excellent NIR emission [29]. As-synthesized sub-10 nm-sized β-NaLuF₄:Gd/Yb/Tm (24/20/1 mol %) displayed bright UCL with high QY estimated to be 0.47 ± 0.06 %. In comparison with 20 nm-sized NaYF₄:Tm/Yb (1/20 mol %), approximately 11-fold enhancement in NIR emission could be achieved. The successful suppression of nonradiative process has been suggested as the main reason behind such enhancement.

Epitaxial growth of an inert protective shell over pre-prepared NaYF₄:Tm/Yb core has been accepted as another useful strategy for minimizing the surface defects for preserving the optical integrity of UCNPs [30, 31]. Although cation-exchange and Ostwald ripening methods have been developed as alternative options recently [47, 48], seed-mediated growth still remains the most adopted technique for synthesis of such core@multi-shell structured UCNP [23]. By coating α-NaYbF₄:Tm (0.5 mol %) with a well-selected CaF₂ protective shell, Chen et al. have developed a novel and biocompatible α-NaYbF₄:Tm (0.5 mol %)/Yb@CaF₂ with highly efficient NIR-to-NIR upconversion property (Fig. 22.3a) [49]. CaF₂ was chosen as the epitaxial shell due to its low lattice mismatch with α-NaYbF₄:Tm, good optical transparency, high crystallizability, stability, and biocompatibility. As-design UCNP exhibited an impressive high QY of 0.6 ± 0.1 % under low power density excitation (0.3 W/cm²). Thanks to the suppressing of surface quenching effects via hetero-epitaxial growth of CaF₂ shell, an approximately 35-fold enhancement in UCL intensity could be achieved in comparison to the noncoated α-NaYbF₄:Tm (Fig. 22.3b). The authors also demonstrated the possibility of imaging deep tissue by using α-NaYbF₄:Tm(0.5 mol %)/Yb@CaF₂ nanoparticles. Clear NIR signal could be detected even through 3.2 cm pork tissue (Fig. 22.3c).

UCNP with strong single-band red emission has been emerged as another interesting imaging probe for in vivo imaging. Recently, studies on Yb³⁺/Er³⁺ co-doped MnF₂ and KMnF₃ nanoparticles have demonstrated a substantially enhanced red-to-green (R/G) emission ratio owing to the energy transfer between the Er³⁺ and Mn²⁺ [50, 51]. Inspired by these, Wang et al. have developed cube-shaped KMnF₃:Yb/Er (18/2 mol %) with extremely high red emission and completely disappeared blue and green emissions (Fig. 22.4a, b) [44]. Such single-band upconversion emission has been ascribed to nonradiative energy transfer from the ¹H_9/2 and ⁴S_3/2 levels of Er³⁺ to the ⁴T₁ level of Mn²⁺, followed by back-energy transfer to the ⁴F_9/2 lever of Er³⁺. Pure NIR emission of KMnF₃:Yb/Tm (18/2 mol %) could also be achieved by applying the same idea (Fig. 22.4c). In a followed-up study, Tian et al. presented a hydrothermal synthesis of Mn²⁺-doped NaYF₄:Yb/Er nanoparticles with a strong single-band red emission [42]. Surprisingly, as-designed nanoparticles could have a R/G ratio of 163.78 after increasing the doping level of Mn²⁺ up to 30 mol %. The doping of Mn²⁺ also resulted in significant enhancement (about 15 times) in overall optical intensity in comparison with Mn-free sample (Fig. 22.4d). As a proof-of-concept experiment, the author injected water-soluble PEGylated NaYF₄:Yb/Er/Mn at the foot, back, and upper leg regions of white Kunming mice and demonstrated a clear tissue penetration and high-contrast photoluminescent imaging of the red emission (Fig. 22.4e).

The engineering of UCNP nanostructures for excellent NIR- and red-band emissions by doping with suitable amount of Mn²⁺ ions will continue to attract increasing interests. More importantly, selectively doping UCNP with other lanthanide ions, such as Gd³⁺, Lu³⁺, Yb³⁺, and ¹⁵³Sm³⁺ might endow UCNP with useful imaging modalities, such as magnetic resonance imaging (MRI), CT, and positron emission tomography (PET).

Although combination of super paramagnatic iron oxide nanoparticles (SPIONs) with UCNP could result in bimodal imaging probe with UCL and MR modalities [39, 56], doping paramagnetic Gd³⁺ ions (or Mn²⁺) in UCNP crystal lattice should represent a better strategy for introducing MR contrast imaging capability without sacrificing the UCL intensities [11, 12]. Kumar et al. reported the first such example by doping Gd³⁺ ions into NaYF₄ and demonstrated the combination of UCL and MR bimodal imaging in one single nanocrystal [12]. The first in vivo demonstration of such bimodal UCL/MRI imaging was later achieved using hexagonal-phase carboxylic acid-functionalized NaGdF₄:Tm³⁺/Er³⁺/Yb³⁺ nanoparticles [13]. However, because the majority of the Gd³⁺ ions are doped into the rigid crystal lattice and have limited chance to alter the relaxation time of surrounding water protons, only low MR sensitivity could be achieved in most of the previous reports [7, 12, 57]. The nanostructure re-engineering and relaxivity mechanism probing of the Gd³⁺-doped UCNP might hold a great chance to enhance its MR sensitivity.

Recently, we have designed three different types of Gd³⁺-doped UCNPs with various Gd³⁺ ion locations and surface densities to probe the roles of surface and bulk Gd³⁺ ions on shortening the T ₁-relaxation time of surrounding water molecules [11]. Our systematic research have demonstrated that it is only the surface Gd³⁺ ions in Gd³⁺-doped UCNP are responsible for shortening the T ₁-relaxation time of water protons, and bulk Gd³⁺ ions could have little impact even with high concentration (Fig. 22.5). A remarkable increase in r ₁-relaxivity (up to 6.18 mM⁻¹s⁻¹/Gd³⁺) has been achieved by further decreasing the NaGdF₄ shell thickness down to <1 nm [11]. By using ultrasmall NaGdF₄ nanocrystals as models, Johnson et al. have shown the similar observation of increased r ₁-relaxivity per Gd³⁺ ion (from 3.0 to 7.2 mM⁻¹s⁻¹) with decreased NaGdF₄ particle size (from 8 to 2.5 nm), again demonstrating the role of surface Gd³⁺ ions in relaxivity enhancement [58].

In an attempt to understand the relaxivity mechanism of Gd³⁺-doped UCNP, we further introduced mesoporous and dense silica coatings for systematically investigating its possible relaxivity mechanisms [59]. A surface-dependent relaxivity mechanism has been proposed based on our results. Specifically, we showed a co-existing of inner- and outer-sphere r ₁-relaxivity mechanism in ligand-free Gd³⁺-doped UCNP, while only main contribution of outer-sphere mechanism in silica-shielded probes. The origin of r ₂-relaxivity is inferred mainly from an out-sphere mechanism with the r ₂ value being surface state related. Also, key factors for tuning r ₂/r ₁ ratio have been found to be highly dependent on the thickness of NaGdF₄ shell and surface modification strategy. Based on these, we showed a 105-fold enhancement in r ₁-relaxivity (14.73 mM⁻¹s⁻¹/Gd³⁺) in comparison with the first-reported same value (0.14 mM⁻¹s⁻¹/Gd³⁺). These new findings provide not only deeper insight into the origin of contrast enhancement of water-soluble Gd³⁺-doped UCNP, but also useful strategy for achieving both high MR sensitivity for future biological imaging applications.

With the presence of heavy metal ions like Gd³⁺, Yb³⁺, and Lu³⁺ in NaYF₄ matrix, lanthanide ions-doped UCNPs also have been considered as an inherent CT contrast agent recently [14–16, 60]. Ultrasmall NaGdF₄:Er/Yb nanocrystals have firstly been developed via an interesting two-phase system and been demonstrated as UCL/MR/CT triple-modal imaging contrast agent [14]. In a followed-up study, Liu et al. reported a Yb-based NaYbF₄:Er/Gd nanoparticles with improved CT contrast capabilities [15]. The doping of Gd³⁺ ions has been demonstrated vital to control both the morphology and R/G ratio of NaYbF₄:Er/Gd nanoparticles (Fig. 22.6a, b). As-synthesized water-soluble polyethylene glycol (PEG)-modified NaYbF₄:Er/Gd nanoparticles have been found to be the best CT contrast agents among iobitridol, Au-, Pt-, Bi-, and Ta-based nanoparticulate agents under the same testing conditions. In vivo imaging study demonstrated the capability of PEG-modified NaYbF₄:Er/Gd nanoparticles as an excellent CT contrast agent (Fig. 22.6c, d). Water-soluble NaLuF₄:Tm/Yb nanoparticles might represent the third type of inherent CT agents. After coating with iron oxide shell, or modified with Gd³⁺-complex, such modified NaLuF₄:Tm/Yb nanoparticles could also be probes with well-combined UCL/MR/CT triple modalities [16, 61, 62]. Although other matrixes, such as BaYbF₅ and BaGdF₅ [63, 64], have also been reported as inherent CT contrast agents, they might not be a good choice for efficient upconversion optical imaging.

Another way for adding a CT contrast capability is by decorating Gd³⁺ ions-doped UCNP with CT contrast nanoparticles, such as tantalum oxide (TaO_x) and ultrasmall gold nanoparticles (Fig. 22.7) [60, 65]. Starting from NaYF₄:Yb/Er/Tm@NaGdF₄ nanocrystals, we recently demonstrated an efficiently way to add CT imaging modality by decorating with a fluorescence-transparent TaO_x shell [65]. As-design water-soluble litchi-shaped trimodal imaging probe showed an extraordinary high r ₁ (11.45 mM⁻¹s⁻¹) and r ₂ (147.3 mM⁻¹s⁻¹) values thanks to the ultrathin NaGdF₄ shell. An obvious CT contrast enhancement could be obtained by the combined effect of radiopaque TaO_x shell and UCNP itself. Both the in vitro and in vivo trimodal imaging have been demonstrated, providing us a new possibility for potential early cancer detection.

Suitable surface engineering and isotopes labeling of UCNP could make it also a PET/SPECT (i.e. single-photon emission computed tomography) tracer. To date, research on synthesis and application of radioactive UCNP (r-UCNP) is still in its infancy. Two techniques have been developed for the synthesis of radioactive UCNP. They are surface ion reaction (or exchange) method and direct isotope(s) doping strategy.