Bone analysis using an aggregation‐induced emission‐active iridium complex

Fluorescent analysis of bone provides valuable insights into bone structures. However, conventional dyes suffer from low specificity on bone tissue, small stokes shift, short fluorescent lifetime, and aggregation‐caused quenching effect, which result in low efficacy and artifacts. In this work, we design an aggregation‐induced emission (AIE)‐active iridium(III) complex (Ir‐BP2) as a highly selective, convenient, nondestructiveness, and dual‐mode staining agent for bone analysis. Ir‐BP2 containing phosphonate groups selectively binds to hydroxyapatites, the main component of bone matrix, and exhibits turn‐on AIE phosphorescence with prolonged lifetime. Ir‐BP2 exhibits promising biosafety and offers higher accuracy in staining calcium deposits than conventional Alizarin Red S staining assay when it is employed in real‐time monitoring of osteogenesis differentiation process. A ready‐to‐use staining spray of Ir‐BP2 is fabricated. By using fluorescent imaging and lifetime imaging, Ir‐BP2 staining provides valuable insights into bone microstructure analysis, microdamage diagnosis, and bone growth state identification. Further, Ir‐BP2 is successfully applied on a human spine vertebra for diagnosing bone invasiveness of eosinophilic granuloma, validating its clinical practice. This work presents a powerful tool in bone analysis and will lead to new approaches for the diagnosis and treatment of bone‐related diseases.

and osteogenesis imperfecta.It has become a meaningful technique used in a wide range of research applications, including bone biology, [1,2] tissue engineering, [3] disease theragnostic, [4] and forensic science. [5]istological analysis, one of the most common methods for bone analysis, usually involves the sectioning of bone tissues and specialized sample processing, which are S C H E M E 1 (A) Chemical structures of phosphonate-containing iridium(III) complexes Ir-BP1 and Ir-BP1.(B) Schematic illustration of Ir-BP1 and Ir-BP2 as aggregation-induced emission (AIE)-active probes for bone analysis using an easy "spray-and-capture" procedure.The immobilization of Ir(III) complexes on the hydroxyapatite surface through phosphonate-Ca 2+ chelation is responsible for the AIE activation.destructive, costly, and time-consuming.Moreover, they are performed postmortem, excluding their practical usage in real-time and longitudinal monitoring, such as monitoring the bone tissue engineering process.Imaging-based bone analysis such as computed tomography (CT) [6,7] and magnetic resonance imaging (MRI) [8] are costly.Therefore, a convenient, nondestructive, and low-cost technique for bone analysis is urgently needed.
Fluorescent analysis possesses high spatiotemporal resolution, nondestructiveness, and ease of operation, and has gained increasing interest in bone analysis.[20][21][22][23] Despite their promising sensing properties, these fluorescent probes had small stokes shifts and short fluorescent lifetime, which posed challenges to distinguishing interfering signals from autofluorescence of bone tissues and nonemissive scattering signals.Aggregation-caused quenching effect of these conventional fluorescent dyes reduced their fluorescence efficacy when attached to a solid bone matrix, which also limited their uses in related applications.
Iridium(III) complexes with AIE features arose to meet these challenges. [24,25][28][29] Long phosphorescent lifetime of Ir(III) complexes offers an additional opportunity to analyze biological fluctuation using photoluminescent lifetime imaging microscope (PLIM). [30]ollowing the general "restriction of intramolecular motion" rule [31] of constructing AIE molecules; however, most AIEactive Ir(III) complexes had extended aromatic ligands with rotatable rings, [24] which were typically hydrophobic with limited water solubility.Consequently, they were prone to aggregate in an aqueous solution, displaying an "always-on" phosphorescent state.
To overcome these limitations, herein we developed two water-soluble Ir(III) complexes with AIE features, Ir-BP1 and Ir-BP2 (Scheme 1).Both Ir-BP1 and Ir-BP2 contained phosphonate groups as the bone binding moiety, which also endowed them with high water solubility.They were soluble in aqueous solution, showing weak emission.When bound to the bone surface, they were immobilized through phosphonate-Ca 2+ chelation and their molecular skeletons were fixed, activating their AIE characteristics for selectively lighting up bone.The unique turn-on mechanism gifted them high phosphorescent selectivity toward hydroxyapatite (HAp) particles.Taking advantage of the excellent properties of Ir(III)-complexes, we applied Ir-BP2 as a representative for monitoring osteogenesis differentiation and analyzing ex vivo bone tissues.

Molecular design
Phosphonate group has a high affinity to HAp due to phosphonate-Ca 2+ chelation. [17]Moreover, phosphonate group is highly hydrophilic because of its polar structure and negative charge under physiological pH, endowing phosphonate-containing compounds with high water solubility.In this work, we designed phosphonate-containing Ir(III) complexes.Aminopropyl phosphonate was incorporated on both para positions of 2,2′-bipyridine through amide bonds, which were then coordinated with iridium precursors to form the phosphonate-containing complexes, Ir-BP1 and Ir-BP2 (Scheme 1A, Figures S1-S9).It was also reported that Ir(III) complexes bearing para-ester bonds showed AIE properties, probably due to twisting and rotatable molecular skeleton. [32,33]Once bound to HAp through phosphonate-Ca 2+ chelation, their molecular skeletons were fixed, activating their AIE properties (Scheme 1B).In this regard, we hypothesized that Ir-BP1 and Ir-BP2 could serve as AIE-active probes with turn-on phosphorescence for bone analysis.
From the density functional theory calculation [34] , the HOMO orbitals dominatingly lay on the coordinated bipyridinyl ligands with a minor contribution on the Ir(III) metal center and the amide bonds in both cases of Ir-BP1 and Ir-BP2 (Figure S12-S14).The LUMO orbitals mainly located on the Ir(III) center and ancillary ligands.The distribution of the excited state locating on the amide bonds significantly increased, as observed in the LUMO+1 orbital for Ir-BP1 (Figure S13) and in the LUMO+2 orbital for Ir-BP2 (Figure S14), respectively.These excited states undergo fast vibrational relaxation or internal conversion (IC) that usually is nonradiative processes [35] .Therefore, high occupation of the excited state on the amide bonds hampered the phosphorescence of Ir(III) complexes.
Indeed, Ir-BP1 and Ir-BP2 were both weakly emissive in the aqueous solution with quantum yield (ϕ) of 0.69% for Ir-BP1 and 0.41% for Ir-BP2 (Table S1), respectively.Upon the addition of tetrahydrofuran (THF) as a poor solvent, however, their emission intensities gradually increased.In a mixed solvent of 98:2 THF/H 2 O (Vol%), Ir-BP1 emitted bright red emission with a 56-fold enhancement in emission intensity (ϕ = 15.3%); while Ir-BP2 showed a remarkable 73-fold emission enhancement (ϕ = 12.2%) and emitted orange emission under a ultraviolet (UV) light irradiation.(Figure 1B,C).Note that slight blue-shit of emission peaks was observed, and Ir-BP2 also displayed a shoulder peak at 560 nm.This suggested the H-type aggregation [36] of Ir-BP2 during THF addition.The phosphorescent lifetime also increased at the aggregated state (Table S1 and Figure S15 and S16).Ir-BP1 exhibited an average phosphorescent lifetime of 199 ns in 98% THF compared with 58.5 ns in 100% H 2 O; and Ir-BP2 exhibited a remarkable prolongation of phosphorescent lifetime increasing from 15.9 ns in 100% H 2 O to 407 ns in 98% THF, respectively.Dynamic light scattering data (Figure 1D) further revealed that in 98% THF they formed nanosized aggregates with hydrated diameters of 123.9 nm for Ir-BP1 and 143.4 nm for Ir-BP2, respectively.These results suggested both Ir-BP1 and Ir-BP2 featured AIE properties.

HAp-sensing behaviors
HAp particles were used as the mimic of bone matrix to test whether it could immobilize Ir-BP1 or Ir-BP2 and activate their AIE properties.As shown in Figure 1E, Ir-BP1 only showed a two-fold increase in emission intensity in the presence of HAp.In comparison, Ir-BP2 showed a relatively high signal-to-noise response to HAp particles with an eight-fold emission enhancement.Emission spectral titration results (Figure 1F) demonstrated that upon the addition of HAp particles, Ir-BP2 displayed gradually increasing emission.When the amount of HAp particles was higher than 120 μg/mL; however, their emission spectra became fluctuated.A similar phenomenon was also observed in Ir-BP1 (Figure S17).This is probably due to HAp particles in high content were prone to precipitate, leading to uneven distribution in the solution.The fluorescence quantum yield of Ir-BP1/HAp and Ir-BP2/HAp were measured as 2.96% and 2.28% (Table S1), respectively.The phosphorescent lifetime of Ir-BP2 also increased to 66.2 ns when bound to HAp (Figure S16).In contrast, HAp-bound Ir-BP1 showed an unexpectedly lower lifetime of 44.8 ns than nonbound Ir-BP1 (Figure S15).Interestingly, both Ir-BP1 and Ir-BP2 could not respond to free Ca 2+ ion (Figure 1G).It is reasoned to speculate that Ca 2+ ion also engages in Ca 2+ -phosphate ionic bonding with Ir-BP2 because phosphonate group has high affinity to Ca 2+ .However, this interaction does not trigger the precipitation or aggregation of Ir-BP2, exhibiting little fluorescent response.Anchoring on a solid calcified matrix such as HAp particles and bone matrix suppresses the skeleton rotation of amide bonds in the complexes, resulting in the recovery of AIE features.Other interference species including amino acids (Arg, Asp, Cys, Gly, Glu, His, Lys, Phe, Tyr, or Thr), metal ions (Cr 3+ , Cu 2+ , Fe 2+ , K + , Li + , Mn 2+ , Na + , Pb 2+ or Zn 2+ ), biomolecules, or biomacromolecules (bovine serum albumin BSA, protamine, gelatin, DNA, RNA, glucose, or sucrose) were also tested (Figure 1G and Figure S18 and S19).The results suggested both Ir-BP1 and Ir-BP2 showed neglectable responses toward these interference species, revealing high selectivity toward HAp particles.
Considering bone tissue exhibits an acidic microenvironment and elevated alkaline phosphatase (ALP) activity during bone remodeling [37] , we also tested the HAp-sensing behaviors of Ir-BP1 and Ir-BP2 under such conditions.The emission intensities of HAp-bound Ir-BP1 were stable at pH values ranging from 4.0 to 8.0 (Figure S20).When the pH value was higher than 9.0, however, the emission intensity decreased, probably because the base facilitated Ir-BP1 dissolving and suppressed its AIE phosphorescence.In comparison, Ir-BP2 showed relatively stable emission after binding to HAp at a wide pH range varying from 4.0 to 10.0 (Figure S21).Both Ir-BP1 and Ir-BP2 demonstrated a notable fluorescence response upon interaction with HAp, and this response remained unaltered in the presence of ALP (Figure S22 and S23).These results suggested the high practicability of Ir-BP1 and Ir-BP2 for bone analysis.Therefore, we could briefly conclude that both complexes exhibited selective turn-on phosphorescence toward HAp particles, and Ir-BP2 had a higher signal-to-noise sensing property.This was probably because Ir-BP1 possessing a lower logP value was more soluble in water than Ir-BP2, suppressing its anchoring on HAp surface.

HAp-binding mechanism
We took Ir-BP2 as the representative to study its HAp-binding mechanism.Zeta-potentials were examined (Figure 2A).Ir-BP2 had a negative charge with a surface potential of −38.8 mV.HAp particles also exhibited a negative potential of −14.6 mV.Ir-BP2 binding significantly reduced its surface potential to −35.8 mV, indicating the successful immobilization of Ir-BP2 to the HAp surface.
To confirm, HAp particles were added to the solution of Ir-BP2 in water and centrifuged to remove the nonbound complex.The pellet was subsequently lyophilized for spectral and microscopic analysis.Fourier Transform infrared (FT-IR) spectrum (Figure S24) of these lyophilized particles showed C=O stretching vibrations at 1666 cm −1 , C=N stretching vibrations at 1548 cm −1 , and C-H bending vibration at 765 cm −1 that were characteristic vibration peaks of Ir-BP2, supporting the successful attachment of Ir-BP2 on HAp particles.Due to the strong vibration peaks of phosphate of HAp, however, we could not directly identify the binding of phosphonate-Ca 2+ through FT-IR spectra.X-ray photoelectron spectroscopy (XPS) was further conducted.XPS spectrum (Figure 2B) showed evident binding energies at 65 eV contributed to Ir4f and 400 eV contributed to N1s, which further confirmed the surface immobilization of Ir-BP2 to HAp particles.The atomic ratio of Ir/Ca was calculated as 1/132.5 (Table S2).Given that HAp has a typical formula of Ca 10 (PO 4 ) 6 (OH) 2 , the immobilization efficacy of Ir-BP2 on HAp was approximately estimated as 7.5%.Compared to nonbound Ir-BP2, HAp-bound Ir-BP2 exhibited stronger binding energy of Ir4f with a 0.4 eV increase (Figure 2C).Meanwhile, HAp exhibited a lower binding energy of Ca2P after Ir-BP2 was attached to the surface (Figure 2D).
To visualize the surface attachment of Ir-BP2 on HAp particles, transmission electron microscope (TEM) imaging and elemental mapping experiments using energy dispersive X-ray spectroscopy (EDX) was carried out.As shown in Figure 2E, TEM images of HAp crystal nanoclusters were captured and showed rich Ca, O, and P distribution abundance.Importantly, N and Ir elements as characteristics of Ir-BP2 mapping on HAp nanocluster were also observed.EDX spectrum (Figure S25) also supported the existence of N and Ir elements on these HAp particles.These results confirmed the immobilization of Ir-BP2 on the HAp surface, contributing to the activation of its AIE properties.

Monitoring osteogenesis differentiation
We next evaluated the capacity of Ir-BP2 for HAp imaging.As shown in Figure 3A, after being stained with Ir-BP2 for 10 min, HAp particles emitted bright fluorescence signal, which could be imaged under a confocal microscope.Large HAp particles exhibited brighter fluorescence than the small particles, due to the high amount of Ir-BP2 absorption.
In contrast, nonstained HAp particles were non-detectable on the fluorescent channel (Figure S26).Specific staining of HAp at the cellular level was also verified.Ir-BP2 at concentrations ranging from 12.5 to 200 μM was tested for low toxicity against several cell lines, including cancerous MCF-7, HeLa, and Saos-2 cells (Figure S27).More importantly, Ir-BP2 also showed little cytotoxic effect on bone-relevant cells, such as human chondrocytes C28/I2 cells, mouse embryonic fibroblasts NIH-3T3 cells, and murine coralline 3T3 pre-osteoblasts (MC-3T3-E1) cells (Figure S28).Ir-BP2 with negative surface charges could hardly permeate the cell membrane, which also exhibits negative charges.Treating Ir-BP2 with preosteoblast MC3T3-E1 cells alone showed neglectable intracellular fluorescence (Figure S29).In comparison, supplementary HAp particles to cultured MC3T3-E1 cells could be specifically lit up by Ir-BP2 (Figure 3B).MC3T3-E1 cells having a high potential to differentiate into mature osteoblast cells were induced for osteogenic differentiation for 14 days.The generated HAp particles in situ could also be selectively stained by Ir-BP2 (Figure 3C).Specific staining of HAp at the cellular level by Ir-BP2 inspired us to further evaluate its feasibility in monitoring the osteogenesis process, during which mineralization occurs to produce a calcified bone matrix.Preosteoblast MC3T3-E1 cell is a typical cell model for studying osteogenesis [38] because it exhibits a high level of osteoblast differentiation into mature osteocytes and generates mineralized bone-like nodules after grown in an osteogenic medium in vitro.We first evaluated the long-term effect of Ir-BP2 on the proliferation of MC-3T3-E1 cells.As shown in Figure 4A,B, MC-3T3-E1 cells that were treated with Ir-BP2 showed no difference on the proliferation on day 3, day 5, and day 7 compared to the ones treated with the cultured medium.The cell density oversaturated after 7 days' culture in both groups, indicating that Ir-BP2 had little effect on the long-term proliferation of MC3T3-E1 cells.When MC3T3-E1 cells were grown in the osteogenic medium, they differentiated into osteoblasts and synthesized bone matrix.To assess the effect of Ir-BP2 on osteogenic differentiation, MC3T3-E1 cells were induced in the osteogenic media in the absence or presence of Ir-BP2 for 3, 7, or 14 days, and the expression of ALP (Figure 4C,D), one of the osteogenic markers, [37] and calcium deposits (Figure S30) were quantified.The results showed little quantitative difference between the control group and Ir-BP2 treated group, suggesting that Ir-BP2 did not affect the osteogenesis differentiation of MC3T3-E1 cells.
After verifying the biosafety of Ir-BP2 and its selective turn-on phosphorescence by HAp, we then validated its real-time monitoring of osteogenesis differentiation.MC3T3-E1 cells were cultured in osteogenic media containing Ir-BP2.The production of calcified minerals was imaged on day 3, 7, and 14, and fluorescently quantified.As shown in Figure 4E,F, on day 3, only a small amount of calcified minerals could be observed; on day 7, more individual calcified mineral dots were stained fluorescently, accompanied by higher fluorescence readout than on day 3. On day 14, a significant elevation of fluorescence signal was detected, and large calcified nodules were observed.In contrast, Alizarin Red S (AR) staining as the conventional calcium deposits staining assay showed nonspecific intracellular staining (Figure S30), because AR is a Ca 2+ -sensitive dye that can also stain free Ca 2+ .It should also be noted that AR staining assay is performed post-mortem at each time-point with cells being fixed.The osteogenesis differentiation process was irreversibly terminated for each staining.Ir-BP2 provided a convenient method for nondestructively monitoring osteogenesis differentiation without any washing steps.

Fluorescent analysis of ex vivo bone tissue
Encouraged by the promising performance of Ir-BP2 in imaging bone minerals at the cellular level, we further investigated its clinical practice for structural analysis of bone tissue.An easy-to-use staining spray comprising Ir-BP2 staining solution was made (Figure S31).A fresh rat femur was collected.After cleaning, the ex vivo bone tissue was sprayed and imaged without washing steps under a confocal microscope (Figure 5A).The penetration depth of the spray-ing Ir-BP2 could reach 70 μm into the cortical bone (Figure S32), which is competent for bone surface analysis.Fluorescent images of the bone surface were captured through Z-stack imaging.The remodeling 3D image (Figure 5B) clearly showed the rough surface structure of the femur, on which the parallel HAp minerals aligning longitudinally and the Volkmann's canals that transmit blood vessels inside bone tissue were identified.The high spatial resolution of fluorescent analysis allowed precise diagnosis of bone fractures.Through surface scanning, we found a microcrack with an approximate diameter of 2.2 μm (Figure 5C) on the bone tissue.Such tiny fracture was almost invisible using conventional diagnostic techniques such as CT [6] and MRI [8] .Interestingly, the number of Volkmann's canals in the fractured area increased, illustrating blood capillary hyperplasia phenomenon which allows high nutritions supply during bone fracture recovery.A cross-section of the compact tissue of the rat femur was also imaged after staining with Ir-BP2.The 3D image (Figure 5D) showed a much more intense fluorescent signal at the compact bone, which is highly calcified.Lamellar structure contributed to the external circumferential lamellae, and an osteon unit could be identified.
In addition to the structural analysis, Ir-BP2 staining could also provide valuable clues, for example, to distinguishing new bone matrices from old ones.Ir-BP2 exhibited an increasing fluorescent lifetime after immobilizing on the bone surface (Figure S16).From the PLIM images, we found that the specimen emitted phosphorescence with a relatively long lifetime (Figure 5E), eliminating the artifact signals, which usually emitted short lifetime fluorescence.The lifetime distribution histogram (Figure S33) indicated that the overall lifetime distributed within a wide range from 30 to 45 ns, with an average lifetime of 37 ns.Analysis of the microstructure and lifetime mapping suggested the bone matrix exhibited a diverse lifetime (Figure 5E,F).We assumed that the newborn bone matrixes (selected area 1) exhibited a relatively shorter fluorescent lifetime after Ir-BP2 staining than the mature ones (selected area 3), because the new bone matrix possessed fewer contents of highly calcified minerals than the mature bone matrixes.
The translational application of Ir-BP2 was further evaluated on a real bone disease sample.A patient who was diagnosed with eosinophilic granuloma (EG) at the C4 spine vertebra was treated surgically, and the lesion spine vertebra was excised for analysis.After applying an easy "spray-andcapture" procedure with Ir-BP2 staining spray, the structural analysis in Figure 5G showed that spongy bone structures could be seen at the lesion site, suggesting EG had invaded deeply and caused massive vertebral destruction.In contrast, the spine at the nonlesion site showed an intact vertebral body surface with high integrity.These results highlighted the practical potential of Ir-BP2 in the clinical diagnosis of bone disease.

CONCLUSION
In summary, we developed a water-soluble Ir(III) complex as the AIE-active staining agent for bone analysis.The AIE phosphorescence of Ir-BP2 was specifically activated to light up the bone matrix, due to the immobilized fixation of its molecular skeletons when bound to Hap particles as the mimics of the bone matrix.Its HAp sensing behaviors were investigated, and the HAP-binding mechanism was verified through spectral and microscopic analysis.Ir-BP2 showed good biosafety with little effect on the proliferation and differentiation of preosteoblast MC-3T3-E1 cells.Therefore, we employed Ir-BP2 for real-time monitoring of osteogenesis differentiation.It offered higher accuracy for staining calcium deposits than the conventional AR staining assay.Moreover, a ready-to-use spray composed of Ir-BP2 working solution was fabricated for wash-free staining of bone tissue.The microstructure of ex vivo bone tissue was analyzed using both fluorescent imaging and PLIM imaging.This bone probe could not only detect microdamage of bone fractures but also could distinguish newborn bone matrixes from old ones using phosphorescence lifetime imaging analysis.In the final, we verified its translational application on a human bone sample diagnosed with EG and successfully diagnosed the invasiveness of EG.Ir-BP2 as a powerful tool offered a convenient and accurate analysis of bone structure, showing great significance in the bone analysis and clinical diagnosis of bone diseases and advancing our understanding of bone biology and pathology.
It should be noted that Ir(III) complexes have been widely explored as photosensitizers in phototherapy. [39,40]he specific light-up of bone tissue by Ir-BP2 we presented here also shows high potentials in light-guided or lightinduced therapies for bone diseases, for example, to eliminate unwanted sarcoma cells for treating osteosarcomas, or to suppress osteoblastic cells activity for treating osteoporosis.We believe the presenting work hopefully promotes the development of bone analysis and bone disease diagnosis, and also broadens horizons in treating bone diseases.

A C K N O W L E D G M E N T S
This work was supported by financial support from the National Natural Science Foundation of China (grant number: 22107087), the Yong Talent Support Plan of Xi'an Jiaotong University (grant number: YX6J024), the Science and Technology Planning Project of Guangzhou (grant number: 202002030089), and the Key Projects of Social Welfare and Basic Research of Zhongshan City (grant number: 2021B2007).The authors acknowledge Chao Li at the Instrument Analysis Center of Xi'an Jiaotong University for the assistance in TEM imaging and EDX mapping experiments and Baochang Lai at the Cardiovascular Research Center of Xi'an Jiaotong University for the assistance in confocal microscope imaging experiments.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E T H I C S S TAT E M E N T
Animal experiments were performed under the approval of the Animal Ethics Committee of Xi'an Jiaotong University (number: 2021-1123).The human spine sample was collected and examined under the approval of the Medical Ethics Committee of Honghui Hospital, Xi'an Jiaotong University (number: 202304009).The use had been approved by the patient.

F I G U R E 3
Specific imaging of HAp by Ir-BP2.(A) Confocal images of HAp incubated with 20 μM Ir-BP2 at room temperature for 20 min.(B) Confocal images of MC-3T3-E1 cells supplied with 100 μg/mL HAp particles and co-stained with Cell Mask and Ir-BP2.(C) MC-3T3-E1 cells were induced for osteogenesis differentiation for in situ production of HAp for 14 days.The cells were fixed and stained with an F-Actin staining kit and Ir-BP2.Ex/em 405/625 ± 25 nm for Ir-BP2 staining; ex/em 488/520 ± 20 nm for F-actin staining; ex/em 645/670 ± 20 nm for Cell Mask staining, respectively.

F I G U R E 4
Real-time monitoring of osteogenesis differentiation.(A) Representative micrographs of Crystal Violet staining of MC-3T3-E1 cells cocultured with Ir-BP2 (20 μM) at day 3, 5, and 7, and (B) the corresponding quantitative analysis of cell viability.(C) Representative micrographs of ALP staining of MC-3T3-E1 cells after culture in osteogenic differentiation media containing 20 μM Ir-BP2 at day 3, 7, and 14, and (D) the corresponding quantitative analysis of ALP expression level.(E) Confocal images of MC-3T3-E1 cells after culture in osteogenic media containing 20 μM Ir-BP2 at day 3, 7, and 14.Fluorescent images and bright-field images were overlaid.(F) Corresponding quantitative analysis of fluorescent intensity at day 3, 7, and 14, as an indication of in situ HAp production.***p < 0.001.

F I G U R E 5
Microscopic analysis of ex vivo bone tissues.(A) Schematic diagram of Ir-BP2 spray for staining a flesh rat femur.(B) 3D remodeling confocal image of the femur cortical surface after spraying with Ir-BP2 for 10 min.(C) 3D remodeling confocal image showed a tiny crack (yellow arrows) after spraying with Ir-BP2 for 10 min.White stars indicated the Volkmann's canals.Insert: Enlarged image and intensity profile clearly showed the crack size.(D) 3D remodeling confocal image of the femur cross-section after spraying with Ir-BP2 for 10 min.Dash lines indicated the external circumferential lamellae; the cylinder indicated an osteon unit.(E) Photoluminescent lifetime imaging microscope (PLIM) image of the femur section after spraying with Ir-BP2 for 10 min, and (F) histogram distributions of fluorescent lifetime in the selected areas.(G) Z-stacking imaging of an ex vivo spine vertebra diagnosed with eosinophilic granuloma after spraying with Ir-BP2 for 10 min at the lesion site or at the normal site.The images were stacked and shown in max-intensity projection.