Recent progress in the green synthesis of rare-earth doped upconversion nanophos

Yuan Pu ,Jingning Leng ,Dan Wang ,*,Jiexin Wang ,Neil R.Foster ,Jianfeng Chen

1 Research Center of the Ministry of Education for High Gravity Engineering and Technology,Beijing University of Chemical Technology,Beijing 100029,China

2 State Key Laboratory of Organic-Inorganic Composites,Beijing University of Chemical Technology,Beijing 100029,China

3 Department of Chemical Engineering,Curtin University,Perth,WA 6845,Australia

Keywords:Green synthesis Upconversion luminescence Nanomaterials Biomedical engineering Imaging

ABSTRACT Rare-earth doped upconversion nanophosphors(UCNPs),which convertlowenergy near-infrared(NIR)photons into high energy photons such as ultraviolet,visible light and NIR light,have found various applications in optical bioimaging.In this review article,we summarize recent advances in the synthesis and applications of UCNPs achieved by us and other groups in the past few years.The approaches for the synthesis of UCNPs are presented,with an emphasis on the role ofgreen chemistry in the advancementofthis field,followed by a focused overview on their latest applications in opticalbioimaging from subcellular structures through cells to living animals.Challenges and opportunities for the use of UCNPs in biomedical diagnosis and therapy are discussed.

1.Introduction

Optical bioimaging uses an optical contrast such as a difference in light transmission,reflection,and photoluminescence(PL)between the region to be imaged and the surrounding region[1,2].PL based bioimaging is among the most widely used techniques for optical bioimaging,which can be used to visualize morphological details for bio-species,ranging from living cells to animals[3-7].Compared with other imaging techniques such as ultrasonic imaging,X-ray computed tomography(X-Ray CT),positron emission tomography(PET),singlephoton emission computed tomography(SPECT)and magnetic resonance imaging(MRI),PL imaging has the advantage of providing a high signal-to-noise ratio,which enables us to distinguish spatial distributions of even low concentration species and exhibits no harmful radioactivity[8].For PL bioimaging,labeling of the biological structure with exogenous fluorophores is usually needed since there are many biological structures and processes which cannot be imaged or probed by using intrinsic luminescence[9].The most commonly used exogenous fluorophores are organic molecules in the past century,though they are suffered from some drawbacks,such as broad emission width and poor photostability,which limit their applications for multiplexed and long-term fluorescent imaging[10-14].The development of nanotechnology has opened up new frontiers in materialscience and engineering[15-17]and the luminescent nanomaterials that came with it such as semiconductor quantum dots[18,19],organic-dye doped nanoparticles[20-22],and fluorescent carbon nanodots[23-25],have found many applications in biomedical fields.Most of these conventional luminescent nanomaterials for bioimaging are based on the Stokes-shifted emission with excitation in the ultraviolet(UV)or blue-green visible spectral ranges[18-26],which are limited by at least two disadvantages,namely low signal-to-noise ratio(SNR)due to the auto fluorescence from the biological samples when excited at high energy light;and low penetration depth of excitation light and/or emission light in tissues especially for animal imaging[27,28].

Upconversion luminescence refers to a series of nonlinear optical processes that convert two or more lower-energy pump photons,usually near-infrared(NIR)light,to one higher-energy output photon(UV,visible light and/or NIR light)[29-33].Compared with UV/visible light excitation,the use of NIR excitation light for bioimaging allows deeperlightpenetration,weakerauto fluorescence and lowerphototoxicity[33].Multiphoton absorption and high-harmonic generation based on ultrashort pulsed lasers(e.g.femtosecond laser)excitation are two general approaches to achieve photon upconversion in organic dyes and QDs for NIR light excited bioimaging[34,35].However,ultrashort pulsed lasers have the disadvantages of high cost,complex in maintenance and strict working environment requirement[36].Rare-earth doped upconversion nanophosphors(UCNPs),by contrast,can realize photon upconversion of continuous wave(CW)NIR light,through the use oflong lifetime and realladder-like energy levels oftrivalentlanthanide ions[29-33,36,37].The photon upconversion emission in UCNPs is attributed to the transitionsbetween physically existing states,which is different from nonlinear multiphoton absorption or high-harmonic generation involving the simultaneous absorption of two or more photons through virtual states[35].Therefore,the efficiency of photon upconversion process based on UCNPs is much higher than that of nonlinearmultiphoton absorption and can perform using a low costCWNIR laser rather than the use of ultrashort pulsed lasers[37].In addition to the unique photon upconversion properties,UCNPs show a sharp emission bandwidth,long lifetime,tunable emission,high photostability,and low cytotoxicity,which render them particularly useful for bioimaging applicationsincluding celltracking,tumor targeting and vascular imaging[38-42].Recently,these UCNPs were shown to act as efficient probes for super-resolution nanoscopy[43,44].Along with others,we have also demonstrated the potential bio-related applications of UCNPs in optical liquid marbles[30],photodynamic therapy[45-48]and optogenetics[33,49-52].Although tremendous progress has been achieved and several review articles focusing on the physical properties,synthetic strategies and applications of UCNPs have been provided in the past years[53-57],the “chemical engineering part”of the UCNPs story has only just begun.The development of robust mass-scale UCNPs synthesis methods is one of the key materials challenges and the dialogue between chemical engineers and other researchers(chemists,physicists,and biologists)is necessary.

In this perspective review,we present the significant advances achieved by us and other groups in the past few years on the design,synthesis and applications of rare-earth doped UCNPs.The approaches for the synthesis of UCNPs are presented,with an emphasis on the role of green chemistry(green solvents,novel reactors and process intensification technologies)in the advancementofthis field.The promise of UCNPs for optical bioimaging in both cells and animals is systematically discussed.Some challenges and opportunities for the use of UCNPs in biomedical diagnosis and therapy will be put forward based on our own understanding of this field.

2.Materials Synthesis

UCNPs are kinds of guest-host systems where trivalent lanthanide ions are dispersed as a guestin a hostlattice with atleastone dimension being less than 100 nm[29].Desirable host materials should have low lattice phonon energies to minimize non-radiative losses and adequate transparency within the wavelength range of interest[58].Various kinds of host materials have been developed,which can be mainly classified into fluorides(NaYF4,NaYbF4,NaGdF4,LaF3,GdF3,etc.)and oxides(Gd2O3,Y2O3,La2O3,Lu2O3,etc.)[59].Ideal host materials need to have close lattice matches to rare-earth dopant ions for achieving high doping levels[29,59].In these regards,NaYF4has been the most popular host materials due to its low phonon energy and high chemical stability[30,60].The doped lanthanide ions in UCNPs can be either activators that emit the radiation or sensitizers that absorb the excitation irradiation and transfer the absorbed energy to the activators.By selecting various kinds of lanthanide dopants,the UCNPs can show emission in a wide range of light from UV through visible(blue,green,red)to NIR(Fig.1)[61].Rare-earth ions such as Er3+,Ho3+,and Tm3+have been well suited for the photon upconversion due to their equally spaced energy levels that facilitate photon absorption and energy transfer steps involved in upconversion processes.Yb3+with a larger absorption cross-section in the NIRregion is usually doped as a sensitizer in combination with the activators to enhance the upconversion efficiency.

Fig.1.Upconversion luminescence spectra ofEr3+,Ho3+and Tm3+doped UCNPs with red,green and blue emission,respectively.The insets show the visible photoluminescence imaging of the UCNPs colloidal suspension excited by a NIR laser.

To date,a variety of methods have been developed for the synthesis ofrare-earth doped UCNPs,including butnotlimited to thermaldecomposition,co-precipitation,and hydrothermal synthesis[62-64].Considerable effort has been devoted to prepare highly crystalline structures for highly efficient upconversion emission[65].Suitable size and uniform shape of the nanocrystals are the primary prerequisites for bioimaging,in particularly for targeted in vivo imaging in animals.The nanoparticles generally have delayed clearance and are mostly excreted through the liver without significant metabolism[66,67],unless they can be made smallenough to be excreted via the kidney[68].In another hand,the upconversion luminescence efficiency is ofcriticalimportance to improve the SNR of the imaging.In most cases,the upconversion luminescence efficiency can be enhanced by increasing the size of UCNPs,since larger UCNPs mean lower surface area and less surface defects for the non-radiative decay which disturbs the upconversion luminescence.Coating of an inert crystalline shell around each particle or employing organic dyes as antennae have also proven to be effective ways to improve the upconversion efficiency of UCNPs,which will be described in the subsequent section[69-73].In as word,the synthesis of UCNPs with desirable sizes that are large enough to take the advantage of the unique features of the particles(e.g.quantum yield)while minimizing the toxicity by using particles small enough to be excreted via the urinary tract is a major challenge[74].To date,considerable efforts have been devoted to exploring the efficiency of various approaches for preparing UCNPs with tunable sizes from sub-10 nm to sub-μm[37].This section does not aim to give a systematic list of all the synthetic approaches of UCNPs reported.The interested readers are referred to specialized review articles for comprehensive knowledge of UCNPs synthesis[32,37,53,54,56,58,59,65,75-77].Here we show several selected examples mainly with references to green and scalable preparation of UCNPs based on green solvents,novel reactors and process intensification technologies,to highlight the research area on which already various studies are present,but which should be further intensified.

The thermal decomposition method has been a common route to synthesis high quality UCNPs with highly crystalline phase and narrow size distribution[78].For the typical synthesis of Yb3+and Er3+codoped NaYF4UCNPs through the thermal decomposition method,metallic tri fluoroacetate precursors are heated over 300°C in the presence of surfactants and high boiling organic solvents.The rapid decomposition of metallic tri fluoroacetate at high temperature yields monomers,which are then crystallized and grow into monodispersed UCNPs.The oleic acid and/or oleylamine were usually chosen as surfactants to prevent nanoparticle aggregation and the most commonly used organic solventwas octadecene(ODE)due to its high boiling point.The thermal decomposition method has been extended to synthesize several types of monodisperse UCNPs with narrow size distribution,good crystallinity,and desirable optical properties[79].However,there are two limitations forthis approach,including the relatively hash reaction conditions and generation of toxic byproducts of fluorinated and oxy fluorinated carbon species during the pyrolysis of tri fluoroacetates(Fig.2)[80],which make the thermal decomposition method unpractical for the large-scale synthesis of UCNPs.Recently,we have reported the replacement of commonly used ODE with paraffin liquid as the high boiling non-coordinating solvent for thermal decomposition synthesis of NaYF4:Gd3+,Yb3+,Er3+nanophosphors35and NaYF4:Yb3+,Tm3+microphosphors[80].Since paraffin liquid is a more environmentally friendly and much cheaper chemical than ODE[15],the modified method is useful for the development of greener thermal decomposition route for scalable preparation of UCNPs.

Co-precipitation is one of the most promising techniques for largescale production of nanoparticles[81-83].In contrast to thermal decomposition method,the co-precipitation synthesis of UCNPs does not need hash reaction conditions or complex procedures and the reactions can be completed in aqueous solutions rather than in organic chemical[84].For instance,in a typical route for the synthesis of NaYF4:Yb3+,Er3+UCNPs,the particles formed by co-precipitation of the rare-earth chlorides with NaF in the presence of ethylenediamine tetraacetic acid(EDTA),showing spherical shapes with narrow size distribution of 32-46 nm in diameter[85].Spherical Gd2O3:Yb3+,Er3+UCNPs can be prepared via a simple homogenous co-precipitation of rare earth nitrates with urea in aqueous solution and the particle sizes of were controllable by adjusting the molar ratios of rare earth ions and urea[86].The co-precipitation method has proven to be a convenient method for the preparation of UCNPs,despite the fact that UCNPs synthesized via the co-precipitation process usually require additional post-heat treatment or annealing at high temperatures to sharpen the crystal structure or prompt the phase transfer to obtain products with high upconversion efficiency[87].During the co-precipitation process,rapid mass transfer and micromixing are critical for the formation of nanoparticles with narrow size distribution[88].However,the efficiency of mass transfer and micromixing of liquids in traditional stirred tank reactors are limited,and these problems will become more prominent in the case oflarge-scale production[89].As one ofthe cutting-edge process intensification,the high-gravity(Higee)technology based on rotating packed bed reactor has been used to produce various kinds of nanoparticles[83,90].In particular,we have recently demonstrated the successfully synthesis of Gd2O3:Yb3+,Er3+UCNPs by Higee reactive precipitation along with a post hydrothermal and calcination process(Fig.3)[91].Due to the intensified mixing by the rotating packed bed reactor,the obtained Gd2O3:Yb3+,Er3+UCNPs exhibited much smaller particle sizes(＜100 nm)than that from the common route(～360 nm).

Fig.2.Schematic diagram of the process and possible chemical reactions involved in the formation of NaYF4:Yb3+,Tm3+particles via thermal decomposition approach.Reproduced from Ref.[80]with permission from Elsevier.

Fig.3.Schematic diagram ofthe routes forthe preparation of Gd2O3:Yb3+,Er3+UCNPs without(a)and with(b)Higee process intensification;typicalscanning electron microscope(SEM)images of Gd2O3:Yb3+,Er3+UCNPs obtained without(c)and with(d)Higee process intensification.

Hydrothermal synthesis of UCNPs involves the mixing of appropriate reaction precursors,solvents and surfactants and then heating of the mixture in a specialized reaction vessel typically known as Te flonlined stainless steel autoclave[92-94].The most representative example of hydrothermal synthesis is provided by Li et al.,who reported on a generalliquid-solid-solution(LSS)strategy for the synthesis ofmonodisperse nanoparticles[95].In this strategy,the nucleation and growth of UCNPs occur at the liquid-liquid or liquid-solid interface[95].The nanoparticles precipitate at the bottom of the reaction vessel,and can be easily recovered and dispersed in nonpolar or moderately polar solvents.The controlled growth of UCNPs of tunable sizes can be theoretically achieved by varying the reaction parameters such as reaction temperature,reaction time and surfactants in the hydrothermalsynthesis approach[96-98].As the reaction conditions are relatively mild,and no particular skills seemto be needed,this method is a prime candidate for large-scale production of UCNPs.However,the stand batch-type reactors do not always allow a fine tuning of conditions,such as fast mixing of the solvents or chemicals,sudden rise or fall of temperatures,when large volumes of solution are involved(i.e.in liters or more),leading to low batch-to-batch reproducibility.This is particularly challenging in moving towards UCNPs for large-scale production and commercial applications.An alternative to a batch reactor is continuous reactor[99,100],in which the reactants are continuously fed and the reaction parameters(e.g.residence time,temperature and pressure)are better controlled than in batch operations.It is highly recommended to use continuous-type reactors such as rotating packed bed reactor[101,102],microchannel reactor[103,104],and spinning disk processor[105],to meet the conditions for scalable and stable production of UCNPs,although few literatures have been reported.As a conclusive note to the synthesis of UCNPs in rotating packed bed reactors,we can certainly say that many syntheses of UCNPs,at least at laboratory scale,can be carried out with a rotating packed bed reactor and the obtained UCNPs possess higher quality,namely with regular shape,uniform size and narrow distribution[91].

3.Surface Modification

As mentioned in the above section,UCNPs are typically prepared in the presence of capping ligands(e.g.oleic acid,oleylamine)to control the particle growth and stabilize the particles against aggregation in solutions,which are generally hydrophobic.In other cases,additional post-heat treatment or annealing are usually needed for the UCNPs obtained via the co-precipitation process in order to sharpen the crystal structure or prompt the phase transfer to obtain products with high upconversion efficiency,which result in bare inorganic powders hard to disperse in liquid solution.For biological applications,the UCNPs are required to be well-dispersed in biologicalbuffers,which are usually aqueous solutions.Surface modification refers to the act of modifying the surface of materials by bringing physical,chemical or biological characteristics different from the original ones.To date,a variety of surface modification methods(Fig.4),including silica coating[106],ligand exchange[107,108],ligand oxidation[109],ligand attraction[110]and layer-by-layer assembly[111,112],have been reported[113,114].For detailed discussions on the various surface modification approaches to UCNPs,the reader can refer to dedicated reviews[29,75-77,115].

Generally,surface modification of UCNPs not only enables the nanoparticles to be dispersed in aqueous phase,but also provides a platform to integrate biomolecules/drugs for multifunctional applications.For instance,we have recently developed a multifunctional nanoplatform by coating NaYF4:Gd3+,Yb3+,and Er3+UCNPs with biocompatible transferrin(TRF),a serum protein with good biocompatibility and tumor targeting ability[45].Protoporphyrin IX(PpIX),a clinically approved photodynamic therapy agent,was loaded into the shell layer of the TRF-coated UCNPs(Fig.5).The final UCNP@TRF-PpIX nanoparticles were demonstrated to be efficient probes for photodynamic therapy with near-infrared irradiation and luminescence bioimaging.It should be noted that the surface modification technique is applicable for preparation of both hydrophilic and hydrophilic UCNPs.We reported the surface coating of UCNPs with polyhedral oligomeric silsesquioxane(POSS)to make the particles highly hydrophobic and describe the preparation of optically and magnetically active bifunctional UCNPs-based liquid marbles as well as their applications as miniature reactors for studying the photodynamic therapy of cancer cells[30].

Fig.4.Schematic illustration ofgeneralstrategies used for surface modification of UCNPs,including ligand exchange,ligand oxidation,ligand attraction,layer-by-layer assembly,and silica coating.

Fig.5.(a)A schematic illustration of the fabrication of UCNP@TRF nanoparticles;TEM images of(b)UCNP and(c)UCNP@TRF nanoparticles;(d)FTIR spectra and(e)TGA curves of TRF,UCNP and UCNP@TRF nanoparticles,respectively;roomtemperature upconversion luminescence spectrum of5 mg UCNPs(f)and UCNP@TRF(g)powderunder NIRlightexcitation with a 980 nm laser at a powder density of 5 W·cm-2.Inset:Photographs of the UCNP and UCNP@TRF samples under NIR light excitation,respectively.

Apart from the improvement of biocompatibility,the surface modification can also improve the optical properties of UCNPs,which is the foundation for further applying UCNPs to bioimaging and other fields.Coating of an inert crystalline shell around each particle has proven an effective way to improve the upconversion efficiency of UCNPs.Excellent demonstrations have been made in various host materials[69,70].For example,Han and co-workers reported the tunable NIR-to-UV upconversion luminescence enhancement in CaF2-coated α-NaYF4:Yb3+,Tm3+core/shell UCNPs[96].Optimization of the four-photon UV luminescence in the(α-NaYF4:Yb3+,Tm3+)/CaF2core/shell system yields the maximum quantum yield of 0.1%,which is over 9 times brighter than the known optimal(β-NaYF4:Yb3+,Tm3+)/β-NaYF4core/shell UCNPs in aqueous phase[70].In other examples,employing organic dyes as antennae to enhance the efficiency of UCNPs has emerged as a new dimension[49,71].Reports have shown that the upconversion luminescence brightness can be increased by a factor of～3300 for NIR dye sensitized β-NaYF4:Yb3+,Er3+nanoparticles and by up to 100 times for NIR dye sensitized(NaYbF4:Tm3+)/(NaYF4:Nd3+)core/shell,compared with original ones[72,73].

4.In vitro Optical Imaging of Cells

The upconversion luminescence is the most fascinating feature of UCNPs that makes them attractive for bioimaging.Compared with the traditional luminescence nanomaterials,the UCNPs possessing spectrally narrow and tunable emission bands excitable by a single wavelength in NIR,can be readily employed as probes for multiplexed bioimaging[116].In addition,as a typical upconversion luminescence imaging system shown in Fig.6a[45],the imaging background produced by the scattering or auto fluorescence of biological samples from the excitation light can be reduced by using short-pass filters.Thus the anti-Stokes character of the upconversion emission excludes the background signals,resulting in a high signal-to-noise ratio[117].Zijlmans and co-workers were the first to use the upconversion luminescence of UCNPs for cellular imaging in 1999[118].Since then,considerable research efforts have been dedicated to exploit UCNPs as fluorescent probes for in vitro imaging of cells,which exhibited signi ficantly low background auto fluorescence[39-45,119-122].Li et al.developed a three-dimensional visualization method of laser scanning up-conversion luminescence microscopy with little photobleaching and no background fluorescence by introducing a reverse excitation dichroic mirror and the confocal pinhole technique,obtaining the first quantitative data for the lack of auto fluorescence under 980 nm excitation[121].Zhan et al.proposed a multi-photon evanescent wave(EW)excitation modality for UCNP-based microscopy and the particles tracking and in vitro cell membrane imaging were performed with ultrahigh contrast and high spatiotemporal resolution[122].Our group demonstrated the efficient uptake of transferrin-coated magnetic UCNPs by MDA-MB-231 cancer cells using upconversion luminescence imaging(Fig.6b),which has instructional significance for further drug delivery studies based on the nanoplatform of UCNPs[45].

Fig.6.(a)AtypicalUpconversion luminescence imaging system;in vitro upconversion luminescence imaging ofMDA-MB-231 cancer cells incubated in 2 ml of DMEM culture media with the addition of(b)nothing and(c)200 μl aqueous dispersion of transferrin-coated NaYF4:Gd3+,Yb3+,Er3+UCNPs(20 μg·ml-1)for 2 h at 37 °C.The scale bars represent 50 μm.

Fig.7.Experimental temperature(°C)distributions after different irradiation times for(a)915 nm laser irradiated mouse skin and(b)980 nm laser irradiated mouse skin and(c)the corresponding temperature line profiles(curves A and B in(c)corresponding to line A in(a)and line B in(b),respectively).

Fig.8.Immuno fluorescence labeling of cellular cytoskeleton protein desmin with antibody-conjugated UCNPs and super-resolution imaging:(a)the multiphoton imaging under 975-nm excitation of some cytoskeleton structures and desmin proteins in HeLa cancer cells incubated with anti-desmin primary antibody and immunostained with UCNPs(11.8±2.2 nm in diameter)bioconjugated with goat anti-rabbit IgG secondary antibody;(b)the same region with a imaged in the super-resolution mode(975-nm excitation and the 810-nm STED laser beam);(c-n)magnified areas selected from(a)and(b)(marked by white dotted squares)and line profile analyses;images in c,f,i,and l are taken from the white dotted squares in a;Images in d,g,j,and m are taken from the white dotted squares in b.e,h,k,n Line profiles analyses of several areas indicated by arrow heads in c and d,f and g,i and j,and l and m,respectively.The scale bars in(a)and(b)represent 2 μm.

Due to the fact that the sensitizer ion Yb3+has a high absorption cross-section in its absorption band,a low-cost commercial CW laser diode with peak wavelength of 980 nm is usually used to excite the UCNPs for upconversion luminescence in bioimaging due to the fact that the sensitizer ion Yb3+has a high absorption cross-section in its absorption band.However,the high absorbance of 980 nm light by water,results in significant heating effect of a biological tissue under irradiation of 980 nm light[123].He and co-workers investigated the proposed the use of the 915 nm laser,to excite Tm3+/Er3+/Ho3+-doped NaYbF4UCNPs in the upconversion luminescentbioimaging[61].The comparison ofimaging at980 nmand 915 nm excitation,along with evaluation ofthe thermal effect produced by both wavelengths,demonstrated the 915 nm light was better choice(Fig.7)[61].Later,Zhong et al.reported the use of Nd3+-sensitized UCNPs as luminescent labels for cell imaging,which are excitable at 800 nm due to the incorporation of Nd3+ions into the outer shell[124].The low water absorption of the 800 nm laser source not only helps to minimize tissue overheating,but can also result in a deep penetration depth for potential in vivo imaging[125].

Recently,along with the rapid development on super-resolution optical microscopy,the use of UCNPs for in vitro optical imaging has been extended to stimulated emission depletion(STED)microscopy[43,44,126,127].Kolesov et al.demonstrated the background-free subdiffraction optical microscopy with praseodymium-doped yttrium aluminum garnet(Pr:YAG)UCNPs and achieved all optical resolution of 50 nm[126].Due to the fact low-efficiency YAG:Pr3+UCNPs emitting toxic UV light under a complicated excitation scheme,their bioimaging applications were limited.Later,using NaYF4:Yb3+,Er3+UCNPs,Zhan et al.proposed an optical luminescence depletion mechanism and explore their potential for multi-photon STED-like microscopy[127].Very recently,Liu et al.realized low-power super-resolution stimulated emission depletion(STED)microscopy based on high concentration Tmdoped(8%)NaYF4:Yb3+,Tm3+UCNPs,and obtained optical resolution of 28 nm,namely,1/36 of the wavelength[43].Zhan and co-workers reported the use of ion-ion interaction to suppress the enhancement effect and enhance the depletion effectofthe depletion laser beam,resulting in large opticaldepletion ofupconversion luminescence in a majorgroup of UCNPs.reported the super-resolution imaging of immunostained cytoskeleton structures of fixed cells(resolution～82nm)using high Tm3+-doped UCNPs(NaGdF4:18%Yb3+,10%Tm3+)as luminescent labels(Fig.8)were performed[44].These results demonstrated the ability of UCNPs-mediated super-resolution optical microscopy technique for imaging subcellular structures.

Fig.9.Targeted in vivo NIR upconversion luminescence imaging of subcutaneous MGC803 tumor(indicated by short arrows)after intravenous injection of silica coated NaYbF4:Gd3+,Tm3+UCNPs(a)with and(b)without folic acid modification.

5.In vivo Optical Imaging of Animals

In the case of animal imaging,the limitations of fluorescence imaging based on short-wavelength light excitation are more prominent,due to the Rayleigh scattering effect and significant background signal associated with auto fluorescence of biological tissues such as fur,skin,and food in the subject studied.Following an intravenous injection of 3-mercaptopropionic acid coated NaYF4:Yb3+,Tm3+UCNPs into Balb-c mice,Prasad et al.[128]performed in vivo whole-body imaging of mice,in which the NIR upconversion luminescence signal of UCNPs peaking at～800 nm was readily detectable through the skin(without hair removal)of the mice.The obtained high contrast images clearly demonstrate the feasibility to image and spectrally distinguish the characteristically emitting UCNPs.Later,the advantages of upconversion luminescence imaging with UCNPs were revealed in in vivo animals[37,129-134].For instance,as shown in Fig.9,Pan et al.[134]performed in vivo whole-body images of mice injected with silica coated UCNPs with and without folic acid modification.The upconversion luminescence signals were collected through the skin of mice(without hair removal),from which the gastric cancer tissues were identified in vivo due to the targeted ability of folic acid conjugated UCNPs to tumors.

As for in vivo bioimaging,tremendousprogresshasbeen achieved on the development of UCNPs for optical bioimaging in mice using NIR(980 nm)-to-NIR(800 nm)upconversion luminescence[135-137].However,the optical imaging technique(even for NIR-to-NIR luminescence imaging)is still limited by their relatively low penetration depth in animals(maximum to several centimeters)[37].There is a growing interestin the developmentof UCNPs that can function as multiple contrast agents forsimultaneous use in differentmedicinal imaging modalities(e.g.,X-Ray CT,PET,SPECT,and MRI)to achieve “real”deep imaging[138-140].In addition to the ability for luminescence diagnosis,UCNPs also hold greatpotentials to “light-up”the cells in deep tissues for other photoactivation applications such as photodynamic therapy,and optogenetic studies[49-52,141,142].Since the topic of this review article is focused on the optical bioimaging based on photon upconversion,we willnotinclude detailed discussions on the applications ofUCNPs for multimodal imaging,photodynamic therapy and optogenetic studies,which nevertheless should be interesting topics in future directions.It should be mentioned that for in vivo upconversion luminescence diagnosis and photodynamic therapy,the UCNPs are usually required to be given by intravenous injection and be small enough(e.g.sub-10 nm)for long-time circulation in animals.In contrast,for optogenetics applications,the upconversion phosphors are expected to be embedded at a particular location in the neuronal plasma membrane,where the neurons are controlled by the exogenous expression of light-sensitive ion channels.Upconversion phosphors with large size(e.g.a few microns in diameter)and high upconversion efficiency are preferable for applications in optogenetic studies than nanophosphors.Fig.10 shows a recentwork by ourgroup,revealing the in vivo upconversion luminescence imaging of mice after microinjection of NaYF4:Yb3+,Tm3+microphosphors in the brain tissue of mice.The distribution depth of interested cells such as cerebellum and cerebral cortex in small mice were generally less than 800 μm in their brains and NaYF4:Yb3+,Tm3+microphosphors can achieve such a depth for in situ light upconversion from near-infrared to blue.Combined with the fact that microphosphors distribution was concentrated in a very small area(500 μm × 500 μm × 50 μm),this make the particles promising for in vivo optogenetic studies[80].

6.Summary and Prospect

Fig.10.(a)A diagram of microinjection of upconverting particles in a mouse brain;(b)in vivo upconversion luminescence imaging of mice injected with NaYF4:Yb3+,Tm3+microphosphors under excitation of 980 nm light;(c)spectra of the luminescence signal from the phosphors in mice brain observed in(b);(d)a typical upconversion luminescence image of the brain tissue slice from mice post-injected with NaYF4:Yb3+,Tm3+microphosphors for 2 weeks.The blue color revealed the distribution of the particles.

Photon upconversion based on rare-earth doped UCNPs has tremendous attention in many fields,including bioimaging,anti-counterfeiting,photovoltaic devices,photodynamic therapy,and other photoreactions.In this article,we focus on the recent advances of UCNPs for optical bioimaging achieved by us and other groups in the past few years and present an overview on the synthesis,surface modification and bioimaging of UCNPs.Even this short perspective review article has revealed the versatility of UCNPs for bioimaging,from in vitro upconversion luminescence imaging in cells through in vivo macroscopic imaging to microscopic imaging in animals.However,the applications of UCNPs for bioimaging are still at its early stage with many important issues remained to be addressed:

(1)Widely applications of the UCNPs will not be realized if there is no facile and large-scale fabrication capability for UCNPs with well-defined properties desirable for applications.We have remarked,however,the scalable synthetic approaches for mass production ofUCNPs with tailor-made structures are stilllimited.This is a direction in which chemicalengineers should involve.As an alternative to commonly used batch reactor for UCNPs synthesis,continuous reactors are highly recommended to overcame the limitations of batch reactors and meet the conditions for scalable and stable production of UCNPs.

(2)Like many other nanomaterials for biomedical applications,there are concerns about the possible side effects derived from the use of UCNPs.Although rare-earth doped UCNPs possess no toxic elements and are generally considered as biocompatible materials after surface modification[143],nanotoxicity studies of UCNPs(with different components,sizes,shapes,and surface coatings)to human and other biological systems need to be carefully ascertained.Along with the cytotoxicity researches at the cellular level and histological toxicity investigations in animal level,potential hazards of UCNPs to DNA damage(i.e.,genotoxicity)are also important since there is a close correlation between DNA damage and mutation or cancer.

(3)There are already very big improvements on the upconversion efficiency of UCNPs in recent years.The reported upconversion quantum yields of current UCNPs remains rather limited for all in vivo optical bioimaging,especially under low irradiance.The integrations of upconversion luminescence imaging with other biomedical imaging techniques such as X-Ray CT,PET,SPECT,and MRI,enable accurate diagnostics in a reduced time frame.Development of multifunctional probes to cover both diagnosis and therapy is also an interesting topic.However,the costperformance for the design and synthesis of complex UCNPs based nanoplatform should be took into account at the early stage.