Progress with, and prospects for, metal complexes in cell imaging.

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Cite this: Chem. Commun., 2014, 50, 384

Received 11th July 2013, Accepted 30th October 2013 DOI: 10.1039/c3cc45229h

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Progress with, and prospects for, metal complexes in cell imaging b Michael P. Coogan*a and Vanesa Ferna ńdez-Moreira*

This article summarises the state of the art of metal complexes in cell imaging, particularly fluorescence microscopy, and presents prospects for the future development of this area. This article combines discussion of, and examples from, both the d- and f-block which have traditionally been considered separately, presenting the important classes of agents in each case, with a general description of their

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photophysical and cellular behaviour, and comparing and contrasting their properties and applications.

Introduction While there have been reviews of the application of various classes of metal complexes in imaging, these have previously focussed on particular areas. Reviews and many articles have focussed on such as organometallics,1 transition metals2 or the individual transition metals (iridium,3 rhenium3c,4 and ruthenium5 have attracted most interest) or the lanthanides,6 but to the best of our knowledge, so far there has been no review covering all of the metals. This brief

perspective aims to present the state of the art in an overview of all of the metals in imaging, focusing on cellular properties such as uptake, localization and toxicity so as to be useful for readers interested in designing new complexes for in vivo applications. As there are more examples of transition metal complexes which have been used in cell imaging these are discussed in more general terms with patterns and rules, whereas in the case of the lanthanides more specific examples are given as for most classes of agent it is still possible to give a good overview of the examples which have been applied in cellular imaging.

a

Department of Chemistry, Faraday Building, Lancaster University, Bailrigg, Lancaster, LA1 4YB, UK. E-mail: [email protected]; Tel: +44 (0)1534592342 b ISQCH-Instituto de Sıńtesis Quı´mica y Catalisis Homogenea, Facultad de Ciencias, Universidad de Zaragoza-CSIC, C/Pedro Cerbuna, 12, Zaragoza 50009, Spain. E-mail: [email protected]

Mike Coogan was born in Birkenhead (UK) in 1969 and attended Leicester University for his BSc and PhD (with Dr R. S. Atkinson, studying asymmetric alkene aziridination). After postdocs in Nottingham, Cardiff and Sheffield he obtained a temporary lectureship in Organic Chemistry at Durham in 1998. In 2000, he moved to Cardiff initially as an organic, then an inorganic chemist, becoming an Michael P. Coogan inorganic group leader in 2009 before leaving in 2012. In 2013 he moved to the newly formed Chemistry Department at Lancaster University where he continues to study organometallic and coordination chemistry and the biological applications of coordination complexes.

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Background The real strengths of metal-based imaging agents are the distinctive nature of their emission, and this work focuses on

ńdez-Moreira obtained Vanesa Ferna her BSc and MPhil in Chemistry at University of Vigo (Spain) in 2005. Then, she moved to Cardiff University (UK) and completed her PhD (2008) under the guidance of Dr M. P. Coogan and Dr A. J. Amoroso in the area of luminescent MLCT complexes. After two years of post-doctoral work, first with Prof. J.-C. G. ¨nzli at the EPFL (Switzerland) Bu and then with Prof. S. Draper in ńdez-Moreira Vanesa Ferna Trinity College (Ireland), she moved to the ISQCH in Zaragoza (Spain) where she is working as a research associate in the research group headed by Prof. M. C. Gimeno.

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those complexes which emit from metal-related states. Examples of metal complexes which show ligand based singlet emission are excluded from this article as they do not share the features which distinguish the complexes under consideration from organic fluorophores. In addition to useful emission properties other criteria are necessary for a cellular probe which have been previously defined and are only briefly reiterated here.2 Requirements: Photophysics: absorb/emit at wavelengths which pass though the tissue/sample concerned and be differentiated from autofluorescence by Stokes shift or luminescence lifetime. Both long luminescence lifetimes and large Stokes shifts are associated with triplet excited states, which in turn are associated with spin– orbit coupling effects in complexes of the heavier elements, hence the dominance of the 2nd and 3rd transition series and lanthanides in this area. Solubility and stability: remain intact and in solution in physiological media for the life of the experiment without precipitating or aggregating. Toxicity: be non-toxic for in vivo work and non-cytotoxic for in vitro cellular work, at least for the timescale of the experiment. Uptake and localisation: cross lipophilic cell membranes and barriers and localise in the site of interest. Sensing action: ideally a probe shows a detectable response to environment reporting on e.g. pH or [O2] allowing combined imaging and sensing. While many metals are regarded as intrinsically toxic, and many classes of metal complexes are thought of as intrinsically unstable to air/water (e.g. organometallics) there have now been reports of cell imaging involving the p-block elements, the d-block transition metals across the series from d0 to d10 configurations, and from the f-block, and each of these areas will be described, before being critically compared and contrasted.† How the photophysical properties, toxicity, etc. of a given metal can be tailored to allow applications in cell imaging varies dramatically with the metals in question and each will be assessed in turn, by the metal class, and within the classes of metals either by the element or by the ligand class as appropriate. The similarities and differences between the approaches will be highlighted and prospects for future development discussed.

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the system and thus enhances emission. Other p-block containing lumophores include the BODIPY fluorophores and other examples of essentially organic dyes which contain the less common p-block elements, but these are best regarded as organic compounds with unusual substituents than metal complexes and so are not discussed in detail here. Aluminium phthalocyanines have been widely used in studies aimed at photodynamic therapy as they absorb in the long-wavelength region of the spectrum allowing good tissue penetration, and generate toxic oxygen species. They are easily detected in emission microscopy experiments due to their high quantum yields, and a number of studies have reported their cellular distribution from microscopy experiments.‡ Again, it is likely that the mechanism of emission is more similar to organic fluorophores than phosphorescent complexes.9

The d-block There are a large number of d-block lumophores which have been applied in cell imaging,2,10 and no attempt will be made to refer to each example, instead a summary of the more important features and families will be presented, along with certain selected important recent developments. Most transition metal based cell imaging agents to date are based on mid- to latetransition metals in low oxidation states, usually emitting from MLCT or mixed IL/MLCT states. The most studied systems are the d6 complexes of rhenium, ruthenium and iridium. d8 and d10 platinum and gold complexes have also been used, with interesting Au–Au orbitals being involved in the emissive processes of certain Au complexes. Finally, a single report of the application of d0 early transition metal lumophores in cell imaging has recently emerged (Fig. 1). It has become apparent that the best way to discuss transition metal imaging agents is by the periodic group, with ligand families as subgroups within these major classifications while the opposite is true for the lanthanides. This reflects the relative importance of the d- and f-electrons in

The p-block One important example of p-block heavy metals in cell imaging is the use of organoarsenic lumophores in the imaging detection of thiol-containing proteins. In these systems the lumophore is essentially non-emissive in the native state, but reacts with proteins containing two thiols in close proximity to form an As(SR)2 unit. The luminescence is greatly enhanced upon this modification allowing an imaging detection of the presence of such groups.7,8 However, it is not clear that As plays a significant role in the emission of such compounds, and may instead be simply responsible for reaction with the proteins which rigidifies † Corresponding author for d/p-block MPC; corresponding author for f-block VFM.


Fig. 1 Basic structural forms of the types of Re(I), Ru(II), Ir(III), Pt(II), Au(I) and Zr(IV) complexes used as imaging agents.

‡ The term ‘fluorescence microscopy’ is widely used in biology, but in this work emission comes not from fluorescence but from a different photophysical mechanism, phosphorescence. We suggest the term ‘emission microscopy’ be used to include all photoluminescence imaging.

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Fig. 3 Cell imaging with zirconocenes. Reprinted with permission from ref. 15 Copyright (2013) American Chemical Society. Fig. 2 Simplified Jablonski diagram of a 3MLCT emissive complex. A = absorption; F = fluorescence; P = phosphorescence; S = singlet; T = triplet; ISC = inter system crossing; small line separations = vibrational energy levels; right hand labels = orbital character.

the chemistry of the these metals, with the d-occupancy being of prime importance to the geometry and reactivity of transition metal complexes, while the f-occupancy has less influence on these factors in the lanthanides. Photophysics of d-block imaging agents The classic mechanism for emission from transition metal complexes involves metal-to-ligand charge transfer (MLCT) processes from electron-rich low oxidation state mid- to late transition metals and conjugated aromatic heterocycles which can easily accept electrons. However, such complexes also show intra- (or inter) ligand (IL) electron transfer transitions and in many cases there are excited states which mix MLCT and IL character.11,12 Typical examples of the ligands involved include neutral chelating N4N ligands such as bipyridine (bpy) and related ligands (typically with Re(I), Ru(II)) and formally anionic cyclometallating C4N ligands such as phenylpyridines (ppy) (usually with Ir and Rh). Alkynyl and thiolate ligands are also important in the later d8 and d10 systems, and these can be involved in LMCT, LMMCT (see below) and IL transfers. MLCT in such a complex typically involves transfer of an electron from metal – based d-orbitals, of t2g nature in octahedral Re(I), Ru(II), and Ir(III) species, to vacant ligand based p* orbitals. IL processes usually involve electron transfer from occupied p orbitals to vacant p* orbitals, although the donors can also be s or non-bonding orbitals. The initially formed singlet excited state (1MLCT) is converted by spin orbit coupling mediated intersystem crossing to a triplet before 3MLCT emission occurs. As this emission is forbidden the lifetimes associated with this process are long, and the energy lost in the conversion to the triplet and in associated geometrical reorganisation gives the large Stokes shifts (Fig. 2).

Overview of d-block imaging agents by groups Group IV: zirconium and hafnium Metallocene derivatives of the general formula Cp2MCl2 (M = Zr(IV)/ Hf(IV)) have been reported to emit from triplet LMCT states, involving the transfer of electron density from the electron-rich Cp rings to the formally d0 metal. In most cases the emission is limited to extremely low temperatures,13 but in certain cases

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involving highly rigid ansa-metallocenes intense room temperature emission is observed, which is assigned to be a result of reduced vibrational quenching at either low temperature or in the rigid systems.14 Very recently a simplified example of a rigidified system based on polyphenyl cyclopentadienes was reported which retains room temperature emission due to the retarded vibrations in these sterically hindered cases.15 The steric hindrance also leads to a reduction in the moisture sensitivity of the complexes, which has allowed application in cell imaging, (Fig. 3) although this work is at too early a stage to allow any general rules regarding patterns of uptake, localisation of toxicity to be deduced. Previous studies of the cytotoxicity of zirconocenes,16 and the existence of a long-lived PET-active isotope 89Zr17 suggest potential for bimodal imaging or combined imaging and therapy with this group. Group VII: rhenium and technetium Rhenium(I) lumophores are most commonly based on the fac-[Re(CO)3(N4N)]+ core (N4N = any chelating diimine ligand such as bipyridine, phenanthroline etc.).2a Other complexes based on bisquinoline amines related to the dpa family of ligands have been used in imaging, and are the basis of the Tc(I) analogues used in radioimaging studies and for bimodal applications.18 fac-[Re(CO)3(N4N)]+ itself is stable,2a shows luminescence with the properties required for imaging2a and is accessible with a range of substituents on the pyridine or the related ligand in the 6th position allowing tuning of biological properties for specific applications.19 There have been extensive studies of the uptake and localisation of rhenium complexes,3c and it is generally possible to endow rhenium complexes with good cellular uptake by the addition of any additional hydrocarbon-containing ligand to the basic core.4a The work of Lo and co-workers has been particularly significant in the area with seminal studies of localisation and toxicity of Re (and Ru and Ir) complexes in a range of cell lines. Several examples of good control of cellular localisation have been reported which usually rely on the addition of targeting vectors to the 6th (pyridine) ligand.4b Re(I) and Tc(I) have a d6 low-spin electronic configuration, which gives kinetic stability moderating their toxicity, but while most examples have low toxicity certain rhenium complexes are cytotoxic as a result of the ligands rather than the metal itself.20 Another group of rhenium imaging agents has emerged based on neither of the previously mentioned groups, these are dinuclear Re diazine (pyridazine) complexes of the general formula [Re2(m-Cl CO)6(m-1,2-diazine)] (Fig. 4).21 These complexes show visible MLCT emission even though the ligand is only monocyclic. They have been prepared


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Fig. 6

Molecular structure of DNA sensor [Ru(bpy)2(dppz)]2+.30

Fig. 4 [Re2(m-Cl CO)6(m-1,2-diazine)] PNA.21

Fig. 7 Cell imaging with [Ru(bpy)2(dppz)]2+. Reproduced from ref. 30 with permission from The Royal Society of Chemistry.

Fig. 5 Cell imaging with [Re2(m-Cl CO)6(m-1,2-diazine)] PNA filtered through 485/30 (a), 535/50 (b), and 600/40 (c) bandpass filters and superposition (d). Reprinted with permission from ref. 21 Copyright (2012) American Chemical Society.

with PNA units and showed good cell uptake, and interesting environment-dependent emission, with the presence of DNA apparently shifting emission maximum (Fig. 5). Group VIII: ruthenium and osmium Ruthenium(II) imaging agents are typically based on the [Ru(bpy)n]2+ core,5 with usually two simpler bpy or phen ligands and one which has been substituted with groups designed to endow some specific properties for the application in question. The [Ru(bpy)n]2+ core has ideal photophysical properties for imaging applications,22 but suffers from limited membrane permeability,23 and in some cases applications have been limited by the need for e.g. electroporation to assist uptake. Addition of lipophilic chains or extended hydrocarbon ligands tends to improve the uptake of Ru(II) complexes and a large range of related complexes have been reported.24 Amongst the most important imaging applications of Ru(II) complexes are in the fields of combined imaging and sensing, particularly sensing molecular oxygen by lifetime methods, as the luminescence lifetime of Ru(II) is reduced by triplet quenching of O2,25 and DNA detection with extended heterocyclic ligands such as dppz (dppz = 2,5-di-2-pyridylpyrazine) which forms complexes that are essentially non-emissive when free but highly emissive in the presence of DNA (Fig. 6 and 7).26 Both of these areas have been reviewed previously and here it is only necessary to note that lifetime methods are attractive as they are independent of concentration, while methods such as emission enhancement with dppz complexes suffer from problems of unknown local concentration in imaging, if uniform cellular distribution cannot be assumed, while a bright spot indicates the presence


of a probe and DNA, a dark spot may be a DNA-rich area which is inaccessible to the probe. These problems have recently been highlighted with related Re(I) dppz complexes, the localisation of which is easier to control than with Ru(II).27 While the application of Os(II) in imaging is almost unknown,28 the limited number of examples suggests that it should be applicable in the same way as Ru(II), and the similarity in geometry and size between 2nd and 3rd row complexes suggests that similar behaviour should be observed. Ru(II) and Os(II) complexes exist as chiral tris-chelate octahedral and thus as L and D isomers. In biological studies these should be regarded as different materials and resolved, although this is rare. As for Re(I), Ru(II) complexes are low spin d6 and thus metal-based toxicity is tamed, although some of the larger heterocyclic ligands form complexes which show appreciable cytotoxicity.29 Group IX: iridium and rhodium The commonest form of Ir based imaging agents are complexes based on Ir(III) with two cyclometallated ligands such as phenylpyridine (ppy) or similar with a third chelating ligand such as bpy, to form monocationic complexes of the general formula [Ir(ppy)2(N4N)]+ which exist as a pair of optical isomers.3 The very few Rh(III) complexes reported in imaging share this form.31 These complexes show a remarkable range of photophysical properties due to the nature of the excited state being variable as both the cyclometallating and the chelating ligands are changed from pure MLCT to purely ligand based, and a wide range of mixed states. As a result of this, their emission is tunable across the visible spectrum from blue to red, and they display lifetimes ranging from ns to ms. Iridium complexes typically show good cellular uptake because of the cationic charge and lipophilicity associated with the Ir(III) biscyclometallate core. It is often difficult to control the localisation of Ir(III) species due to this high lipophilicity, and while the lowspin d6 configuration leads to stable complexes which do not show toxicity related to the metal ion, certain complexes show ligand-dependent cytotoxicity.

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Group X: platinum

The f-block

Pt(II) forms luminescent complexes with a range of neutral N4N4N, formally anionic cyclometallating N4C4N and related ligands which can show high quantum yields.32 The photophysics of these complexes is interesting with the typical properties of triplet emitters being complemented with, in certain cases, the ability to be excited with low energy irradiation utilising their high two photon absorbance.33 Although simple Pt(II) terpyridine complexes are non-emissive, their alkynyl derivatives have been shown to self-associate and show 3MMLCT based emission in the NIR which has been used in cell imaging.34 These Pt(II) complexes with a d8 electronic configuration are usually square planar and can suffer from low solubility, but are typically stable and the few examples reported in imaging so far do not appear to be highly cytotoxic. Pt cyclometallates33a and Pt(II) porphyrins35 have both been used in time-gated studies, taking advantage of their extremely long lifetimes.

The bioactivity of rare earths has been known since the 19th century when cerium oxalate was used as antiemetic drug. Since then, lanthanide ions have been shown to have anticoagulant and antimicrobial properties40 and been used in multiple assays (immunoassays, protein staining, etc.). Their main current application is in MRI contrast agents, where Gd(III) chelates are widely used.41 Recently, advances in time resolved microscopy have stimulated interest in Eu(III) and Tb(III) species as cell imaging agents.42 In this section, the focus will be on those visible-lanthanide species, Eu(III) and Tb(III), and their contribution to the further development of lanthanide bioprobes in cell imaging.

Group XI: gold Several different structural types of gold complex have been used in cell imaging, with as yet no obvious patterns of localisation or toxicity to be identified. Much of the interest in imaging with gold has been driven by a desire to understand the cellular localisation of gold species which were designed as pharmaceutical agents, rather than in an attempt to develop useful imaging agents. Most of the complexes so far reported are organometallic complexes of Au(I) in which a C-donor (either NHC or alkynyl) forms a complex with the coordination sphere of the linear d10 Au(I) complex being completed with a neutral phosphine ligand. One particularly interesting example involves a dimeric NHC gold cyclophane in which in an attempt to understand the cytotoxicity of an Au(I) chemotherapy target, the structure was tuned to bring two Au(I) nuclei into close proximity and thus use Au–Au interactions to give a visible wavelength lumophores (Fig. 8 and 9).36 Other examples of Au complexes in imaging have been reported37–39 but it is not always clear that the emission is metal-based.

Photophysics of f-block imaging agents In the area of cell imaging, lanthanide compounds offer great photophysical advantages over conventional organic dyes in terms of Stokes shifts and luminescence lifetimes, which also exceed those of the d-block agents described above.43 Emission from lanthanide ions involves intra-configurational 4f–4f transitions, which are orbitally-forbidden transitions, so the excited state lifetime is long enough (ms–ms) to allow the use of time resolved techniques. In addition, their emission patterns are easily recognizable and the pattern barely affected by the environment as the 4f n electronic configuration is protected from the outside interactions by the more extended subshells (5s2 5p6). Also as a consequence of the orbitally-forbidden nature of the 4f–4f transitions, the absorption coefficient of lanthanide ions is very low and must be increased by a process called ‘‘luminescence sensitization’’ (Fig. 10). An antenna or a chromophore is grafted to the surroundings of the lanthanide species, which efficiently absorbs UV-visible radiation and transfers it to the emissive excited state of the lanthanide ion. In this way, the molar extinction coefficients could be increased from 10 mol 1 L cm 1 (uncomplexed trivalent lanthanide) to 104–105 mol 1 L cm 1 in a typical lanthanide antenna-sensitised complex.44 Several photophysical factors need to be controlled in order to optimize the sensitization process (Fig. 11): (a) Spectral overlap between the donor state of the chromophore and the absorption of the lanthanide is crucial. (b) The overlap with the emission of the lanthanide should be minimized to prevent back transfer processes and thus maximize signals. (c) Control of the coordination sphere is important for sensitization. Saturation of the inner coordination sphere prevents non-radiative deactivation of the excited state (i.e. quenching by groups such as O–H and N–H).

Fig. 8 Au–Au dimeric probe.36

Fig. 9 Cell imaging with the Au–Au probe. Reproduced with permission from ref. 36.

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Fig. 10 Representation of the sensitization process in lanthanide complexes.


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Fig. 11 Jablonski diagram of Eu3+.

Requirements for the design of lanthanide bioprobes In addition to the general requirements for imaging agents alluded to above, there are specific considerations which are important in lanthanides due to the electrostatic bonding, and the requirement for antennae: (a) high thermodynamic stability, (b) kinetic inertness, (c) intense antennae absorption at long excitation wavelengths, (c) an efficient energy transfer process, (d) long excited state lifetimes, (e) water solubility, (f) ability to cross cell membranes (high speed and low egress), (g) ability to localize within an uniform distribution in a particular organelle and, when relevant, (h) ability to conjugate with bioactive molecules. Not to perturb the cellular hemeostasis45 and to resist enzymatic degradation are also premises desirable. A more technical concern will be to design the bioprobe whose antenna can be excited at an already accessible wavelength, i.e. 337, 365, 405 nm or by two photon excitation close to the NIR optical window (700–820 nm), which best permeates cells and tissues. Lanthanide ions have large coordination numbers, in the range of 6–12, with 8–10 being the most common. They are hard cations so they have preference for anionic ligands with –O and/or –N as donor atoms. Building robust scaffolds for the lanthanide ions, and thus affording kinetically stable species, also overcomes problems such as their inherent toxicity. Classification of lanthanide probes by ligand families The imaging agents based on d-block metals are best grouped for discussion by metal, as each element has its own distinctive oxidation state, d-electron configuration (and hence preferred geometry) and reaction chemistry, however, as the f-electrons are sheltered the element is less important in the biophysical behaviour of the complexes than the ligand scaffold which surrounds it, and so Ln probes are best classified by ligand family. There are three main families of lanthanide ligands (a) polyaminocarboxylates, (b) b-diketonates and (c) chromophoric chelates (Fig. 12),46 from which polyaminocarboxylates, chromophoric chelates have emerged as leaders in cell imaging. Polyaminocarboxylates Polyaminocarboxylate ligands are the most common class and can be divided into two sub-categories, acyclic and cyclic. Apart from their high dentricity that confers saturation, they can be easily functionalised with antennae or bio-reactive groups. Research groups such as those led by Parker, Faulkner, Pope and Bornhop have driven the development of efficient lanthanide polyaminocarboxylate-based sensors and imaging agents.


Fig. 12

Examples of the different scaffold for lanthanide ions.46

b-Diketonates b-Diketone ligands have two oxygen atoms ready for coordination in a chelate mode. However, their bidentate character does not confer as much stability to the complex as the polyaminocarboxylates, and they tend to form unsaturated 3 : 1 complexes with lanthanides, allowing quenching by water. Despite that, one of the most successful lanthanide immunoassays, DELFIAs,47 is comprised of two b-diketones. Specifically b-naphthoyltrifluoroacetone and 2-thenoyltrifluoroacetone are used for enhancing the luminescence of Eu(III) and Tb(III) ions, although other lanthanide ions such as Sm(III) and Dy(III) can also be detected with this heterogeneous assay. Nonetheless, b-diketone derivatives have not made a great impact in the design of novel cell imaging agents. Some examples of composites based-lanthanide b-diketone derivatives have been reported by Tang and co-workers.48 but as this review is envisaged to focus on Eu(III) and Tb(III) molecules as cell imaging agents, composites and nano-lanthanide species will be deliberately avoided. Chromophoric chelates Chromophoric chelate ligands bind to lanthanides via multiple carboxylates and one or more aza-aromatic groups. The main difference between these and the aforementioned ligand types is that the aza-aromatic group acts as both chelator and antenna. Generally, this approach facilitates a rapid energy transfer and increases the quantum yield.49 Typical ligands are trisbipyridyl cryptates, polycarboxylato-terpyridine derivatives or helicates such ¨nzli and co-workers (bis-tridentate ligands as those developed by Bu based on picolinic acid and benzimidazole derivatives).50 In selecting the antenna, features such excitation wavelength and energy of the triplet excited state are crucial. The preferred sensitizers are those whose excitation wavelength is the longest to avoid excitation of other chromophores in the biological media (e.g. Trp, NADH). Organic dyes such as tetraazatriphenylenes, acridones, azaxanthones, azathiaxanthones and pyrazole-azaxanthones are widely used for this purpose (Fig. 13), especially with Eu(III) and Tb(III). As well as organic dyes, transition metal complexes have proved to be good sensitizers, especially

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Fig. 13

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Depiction of antenna derivatives.

Fig. 15 Representative Eu(III) emission spectra of different ligand environments: (A) Eu3+ in water; (B) Eu(DPA)+ in water; (C) Eu(DPA)33 in water.63 Reproduced from ref. 63 with permission from The Royal Society of Chemistry. Fig. 14

Example of a heteronuclear Eu–Ir species.60

for NIR-emissive lanthanides.51 Van Veggel and co-workers reported the first example of Nd(III) and Yb(III) with a transition metal complex, ruthenium trisbipyridine and ferrocene, as an antenna.52 Since then, a wide range of metal complexes including Pt(II),53 Pd(II) porphyrins,54 Re(I),55 Os(II),56 Ru(II),57 Ir(III)58 derivatives and Cr(III) and Zn(II) helicates59 have been used as sensitisers. To sensitize UV-visible lanthanide ions, a much higher energy is needed, and this has been achieved by De Cola et al. with Ir(III) complexes (Fig. 14).60 However, to the best of our knowledge, there have been no applications of transition metal sensitised lanthanides as cell imaging bioprobes. Lanthanide probes for cell imaging More than 90 Eu(III) and Tb(III) complexes have been investigated as cell imaging agents,61 as a result of several features which make them more attractive than the other lanthanides: their excited states are comparatively less sensitive to vibrational quenching; they have relatively simple emission spectra (see Fig. 15) and a correlation can be made between the relative intensity of the transitions and the ion’s symmetry and speciation; their wellstudied excited states simplify the selection of the sensitizer, i.e. a heterocyclic or an aromatic moiety with a triplet state energy of at least 2000 cm 1 above excited states of Eu(III) or Tb(III), 5D0 (17 200 cm 1) and 5D4 (20 400 cm 1), respectively. So far, the focus of cell imaging has involved two ligand classes: cyclic polyaminocarboxylates and chromophoric chelates, in particular helicate ligands. Parker and co-workers have made an extraordinary effort to clarify the relation between the structure of the cyclic polyaminocarboxylate bioprobes and their behaviour in cells.6e Research groups such as those led by Sames and Nagano have also contributed by developing similar europium and terbium polyaminocarboxylate derivatives. On the other hand, chromophoric chelate derivatives were mainly studied by the ¨nzli (helicate bioprobes).62 Further research groups led by Bu contributions were made by Nishimoto (macrocyclic bipyridine

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derivatives), Yoan (terpyridine-based bioprobes) and Xiao and coworkers (dipicoline based species). Cyclic polyaminocarboxylates Parker and co-workers have contributed extensively in this area and elucidating the relationship between the structure of Eu(III) and Tb(III) bioprobes and their behaviour in vivo has been one of their main goals in recent years. After analyzing many structurally-similar species in mouse skin fibroblasts (NIH-T3 cells), Chinese Hamster Ovarian (CHO) or carcinoma cells (HeLa), they have concluded that neither the complex charge, nor lipophilicity or donor group structure dominate the cell uptake and localisation behavior. In contrast, the nature and mode of linkage of the sensitizing chromophore is the key factor.64 Several experiments to elucidate the uptake mechanism were undertaken using inhibitors or activators of various endocytotic pathways (caveolae, clathin-mediated and macropinocytosis) which proved macropinocytosis to be the dominant/only uptake mechanism for these complexes (further supported by the clear decrease in uptake in cells incubated at 4 1C) (Fig. 16).65 An interesting feature of the macropinocytosis mechanism is that the vesicles formed are considered quite ‘‘leaky’’ which is attractive for the escape of the probe and its further localization. Toxicity assays were also undertaken and revealed that the nature of the lanthanide ion did not seem to affect the IC50 value significantly. Moreover, and as a general rule, neutral probes

Fig. 16 Three endocytotic pathways: (A) macropinocytosis, (B) clathrin mediated endocytosis, (C) caveolin-dependent endocytosis.


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Table 1 IC50 values from several lanthanide probes incubated with NIH 3T365 (see Fig. 17 for complex numbering)

Eu(III) vs. Tb(III)

Cationic vs. neutral

Nature of the antenna


[EuL1c]Cl3 [TbL1c]Cl3 [EuL1a]Cl3 [EuL1a] [EuL1c]Cl3 [EuL8]Cl3 IC50 (mM) 109

131

144

>240

109

72

seem to be less toxic than their cationic analogues, with the triflates being more toxic than the chlorides. The nature of the antenna is the most important feature controlling the toxicity. For instance, species with tetraazatriphenylene antenna seemed to be less toxic than those containing a pyridine or pyrazolelinked azaxanthone. Additionally, the nature of the substituents on the sensitiser plays an important role, probes having an appended tert-butyl group displayed even a higher toxicity (Table 1).66 As general rule, the cyclic polyaminocarboxylate derivatives are not toxic under the conditions of their application in cell imaging (i.e. 1–4 h incubation, r 50 mM) and those that appear to be toxic can be considered exceptions.67 The localization pattern of cyclic polyaminocarboxylate lanthanide derivatives can be divided into four main groups (a) lysosomal (b) mitochondrial (c) simultaneous mitochondria and lysosome localization and (d) nuclear. (a) Lysosomal localization was, by far, the most common localization pattern observed with this type of lanthanide probes preferred by around 80% of the species studied, Fig. 17(A). They have a fast uptake and an egress rate, and no correlation between the specific features of a probe’s structure and lysosome localization has been found so far.68 (b) Mitochondrial localization was seen for a smaller number of complexes at incubation times between 5 min and 12 h, Fig. 17(B.1). When incubation times were longer than 24 h, the localization was observed in the lysosomes instead. Therefore, it seems that longer incubation times promote trafficking processes between the mitochondria and the lysosomes, where degradation of the probe occurs.67b (c) Simultaneous lysosomal-mitochondrial localisation was seen for a series of complexes containing an azaxanthone chromophore linked to the scaffold through a pyrazole or pyridine Fig. 17(C.1).69 Those probes displayed fast uptake but slow egress and no toxicity. (d) Localization in the nucleoli, a protein rich sub-nuclear structure involved in ribosome assembly, was observed with complexes with an N-coordinated azathioxanthone sensitiser, Fig. 17D.70 They showed a relatively slow uptake and egress as well as toxicity (IC50 40–90 mM) attributed to the oxidative sulphur metabolism, as it was absent in their azaxanthone analogues.61 Further studies also indicated that this type of localization is characteristic in cells under stress i.e. when the cell membranes are more permeable. Therefore, probes such as [EuL1C]3+, [TbL2]3+ with a mitochondrial or lysosomal preference localise in the nucleoli or ribosomes after disrupting the cell permeability with surfactants such as saporin or by increasing the probe concentration or incubation times.64 The nature and the number of pendant arms also seemed to affect drastically the localization patterns. Therefore, analogous


species to [TbL7]3+ and [EuL8]3+ bearing only two pendant arms, [TbL9]3+ and [EuL10]3+, localises specifically in the lysosomes instead of the simultaneous localization pattern (mitochondria and lysosome) observed for the three pendant arm analogues, Fig. 17(C.2). Moreover, changes in the nature of the linker also influence the localization pattern. Whereas amide-linked azaxanthone sensitisers (e.g. [EuL5]3+) seem to promote mitochondrial localization,71 their methylene analogues (e.g. [EuL6]3+) accumulate in the lysosomes. Fig. 17(B.2). One method to control localization is the use of moieties with a known affinity for a particular organelle. Parker and co-workers used such an approach for the synthesis of a terbium probe bearing a 2-chlorophenyl-1-naphthyl in the scaffold whose affinity for the mitochondria led to accumulation in this organelle, see Fig. 17E. As a summary of the localization patterns, Fig. 18 shows a series of colocalisation experiments performed with different lanthanide bioprobes, each of them having preference for a different organelle. Recent advances with cyclic polyaminocarboxylates Parker and co-workers have reported a study in HeLa cells of two dibasic Tb(III) species with two trans-disubstituted azaxanthone chromophores, [TbL16]+ and [TbL17], which showed endosomal/lysosomal distribution at high concentrations (10–100 mM) but at much lower concentrations, (1 mM) less than the 10% of the cells, instead showed nuclear localization (Fig. 19).72 The stained cells were shown to be those undergoing division in the mitosis phase, where the membrane integrity was compromised, Fig. 20, and this was used to image changes in the packaging and assembly of chromatin from the prometaphase to metaphase, Fig. 21, suggesting that these bioprobes seem to be extraordinary start points for studying nuclear DNA in mitotic cells. Europium and terbium polyaminocarboxylate derivatives suitable for ratiometric assessment of lysosomal pH or measuring bicarbonate concentration directly in cellular mitochondria and in human serum have been developed.73 Very recently, Parker and co-workers have demonstrated that triazacyclononane with three p-substituted arylalkynyl groups can also be used as alternative lanthanide bioprobes, Fig. 22.74 Such species have greater extinction coefficients and quantum yields than their azacyclododecanyl-azathioxanthone or azaxanthone analogues, and [EuL19b] specifically stained mitochondria of various cell lines, Fig. 23. In addition to these studies, they demonstrated that the replacement of one of the antenna moieties with a methyl sulphonamide pendant arm led to a new probe suitable for the determination of pH changes within the endoplasmic reticulum of living cells, [EuL20] (Fig. 24 and 25).75 Sames and coworkers have also developed cyclic polyaminocarboxylate derivatives with applications in bioimaging. In particular, they have designed a lanthanide scaffold based on a polyaminocarboxylate derivative grafted with a carbostyril 124 chromophore. There is still some controversy over this ligand as Selvin and coworkers76 consider Cs124 one of the most efficient energy transfer donors synthesised for terbium and europium ions whereas Parker and coworkers suggested a

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Fig. 17 General scheme of some examples of lanthanide probes and their cellular localization affinity. Structural features that could be responsible for the localisation profile are highlighted in blue.

much modest efficiency because of a triplet excited state thermal repopulation (Tb) and charge transfer (Eu) deactivation process that reduces its ability as a sensitizer.77 Despite the polemic regarding the effectiveness of Cs124 as a lanthanide sensitiser, Sames and coworkers observed that when aqueous solutions of both Tb and Eu complexes are mixed, an impressive intramolecular energy transfer process from the terbium species to the europium species takes place. This energy transfer process was also demonstrated on the cellular membrane of HEK-293T cells (Fig. 26).78

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Nagano and coworkers also reported some acyclic polyaminocarboxylate species suitable for cell imaging,79 using a series of long lifetime probes, Fig. 27, with a new system for time-resolved long lived luminescence microscopy (TRLLM), managing to visualise cells after injecting [EuL22c] and [EuL22e] and to perform dualcolour imaging with [EuL22e] and [TbL24],80 see Fig. 28. Chromophoric chelates ¨nzli and coworkers have reported a series of homobimetallic Bu triple-stranded helicates with an overall composition of [Ln2(L)3].


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Fig. 18 Fluorescence microscopy images of several lanthanide probes co-localised with the corresponding commercially available dye to visualise the localisation pattern.64 Reproduced from ref. 64 with permission of The Royal Society of Chemistry.

Fig. 19 Depiction of the two trans-related azaxanthone cyclic polyaminocarboxylate terbium derivatives.72

They are highly luminescence due to a tight pseudo-tricapped trigonal prismatic geometry that perfectly protects the lanthanide ion from the environment. Eu(III), Tb(III), Sm(III) and Yb(III)


Fig. 20 Bright field and fluorescence microscopy images of HeLa cells undergoing mitosis incubated with [TbL16]Cl. A schematic cell cycle is also presented. Adapted from ref. 72 with permission of The Royal Society of Chemistry.

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Fig. 21 Fluorescence microscopy images taken every 5 minute intervals of stained chromatin with [TbL16]Cl in a HeLa cell undergoing division. Reproduced from ref. 72 with permission of The Royal Society of Chemistry.

Fig. 22 Depiction of p-substituted arylalkynyl-triazacyclononane europium species.74

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Fig. 25 Fluorescence microscopy images of [EuL20] colocalised with ER-Traker in NIH 3T3 cells. Reproduced from ref. 75 with permission of The Royal Society of Chemistry.

Fig. 26 Fluorescence microscopy images of luminescence and bright field of HEK-293T cells incubated with 20 mM [TbL21] (A–C), 20 mM [EuL21] (D–F), and a mixture of 20 mM [TbL21] and 20 mM [EuL21] (G–I) for 15 min.78 Reprinted with permission from ref. 78 Copyright (2011) American Chemical Society.

Fig. 23 Fluorescence microscopy images of [EuL19b] colocalised with Mito-Traker in NIH 3T3 cells. Reproduced from ref. 74 with permission of The Royal Society of Chemistry.

Fig. 27

Depiction of Eu(III) and Tb(III) species.79

Fig. 24 Scheme of [EuL20] and the sulphonamide coordination as a function of the pH value.75

can be sensitized by those helicates hence offering the possibility of designing bimodal probes, either a dual luminescent probe, i.e. introducing two different luminescent lanthanide ions such as for instance a Tb(III) and Eu(III), or luminescent and magnetic probes.81 During the past fifteen years, the ¨nzli has been tailoring research group headed by J.-C. Bu homo-82 and hetero-83 bimetallic helicates to tune properties such as solubility, luminescence and bioaffinity, in order to design optimal bi-functional probes, see Fig. 29. In addition, a great amount of work has been devoted to testing Eu(III) and Tb(III) helicates in several cancerous cell lines, HeLa, MCF-7,

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Fig. 28 Fluorescence microscopy images of one or two cells injected with: (a) [EuL22e] (left) and [EuL22d] (right) and (b) one cell injected with [EuL22e] and [TbL24] respectively. Adapted with permission from ref. 79b Copyright (2007) American Chemical Society.

HaCat, and noncancerous cell lines such as Jurkat.84 The most relevant outcomes of those cell analysis are: (a) the probes enter the cell by endocytosis, (b) they present slow egress and almost no leakage was observed after 24 h,


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Fig. 33 Time resolved microscopy images of a breast cancer tissue section pressed into a 100 mm wide microchannel loaded with [Eu2(L34)3] and [Tb2(L35)3] and the scheme of the detection. Reproduced from ref. 86 with permission from The Royal Society of Chemistry. Fig. 29 Depiction of the different functionalised helicate species and their overall geometry when coordinated with Ln(III).84

Fig. 30 Fluorescence microscopy images of a colocalization experiment of [Eu2(L27)3], ER tracker and Golgi tracker in HeLa cells and the densiometry traces of the merged image (blue: ER tracker blue, green: Golgi tracker, red: [Eu2(L27)3]). Reproduced from ref. 84a with permission of The Royal Society of Chemistry.

Fig. 31 Fluorescence microscopy images of [Eu2(L27)3] and [Eu2(L27)3] in HeLa cells. Reproduced from ref. 84d with permission of The Royal Society of Chemistry.

Fig. 32

the nucleus, co-localized with the endoplasmic reticulum (Fig. 30) (e) the behaviour of the internalized Ln(III) chelates is not influenced by the nature of the lanthanide ion, see Fig. 31. As selectivity was not seen without bioconjugation, Eu and Tb-helicates were conjugated to specific antibodies, see Fig. 32, in order to detect two biomarkers over-expressed by cells in human breast cancer tissue, ER and Her2/neu,85 which successfully established the potential of this family of probes in the breast cancer diagnosis (Fig. 33). Additionally, the bioaffinity experiment was combined with microfluidic technology promoting a significant reduction in the time-frame, quantities and therefore costs of analysis, features of great interest to make more dynamic breast cancer detection and therapy assignation.86 Following these examples of bioconjugated lanthanide probes within the chromophoric chelate group, Nishimoto and coworkers presented a macrocyclic bipyridine ligand bearing two c(RGDfK) peptides as a tumor-targeting moiety, (c(RGDfK) = Arg-Gly-Asp-DPhe-Lys). They demonstrated the selectivity of such Eu(III) probes for anb3-integrin on the surface of glioblastoma U87-MG cells. Furthermore, they highlighted the importance of the bioconjugation approach to selectively imaging a given analyte, in this particular case anb3-integrin,87 see Fig. 34. Yuan and coworkers also presented some chromophoric chelates with applications in cell imaging.88 They designed a terpyridine based-europium complex that could be used as an efficient singlet oxygen probe in HeLa cells when incubated

Depiction of the bioconjugated Tb and Eu bioprobes.85

(c) they did not show significant toxicity values, i.e. IC50 values are over than 500 mM, (d) their localization seems to be in late endosomes and lysosomes which are predominantly distributed around


Fig. 34 U87-MG cells incubated with [EuL36] (a, b) and incubated with [EuL37] (c, d). (a, c) Phase-contrast images, and (b, d) merged images of fluorescence microscopy and phase-contrast pictures. Reproduced from ref. 87 with permission of Elsevier copyright (2011).

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Fig. 35 (left) Fluorescence microscopy images taken every 5 minute intervals of HeLa cells incubated with [EuL38]Cl and TMPyP and (right) representation of the 1O2 detection process. Reprinted with permission from ref. 88. Copyright (2006) American Chemical Society.

with TMPyP (5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra( p-toluenesulfonate)). The terpyridine based-europium complex and TMPyP formed a cation–anion pair and were internalised in the cell by endocytosis. In the presence of 1O2 the probe forms its endoperoxide, which results in a significant enhancement of the luminescence intensity. Time gating techniques allowed observation of a rapid increase in the luminescence in the nucleus of the HeLa cell, where the 1O2 was being generated (Fig. 35). More recently, Xiao and coworkers have designed six new dipicoline-based species that seemed to localise in the perinuclear region of sertoi cells, Fig. 36.89 A comparison between the luminescence intensity showed the influence of the para substituent of the ligand. The luminescence intensity trend found was –N(Ph)2 > –N(CH3)2 > OCH3. Developments within the area of lanthanide basedchormophoric chelates are still at an early stage. More examples are still needed to make a fair comparison between the structure of the scaffolds and properties such as uptake processes, localization patterns, toxicity values, etc. Up to date, conjugation of the probes with biomolecules seems to be the best way to control all the parameters. As a result of the extensively studied library within the cyclic polyaminocarboxylate lanthanide family, some general ideas could be drawn out that might be helpful to anticipate their behaviour in vivo: (a) Two pendant arm scaffolds seem to favour lysosomal localisation. (b) Azaxanthone linked to the scaffold through a pyridine or pyrazole appears to promote simultaneous mitochondria– lysosome localisation.

Fig. 36 (left) Scheme of ligands L(40–42). (right) Fluorescence microscopy images of [Tb(L42)3]NO3 in steroil cell. Reproduced from ref. 89 with permission of Elsevier copyright (2011).

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(c) N-coordinated azathioxanthone seems to direct the localization towards the nucleoli. (d) Disrupting the cell permeability with surfactants or increasing the probe concentration or incubation time appears to induces localization within the nucleoli and extracellular ribosomes. Such ideas have to be approached very carefully as they sometimes reflect the trend for a specific type of scaffold, e.g. azacyclododecanyl derivatives, and rarely if ever could be considered as an absolute general rule. Overall, however, it seems clear that both within the chromophoric chelate and the polyaminocarboxylate classes it is possible to rationally design highly luminescent and non-toxic (or low toxicity) imaging agents which will be taken up well by considering charge and ligand lipophilicity, and that within new systems there is potential to tune their localisation by either chemical changes to ligand/chromophore structure (polyaminocarboxylates) or bioconjugation (chromophoric chelates).

Prospects for developments of, and recent and innovative developments with, metal complexes in cell imaging and related applications Bimodal imaging agents based on metal complexes Metal complexes have been widely applied in cell imaging, which provides very high resolution microscopic details, but is incapable of deep tissue penetration such as seen with MRI, PET, etc. An ideal imaging technique would provide both full body and cellular level imaging, but this is currently impossible. If more than one imaging mode must be employed it is better if the same contrast agent can be used for both techniques limiting the patient’s exposure to the toxic chemicals. Thus, bimodal agents which work in cell microscopy and other techniques are of great interest.90 As mentioned above, gadolinium is often used in MRI, and d-block complexes have been used to sensitise lanthanide emission. However, a combined d-block gadolinium complex shows not sensitised emission (due to the photophysically inert f7 state) but d-based emission and enhanced relaxivity for MRI, again due to the f configuration and interaction with coordinated water.91 This is one of the few examples of potential MRI/optical bimodal agents, there are far more examples based on optical/radioimaging (PET/SPECT)92 as there are metal complexes which are involved in MLCT mechanisms which have useful radionuclides. In particular, Tc(I) analogues of Re-based cell imaging agents have attracted much attention following the first report of an agent ‘‘breaching the gap’’ in which the radiolabelled Tc(I) species and the luminescent Re(I) analogue were compared and shown to mimic each other’s biological behaviour.93 The Tc–Re analogy has been widely exploited with ligands which allow incorporation into peptides,18 but also more recently with ligands incorporating units such as porphyrins which allow the possibility for combined imaging (in both modes) and therapy, through the generation of phototoxicity


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Fig. 37

Single Core Multimodal Probe for Imaging (SCoMPI).96

Fig. 38 Cell imaging with SCoMPI co-localisation with Golgi stain (LHS) and IR microscopy (RHS). Reproduced from ref. 96 with permission of The Royal Society of Chemistry.

with photodynamic therapy.94 Mixed metal agents are known in which a single complex incorporates both one metal which is used for MLCT-emission and a binding sites for a second metal ion which has radio-imaging properties.95 Finally, one particularly exciting area is the development of agents which utilise M-CO IR/ Raman bands as tissue-penetrating imaging modes, along with luminescence from the metal centre. A Re(CO)3(N4N) complex was recently reported which combines MLCT luminescence with IR bands which were utilised in what the authors coined termed a Single Core Multimodal Probe for Imaging (SCoMPI) (Fig. 37 and 38).96 Prospects for future developments with metal complexes in imaging It is clear that the real strengths of metal complexes in optical imaging lie in the differences between their photophysical properties and those of organic dyes, and in the other properties which the presence of a metal can bring to a biological arena. It is likely that metal complexes will find much of their role in specialist applications requiring particular properties such as large Stokes shifts or long lifetimes, rather than replacing organics as the standard fluorochromes in everyday work. Their advantages will be exploited more as new spectroscopic/detection techniques such as differential excitation and time-resolved methods become more developed. The large range of different photophysical mechanisms of excitation and emission in metal complexes (e.g. metal-based and or triplets) gives a range of excited state reactions and interactions useful for sensing e.g. 3 O2 and charge transfer states available with d-block complexes allow excited state reaction and energy and electron transfer, making sensing of biomaterial possible through deactivation or exchange of excited states (FRET). Extension of the emission wavelength into the Near IR (NIR) gives greater tissue penetration and this is possible with both d- and f-block complexes. Pt porphyrins with intense NIR emission (lem = 758–767 nm,


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F = 0.1–0.12, t = 24–60 ms) have already been used in cell imaging,97 and there are many reports of NIR emitting f-block complexes which have been proposed as bioprobes and imaging agents.98 The toxicity of some metal ions also allows the possibility of combined imaging and therapy by controlled release of the metal ion from an imaging agent. The combination of radioisotopes of metals which are used in cell imaging, and of M-CO IR bands offers great scope for metal complexes to develop as bimodal imaging agents with emission microscopy providing sub-cellular details and the other modalities allowing whole body scanning due to deeper tissue penetration. In addition to the intrinsic toxicity of some metal ions, the charge-separation involved in many of the excited states of metal-based imaging agents can be utilised in combined imaging and phototoxicity by inducing redox reactions with biological chemicals. Small changes in ligand structure can induce dramatic changes in these properties,99 and multielectron shuttles can also be induced100 suggesting many possibilities using the energetics of the excited states in reaction chemistry during imaging experiments. Finally, the range of mechanisms by which metal complexes can emit photons (e.g. chemiluminescence, electroluminescence) as well as by direct photoluminescence and the ease of tuning these properties offer new opportunities for sensing and detection within the framework of luminescence imaging, offering an extension to the modality which complements the potential for multimodal agents.101

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