Near-Infrared Fluorescent Nanomaterials for Bioimaging and Sensing

Transcription

1 Near-Infrared Fluorescent Nanomaterials for Bioimaging and Sensing Philipp Reineck* and Brant C. Gibson A great challenge in noninvasive biomedical imaging is the acquisition of images inside a biological system at the cellular level. Common modalities used today such as magnetic resonance or computed tomography imaging have the advantage that any part of a living organism can be imaged at any depth, but are limited to millimeter resolution and can usually not be employed e.g., for surgical guidance. Optical imaging techniques offer resolution on the 100 nanometer scale, but are limited by the strong attenuation of visible light by biological matter and are traditionally used to image on the surface. Near-infrared light in the biological windows can penetrate much deeper into biological samples, rendering fluorescence-based imaging a viable alternative. In the past two decades, many fluorescent nanomaterials have been developed to operate in the near infrared, yet only few materials emitting above 1000 nm exist and none are approved for clinical use. This review describes recent advances in the development and use of near-infrared fluorescent nanomaterials for biomedical imaging and sensing applications. The physical and chemical properties as well as the bioconjugation and application of materials such as organic fluorophores, semiconductor quantum dots, carbon-based materials, rare earth materials, and polymer particles are discussed. 1. Introduction In a photograph, optical contrast is generated by certain features in the image absorbing more light than others. Differences in attenuation of light of a certain wavelength are still used today in many biological and medical imaging modalities such as bright-field microscopy and X-ray imaging. Many other intrinsic material properties including refractive index (phase contrast microscopy) or nuclear spin (magnetic resonance imaging, MRI) are exploited as well to create two and three dimensional representations of biological systems. Contrast agents are often used to enhance the image contrast or to clearly mark certain areas such as a tumor or vasculature. Clinical examples are MRI contrast agents such as paramagnetic gadolinium, radioactive materials for positron emission tomography (PET), and iodine and barium in X-ray and computed tomography (CT) Dr. P. Reineck, Prof. B. C. Gibson ARC Centre of Excellence for Nanoscale BioPhotonics & School of Science RMIT University Melbourne, VIC 3001, Australia philipp.reineck@rmit.edu.au DOI: /adom imaging. One of the few clinical examples of the use of fluorescent contrast agents for imaging is retinal angiography using organic fluorophores, which has been used for many decades. Only few fluorescent substances are approved by the U.S. Food and Drug Administration (FDA) and only one molecule that fluoresces in the nearinfrared spectral region at about 830 nm, indocyanine green (ICG). [1] Therefore, fluorescence-based imaging technologies are still predominantly used in laboratory settings for diagnostic purposes (e.g., enzyme-linked immunosorbent assay (ELISA), western blot even though optical absorption measurements are still the standard), for imaging of animal models and high resolution imaging in vitro using immunohistochemistry for the fluorescent labeling of biological targets. Fluorescence-based imaging can make use of both intrinsic fluorescence of biological matter (so-called autofluorescence) and fluorescence from materials added to the biological system such as organic fluorophores. Situated between these two approaches are fluorescent proteins, which are synthesized (or expressed) by the cell or organism itself when the appropriate DNA sequence is incorporated into the genome of the organism under investigation. Auto-fluorescence-based imaging has the advantage of minimal perturbation of the system by the imaging process by the excitation light used for imaging itself. However, many structures cannot be clearly delineated and the identification of different endogenous fluorophores inside the biological system, for example based on their fluorescence spectra, is often difficult. The strength of immunohistochemical labeling is the ability of many fluorescent nanomaterials to overcome most biological barriers such as cell membranes resulting in a clear optical separation of labeled molecules, cells or tissues from other structures. In this context one fundamental challenge is the perturbation (e.g., toxicity, cell stress, interference with signaling pathways) of the system under investigation introduced by the fluorescent material itself. Generally, fluorescence-based imaging techniques offer high spatial and temporal resolution compared to MRI, CT, PET or ultrasound imaging. [2,3] For in vitro applications, super resolution microscopy offers optical resolution well below 100 nm [3] and most standard fluorescence microscopes offer fast image acquisition on the millisecond timescale allowing many cellular 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com (1 of 26)

2 processes to be imaged in real-time. A great challenge for in vivo fluorescence-based imaging is the scattering and absorption of light by biological matter. Subcellular structures can be clearly resolved in an in vitro setting because excitation light can be delivered and fluorescence collected with high efficiency (and due to the relatively static nature of the sample compared to a living organism). However, visible light (400 nm 650 nm) is strongly attenuated by collagens, hemoglobin, lipids to name just a few, rendering deep tissue imaging all but impossible in this spectral region. (Here, we will use the term attenuation synonymous with extinction, which is the sum of scattering and absorption.) Furthermore, biologically important molecules such as collagen, NADPH, fatty acids, flavins and porphryns show strong auto-fluorescence throughout the visible spectrum. [4,5] Therefore, for in vivo applications attention has shifted to the two so-called near-infrared biological windows from 650 nm to 950 nm (NIR1) and 1000 nm to 1350 nm (NIR2). In these spectral regions light can penetrate significantly deeper into biological matter (see Figure 1) and is not yet strongly absorbed by water, whose absorption increases steeply towards longer wavelengths. Endogenous fluorescence of biomolecules is significantly weaker in these spectral regions as well. [4,5] Using mostly established fluorescent materials such as organic fluorophores, two-photon microscopy (TPM) makes use of the higher transparency in the NIR1 region and has been used extensively in the past decade for deep tissue imaging applications. [6 8] It is an intrinsically confocal technique and excitation usually occurs between 800 and 1000 nm, while fluorescence is collected in the visible. However, TPM requires costly tunable femtosecond pulsed lasers to locally create extremely high light intensities to enable two-photon excitation of fluorophores. Furthermore, fluorescence below 600 nm is collected, which again is strongly attenuated. This review focuses on materials that fluoresce in the NIR1 and NIR2 spectral regions, while also including some new materials showing red emission between 600 and 650 nm. The Philipp Reineck is a research fellow at the ARC Centre of Excellence for Nanoscale BioPhotonics at RMIT University in Melbourne. He graduated in Physics from the University of Munich (LMU) and received his PhD in Materials Engineering from Monash University in His research interests span many areas including biophysics, fluorescent nanomaterials, plasmonics, nanoparticle chemistry and self-assembly, nano-photonics and bioimaging. His current focus is the development of fluorescent nanomaterials for imaging and sensing applications in biology. Brant Gibson is currently the RMIT University Node Leader of the Australian Research Council (ARC) Centre of Excellence for Nanoscale BioPhotonics. He received his PhD in Photonics from La Trobe University, Melbourne in He has wide-ranging research interests in the areas of diamond, fluorescent nanoprobes, wide band gap materials, single photon sources, quantum information, hybrid nanomaterial integration, fibre optics, photonics, biophotonics, optical, confocal, and atomic force microscopy. Figure 1. Near-infrared optical windows in biological tissues. The effective attenuation coefficient graphed as a function of wavelength shows that absorption and scattering from oxygenated blood, deoxygenated blood, skin and fatty tissue is lowest in the NIR1 and NIR2 spectral regions. Reproduced with permission. [9] Copyright 2009, Macmillan Publishers. ideal fluorescent nanomaterial has a high absorption cross-section coefficient and a fluorescence quantum yield of one (i.e., every absorbed photon results in one photon emitted by the material). It is photostable as well as chemically stable, exclusively interacts with the biological system in the desired way, e.g., attaches to a particular protein or structure, and leaves the system once the imaging or sensing measurement has been carried out. The material is only few nanometers in size to enable fast diffusion, penetration of tissues, homogenous staining and high resolution imaging of cellular and subcellular structures. Yet it should be bright enough to track individual particles in order to investigate for example the active transport of single proteins. Ideally it shows a very narrow fluorescence spectrum and is available in different colors to enable hyperspectral imaging and the simultaneous identification of many different objects inside the biological system. For sensing experiments the material is also sensitive to picomolar concentrations of the analyte with high specificity. Of course, this ideal material does not exist, and all materials have their strengths and weaknesses. The aim of this review is to discuss the stateof-the-art of fluorescent nanomaterials in the NIR1 and NIR (2 of 26) wileyonlinelibrary.com 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3 Figure 2. Illustration and overview of the material classes discussed in this review. Images are artistic impressions only and not drawn to scale. Quantum dot: Reproduced with permission. [15] Copyright 2009 American Chemical Socienty. Metal cluster: Reproduced with permission. [16] Copyright 2009, Royal Society of Chemistry. Rare-earth NP: Reproduced with permission. [17] Copyright 2014, American Chemical Society. spectral regions for bioimaging and sensing applications and to guide potential users of these materials to the materials best suited for their application. Due to the large number of publications in this field, this review is not comprehensive; and in fact a single review on this topic cannot be. Other recent publications in this field include a review by Hemmer et al. on materials emitting above 1000 nm, [10] a general review on nanoparticles for bioimaging by Wolfbeis, [11] a review on nanoparticles as biological sensors, [12] as well as many other more specialized reviews cited throughout this manuscript. The manuscript is structured as follows: Different classes of materials are introduced and their physical and chemical properties discussed including their optical properties and colloidal stability. Then bioconjugation strategies are discussed for the different materials as well as their toxicity. The last section of the review focuses on imaging and sensing applications of all materials. Throughout the manuscript, the wavelength λ em indicates the spectral position of the fluorescence emission maximum of the material discussed. 2. Material Classes Fluorescent nanomaterials can be grouped in classes in many different ways. Often drawing a clear line between two types of materials is difficult and to some extent arbitrary. Nonetheless, classes help to describe general advantages and drawbacks of most materials within a class and to describe trends. For example an advantage of organic fluorophores is their high fluorescence brightness. Of course this is not true for all organic fluorophores, but it is correct that well engineered organic fluorophores remain among the brightest fluorescent materials that exist today in particular relative to their size. [13] Classes also help to clarify material terminology, which is all but consistent in the current literature. The term quantum dot for example can refer to a wide range of materials from graphene based materials and graphitic carbon particles to semiconductor nanoparticles. In this section, we group materials based on the material they consist of, introduce the terminology used throughout this review and very briefly discuss general fluorescence properties and the stage of technological development of the material type. Wang et al. have reviewed commercially available nanoparticles (including fluorescent particles) for stem cell labeling and tracking and list the major companies in this sector and some of their products. [14] The material classes discussed in the following are also illustrated and summarized in Figure Molecules and Polymers Fluorescent Proteins Green fluorescent protein (GFP) was discovered more than 50 years ago, [18] has been used as a biological marker for more than 20 years, [19] and fluorescent labeling protocols are well established. Only recently have NIR1-emitting proteins like mneptune1 (λ em = 650 nm) and TagRFP657 (λ em = 657 nm) been developed. [20,21] The great advantage of fluorescent proteins in general is that they can be expressed endogenously by any prokaryotic or eukaryotic cell. Red fluorescent proteins are structurally very similar to e.g., GFP and consist of the typical beta-barrel scaffold, but with different chromophores in their center. [22,23] To date, fluorescent proteins that emit in the NIR2 region do not exist. Fluorescent proteins can also be used to sense for example metal ions [24] and voltages [25] among others in biological systems. [26] However, the majority of fluorescent proteins used for sensing operate below a wavelength of 600 nm WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com (3 of 26)

4 Organic Fluorophores A vast range of organic fluorophores (OFs) that emit in the NIR1 region exist. Most OFs are commercially available and are the standard for immunohistochemical labeling in biology today. Organic fluorophores fluorescing in the NIR2 spectral region are usually not water soluble, while the first water soluble NIR2 emitting organic dye was reported very recently. [27] Commonly used molecule families include cyanine, squarine and boron-dipyrromethene (bodipy). [28] Most dyes are also available with various functional groups or already conjugated to biomolecules such as the primary antibody immunoglobulin G (IgG) or secondary antibodies (e.g., anti-igg) for targeted imaging. Frequently used OFs in the NIR1 region are Atto 750, DY-780, Alexa 790, Cy7 and IRDye800-CW to name just a few. [29] OFs for the detection of metal ions, reactive oxygen species (ROS), hydrogen peroxide, hydrogen sulfide and enzymes as well as to measure ph and temperature have been developed, which operate in the NIR1 region. [30,31] Polymeric Materials Polymers can either be used as an optically inactive host material for fluorophores (often referred to as a fluorescent bead) or it can exhibit intrinsic fluorescence. Here, we refer to the latter, while fluorophore-doped particles will be discussed in the following subsection. Dendrimers (also called hyperbranched macromolecules) like poly(amidoamine) are one type of intrinsically fluorescent polymer. [32] While these molecules usually fluoresce in the blue and green part of the spectrum, dendrimers emitting well above 800 nm have been reported recently. [33] Conjugated polymer nanoparticles are a fast emerging class of fluorescent nanoparticles, which we will here refer to as polymer dots (p-dots). Common polymers used for the synthesis of p-dots are polyfluorene, poly(p-phenylenevinylene) (PPV) and poly(phenylene-ethylene) (PPE). Most of these polymers fluoresce in the visible part of the spectrum, are not water soluble and therefore must be encapsulated, for example in a water soluble polymer, to be used in bioimaging applications. [34 38] Recently, several p-dots fluorescing in the NIR1 [39 41] and NIR2 [42] have been reported and used for bioimaging. P-dots are not commercially available as yet. OF, fluorescent beads with emission peaks in the NIR1 [43] and NIR2 [44] can be synthesized. Fluorescent beads emitting up to 700 nm are also commercially available. [14,45] Many conjugated polymer particles and fluorescent beads as well as other particles based on lipids, nanogels, micelles and vesicles are often referred to as soft fluorescent nanoparticles. [37] These particles are important for a range of imaging applications since they can be used for multimodal imaging, [38] often use biodegradable matrices doped with fluorescent small molecules or nanoparticles and can be tuned to specifically interact with a biological system e.g., for targeted cell imaging. [37] 2.2. Semiconductor Nanoparticles Cassical compound semiconductor nanoparticles will here be referred to as quantum dots (QDs). The first QDs in a colloidal form were discovered by Brus in 1985 and were ZnS and CdS particles. [46] Today, an astounding number of different types of particles exist in this field, whose fluorescence can be tuned from the visible to the far NIR2 spectral region. They consist of combinations of elements from the transition metal, post-transition metal, metalloid groups as well as phosphorus, sulfur and selenium. [47] Most particles are chemically synthesized from precursor salts in organic solvents. Particles fluorescing from the red to the NIR2 are commercially available, often with biofunctionalization. QDs can be used for e.g., ion [48] and ph sensing. [49,50] However, QD-based sensing often requires an organic molecule as a ligand in combination with electron or energy transfer between QD and the ligand to be efficient Core-Type Quantum Dots Some of the most well-known QDs such as CdSe, CdTe and PbS belong to this particle type. Here, the emission wavelength of the particle is mainly determined by its size. CdSe and CdTe QDs can be tuned from the visible to the red and NIR1 spectral region, while other material combinations such as PbS, InAs, and Ag 2 S show fluorescence up to the NIR2. [47,51] Fluorescent Beads The term fluorescent bead commonly refers to an optically inactive silica or polymer particle matrix with organic fluorophores either incorporated in the particle or attached to its surface. Common host materials are silica, poly(methyl methacrylate) (PMMA), polystyrene and poly(lactic-co-glycolic acid) (PLGA), which can have various shapes and sizes from a few nanometers to several micrometers. [38,43] A wide range of strategies for the incorporation of fluorophores into or onto these particles exist including physical entrapment and covalent attachment of the fluorophore to monomer building blocks [32] as well as emulsion, nano-precipitation and selfassembly. [43] Since the optically active component is usually an Core Shell Quantum Dots The optical properties of these particles are usually still determined by the core. The main function of the shell is to optimize e.g., the photostability and fluorescence quantum yield of the particles. Depending on the relative band gap of core and shell material, the shell can also be used to shift the emission spectrum of the particles. [47] Examples for common core/shell systems emitting in the NIR1 region are CdTe/CdSe, CdSe/ ZnS and InP/ZnS Alloyed and Doped Quantum Dots Another way to engineer the optical band gap of quantum dots is the synthesis of ternary alloys or doped quantum dot (4 of 26) wileyonlinelibrary.com 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

5 particles. For example the emission of CuIn x Se y QDs can be tuned between 600 and 1000 nm by changing the relative content of In and Se as well as the particle size. [52] Similary, the fluorescence of InP QDs can be shifted from the visible to the NIR1 and NIR2 regions by doping with Cu. [53] Silicon Quantum Dots fluorescence wavelength of c-dots is usually size dependent and occurs throughout the visible and red spectral regions, but NIR emitting c-dots have not been reported to date. Unlike most other materials discussed here, the fluorescence wavelength of c-dots depends on the excitation wavelength. C-dots have been used for metal ion as well as ph sensing [65] and are commercially available, but not with biological surface modifications. Silicon QDs show fluorescence from the visible to the NIR1 [54] and can be extended into the NIR2 region by doping with phosphorus and boron. [55] A major advantage of Si QDs is the absence of toxic elements Others There are other fluorescent wide bandgap semiconductor nanomaterials such as boron nitride [56] and zinc oxide, [57] which fluoresce mostly in the visible part of the spectrum and have also been employed for bioimaging applications. [58 60] 2.3. Carbon Materials Nanodiamonds Nanodiamonds can be divided into two groups: detonation nanodiamond and non-detonation diamond. Detonation nanodiamonds are recovered from a detonation soot and are usually around 5 nm in size. [61] Non-detonation nanodiamonds are made through milling of millimeter sized crystalline diamond samples to particles of any size from about 15 nm to several micrometers. [62] Diamond shows only weak intrinsic fluorescence and is generally made fluorescent through a combination of irradiation, annealing and chemical purification processes. [63] The fluorescence originates from so-called color centers in diamond. The nitrogen vacancy (NV) center is the most wellknown and the emission spectrum is not size dependent. Most color centers in nanodiamonds fluoresce in the red and NIR1 region, while fluorescence in the NIR2 region has not been reported to date. Nanodiamonds are excellent temperature as well as magnetic and electric field sensors. [64] However, these measurements generally require an external microwave field to detect e.g., a magnetic field from optically detected magnetic resonance (ODMR) experiments. Fluorescent nanodiamonds of all sizes are commercially available with and without basic biofunctionalizations Carbon Dots Carbon dots (c-dots) also referred to as carbon quantum dots consist mostly of graphitic carbon that contains significant amounts of oxygen, hydrogen and nitrogen. A large number of synthetic protocols have been reported. [65 68] Nanocrystalline graphite is commonly used to describe the particle material. In this review, the term c-dot does not refer to two-dimensional materials or diamond (sp 3 crystalline carbon) particles. The Graphene-Related Materials All graphene-related materials are comparatively novel. Graphene is a single atomic layer of carbon atoms arranged in a honeycomb lattice structure and is not fluorescent. Nanoscale graphene sheets become fluorescent through oxidation or doping e.g., with nitrogen [69] or boron. [70] Single sheets of nitrogen doped graphene quantum dots emit blue light, [69] but through layering [71] or variation of the doping level [72] can be made to fluoresce from the UV to the red and NIR1 regions. Graphene oxide particles dispersed in water show red to NIR1 fluorescence. [73] Single-walled carbon nanotubes (SWCNTs) have a very characteristic and broad fluorescence spectrum in the NIR2 and have been used for deep tissue imaging [74] as well as numerous sensing applications. [75] Fullerenes (C60 and C70) show fluorescence in the NIR1 region in organic solvents, [76] but are poorly water soluble and exhibit visible to red fluorescence when chemically modified to enhance water solubility. [77] Many graphene-related materials are commercially available, but not functionalized for biological applications Metal Clusters Metal clusters usually consist of only several tens of metal atoms like Au, Ag, or Cu and unlike larger metal particles exhibit molecule like properties including discrete energetic states that lead to fluorescence. [78] They are made via wet chemical reduction of metal ions in solution or etching of larger particles [79,80] and are stabilized in solution by ligands. The fluorescence of Au nanoclusters can be tuned form the visible to the NIR1 spectral region [81] and has been used for biological sensing and imaging applications. [79] Ag clusters mostly emit in the visible and up to about 800 nm in the NIR1 [82] and have also been used for bioimaging [83] and sensing, e.g., protein detection. [84] Despite significant recent scientific interest in metal nanoclusters as biolabels and sensors, fluorescent clusters are not commercially available yet Rare-Earth-Based Particles Upconverting Rare Earth Nanoparticles Upconverting nanoparticles (UCNP) consist of a crystalline lanthanide host matrix such as NaYF 4, which is often doped with other lanthanides commonly referred to as sensitizers (e.g., Yb 3+ or Er 3+ ). [85] Upconversion of NIR1 light to higher energy photons can be achieved via a number of complex photophysical mechanisms and strongly depends on the nanoparticle design. [86] The 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com (5 of 26)

6 particles are usually excited at 980 nm and emit light in the visible and up to the NIR1. [85,87] Relatively high excitation intensities (typically on the order of 100 to 1000 W/cm 2 ) are required for their imaging as well as nonstandard excitation sources and emission filters. [88] The fluorescence quantum yield is strongly excitation intensity dependent and increases with increasing excitation intensity. [89] UCNPs have been used successfully in in-vivo [90] and animal imaging [91] as well as a number of chemical sensing applications. [85] UCNPs are commercially available with basic biofunctionalizations for targeted bioimaging Downconverting Rare Earth Nanoparticles Other rare earth materials of very similar composition such as NaYF 4 doped with Yb 3+ and Ho 3+ show downconversion emission (like most other materials discussed here) when excited at 980 nm and emit in the NIR2 spectral region. [92,93] More complex core/shell structures with multiple shells have been reported, which are excited at 800 nm and emit photons above 1500 nm in the far NIR2. [94] Compared to UCNPs downconversion rare earth nanoparticles (DCNPs) have received far less attention. Nonetheless, DCNPs have also been used for bioimaging studies in vivo. [93 95] Li and coworkers have reported a particle system capable of up- and down-conversion upon excitation at 800 nm and 980 nm, respectively. [96] 2.6. Others One well-known material that does not fit into any of the above categories is ruby, i.e., Cr doped aluminium oxide. It shows a narrow emission peak at 693 nm. Ruby nanoparticles have recently been used for bioimgaing. [97,98] 3. Physical and Chemical Properties 3.1. Size The sizes of fluorescent materials that can be used for bioimaging and sensing span the entire nanometer scale (Figure 3). The size of a fluorescent label or sensor, in particular relative to the size of the structure to be investigated, is vital for a bioimaging experiment. From a physical point of view, the size of a particle or molecule determines its diffusion properties. The smaller the substance is, the faster the passive, diffusion-driven distribution of this material throughout a biological sample. In many experiments other factors such as the biochemical surface groups of the fluorescent material and its active and passive transport through cell membranes and tissue will be even more important. The size of the material also determines the smallest feature size that can be resolved in an image. The dimensions of nanomaterials are per definition well below the diffraction limit of visible light (ca. 250 nm for green light). Nonetheless, smaller particles of only a few nanometers in diameter are usually better suited to clearly and homogenously delineate subcellular structures than 100 nm sized particles. This is certainly true when super-resolution techniques are employed, which increases the resolution to well below 100 nm. Organic dyes and metal clusters are the smallest fluorescent labels available today with sizes from below one up to a few nanometers. They are significantly smaller than most proteins, Figure 3. Typical fluorescent nanomaterial sizes compared to important biomolecules and a virus. The red lines show the typical size range for different fluorescent materials. The x-axis positions of biomolecules and virus indicate their approximate sizes for comparison. Note that these are typical sizes only. Biomolecules and virus are not drawn to scale (6 of 26) wileyonlinelibrary.com 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

7 DNA or lipid bilayers. Fluorescent proteins, semiconductor quantum dots and carbon dots are usually 2 10 nm in size and therefore have a size comparable to that of e.g., hemoglobin. However, it is important to note that semiconductor quantum dots are generally not water soluble without ligand molecules or a shell, which significantly increases their size. Hence, the size of quantum dots in biological applications is usually well above 15 nm. The effect of ligands on the size (hydrodynamic radius) of particles will be briefly discussed in the next paragraph. Carbon dots and most quantum dots have spherical shapes, while for example CdSe QDs can also be synthesized in rod shapes. [99] Dendrimers, graphene oxide and doped graphene sheets are usually in the same size range, but can have a wide range of sizes depending on their synthesis conditions, while graphene-based materials are always <1 nm in thickness (unless they are layered) as they only consist of one atomic layer of carbon. Carbon nanotubes are significantly below 100 nm in diameter and can reach lengths of several hundred nanometers. [75] Nanodiamonds are often made from bulk diamond samples via ball-milling and can therefore have any size from more than 200 nm down to about 15 nm, [100] but usually show high polydispersity and are of random shape. Most diamond particles around 5 nm in size are produced via a detonation synthesis and are roughly spherical in shape, but only show weak fluorescence. [61] Fluorescent beads can be synthesized in sizes from several microns to few tens of nanometers and are also usually spherical. Lanthanide based upconversion NPs can be synthesized as small as 5 10 nm, [87,101] while most particles of this type are several tens [102] and up to hundreds of nanometers in size. [103] We have so far focused on the physical size of particles without a ligand, shell or biofunctionalization. However, in many biological applications some or all of these may be required, which significantly increases their hydrodynamic radius, which takes into account the particle size increase due to ligands and interactions with solvent molecules. The hydrodynamic radius is commonly determined in dynamic light scattering experiments, while the bare particle size is determined for example via transmission electron microscopy. The hydrodynamic radius is also very useful for the size quantification of biomolecules. [104,105] Another factor complicating the precise size determination of nanoparticles in biological experiments is the protein corona. [106] In most complex fluids such as serum or blood, proteins present is that fluid will adsorb to the nanoparticle surface and create a protein corona around the particle, which also affects the particle's hydrodynamic radius, its diffusion properties and its faculty to specifically target other molecules. [106] visible as well), but the physical origin of this fluorescence is very different among them. Delocalized π electrons in aromatic groups of organic dyes result in well-defined electronic ground and excited states that give rise to fluorescence. Quantum confinement in semiconductor particles a few nanometers in size creates size-dependent photon emission in the otherwise nonfluorescent materials. Optical transitions of defects and dopants in materials like diamond or alumina also result in very stable emission of photons upon light excitation. The emission of most particles or molecules can be tuned across a certain wavelength range (Figure 4). Carbon dots and fluorescent proteins mostly fluoresce throughout the visible part of the spectrum, [65,107,108] while carbon dots exhibit a much broader emission (>100 nm full width at half maximum (FWHM)) compared to fluorescent proteins. Graphene oxide fluorescence covers a similar spectral range. [73,109,110] The emission of both c-dots and graphene oxide shows a strong excitation wavelength dependence. [65,110] N-doped graphene has been reported to emit up to the NIR1 region, [71] but the emission usually occurs in the visible part of the spectrum. [71,72,111] Organic fluorophores can be designed to emit from the visible well into the NIR2 spectral region (e.g., the dye IR-26), [112] but those emitting in the NIR2 are mostly not water soluble. Only recently has an NIR2-emitting fluorophore (λ em = 1050 nm) been reported. [27] Common dyes emitting in the NIR1 region are IR-dye800 and Cy7. [29] Since fluorescent beads are based on organic fluorophores, they emit in a similar spectral window. A notable exception is the common NIR2-emitting dye IR-1061, which has been successfully embedded in a polymer matrix to make it water-soluble. [44] Gold nanoclusters can be synthesized to emit between 400 and 850 nm, [81,113] very similar to Ag clusters. [80,82] Both Ag and Au clusters generally show a rather broad emission with about 100 nm FWHM. [79,82] The fluorescence of nanodiamonds between 500 and 800 nm cannot be 3.2. Optical Properties All materials discussed here show fluorescence emission in the red, NIR1 or NIR2 spectral ranges (and many throughout the Figure 4. Fluorescence emission range of selected material types. The blue bars indicate the spectral range within which the fluorescence peak positions can be varied WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com (7 of 26)

8 continuously tuned like many other materials. Here, different color centers like nitrogen-vacancy (λ em = 690 nm), nitrogenvacancy-nitrogen (NVN, λ em = 530 nm) or silicon-vacancy (λ em = 738 nm) show very distinct emission spectra from the visible to the NIR1, some with broad (NV, FWHM > 100 nm) others with narrow spectral features (silicon vacancy, FWHM < 20 nm). Upconversion nanoparticles are usually excited at 980 nm and emit light at shorter wavelengths. This emission can be tuned by a number of parameters from particle composition to excitation pulse width and is generally spectrally narrow (< 50 nm FWHM). [86] The most versatile particles in terms of the tunability of their emission wavelength in the NIR are certainly polymer dots, [36,42] quantum dots [47,114] and rare earth downconverting [93,115] particles. They can emit from the visible to the NIR2 spectral region and in case of QDs and DCNPs even beyond 1400 nm. Quantum dots usually exhibit a narrow emission with a FWHM < 50 nm, in some cases even up to the NIR2 region as in the case of Ag 2 S. [116] The spectral width of rare earth DCNPs strongly depends on the dopants used, but is generally above 50 nm FWHM in the NIR2 region. [92,93] Only very few p-dots emitting above 1000 nm have been reported to date and their emission spectrum is broad (>100 nm FWHM). [42] The Stokes shift also varies significantly between materials. While organic fluorophores and quantum dots usually have a fairly small Stokes shift of less than 50 nm, [117] other materials like nanodiamonds and rare earth downconverting NPs have a shift of more than 100 nm and several hundred nanometers respectively. [64,93] A large Stokes shift is a desirable property in many applications since it can help to reduce the background signal from autofluorescence. Upconversion nanoparticles exhibit blue-shifted (anti-stokes) emission, which also minimizes background signals. The fluorescence brightness (B) of a material is critical to create optical contrast in a fluorescence microscopy image. It is generally defined as the molar absorption coefficient (ε) multiplied by the fluorescence quantum yield (Γ) of a material: B = Γ (1) It is important to note that using this definition of brightness to compare different nanomaterials, one particle or molecule of material A is directly compared to one particle or molecule of another material B (since the molar absorption coefficient is a particle number based quantity). For example, the molar absorption cross-section of a NIR1 fluorescing organic dye is usually on the order of m 1 cm 1, while that of a 100 nm sized nanodiamond is on the order of m 1 cm 1. Using typical values of 0.2 and 0.9 for the quantum yield of a NIR organic fluorophore and a nanodiamond, respectively, in Equation (1), this leads to a more than 10 times higher brightness value for nanodiamonds. This however does not take into account that the volume and mass of a 100 nm nanodiamond is about 5 orders of magnitude larger than that of the dye. On an equal mass basis, the brightness of a common dye exceeds that of a 100 nm diamond by 4 orders of magnitude. [13] The fluorescence quantum yield of a nanomaterial can be a misleading quantity. A quantum yield of 1, i.e., every photon absorbed by the material yields one emitted photon, is often understood to indicate high fluorescence brightness, which is incorrect as Equation (1) shows. If the absorption coefficient of a material is low, wich means that it does not efficiently absorb photons, it will not show bright fluorescence even if the fluorescence quantum yield is close to 1. Both the absorption coefficient and the fluorescence quantum yield vary by several orders of magnitude between different materials and are equally important to generate high fluorescence brightness. Typical values for the molar absorption coefficient and the quantum yield of selected materials are shown in Table 1. It is important to note that the absorption coefficient is strongly excitation wavelength dependent, while the quantum yield is usually independent of the excitation wavelength. Here, we focus on the maximum absorption coefficient of materials. For NIR fluorescing materials, this maximum is commonly located in the red or NIR1 spectral region. However, several materials discussed here (e.g., NV nanodiamonds, carbon dots, gold clusters, graphene oxide) only fluoresce in the red and NIR1, have relatively large stokes shifts and are most efficiently excited in the visible. Table 1. Comparison of the optical properties of selected fluorescent nanomaterials. Excitation range [nm] ε [M 1 cm 1 ] Γ [%] Fluorescence lifetime Time-gating Blinking Photostability Quantum dots ns 5 µs Yes Yes High Polymer dots ns No No Medium Nanodiamonds (NV)* ns Yes* No Very high Organic dyes <1 6 ns No Yes Low Carbon dots ns No No Medium Gold clusters < ns Yes Yes Medium Carbon nanotubes** <<1 7 1 ns No No High Graphene oxide <1 5 1 ns No Yes Medium UCNPs <1 7 >100 µs Yes No High * Nanodiamond here refers to a fluorescent nanodiamond > 60 nm. Time-gated imaging of nanodimaonds has been demonstrated, [118] however for many applications the fluorescence lifetime is not long enough for efficient time-gating. **Absorption coefficient is proportional to nanotube length. The value given here is estimated based on Hertel et al. [119] for a tube length of 200 nm. See Supporting Information for a complete list of references for the numbers quoted here (8 of 26) wileyonlinelibrary.com 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

9 Quantum dots and polymer dots have very high molar absorption coefficients, [35, ] often on the order of 10 6 m 1 cm 1 as well as relatively high quantum yields [34,35,112,122,123] even in the NIR resulting in a very high molar brightness. However, due to the extremely high absorption coefficient for their small size, organic fluorophores generally have the highest mass brightness. [13,28] Nitrogen-vacancy centers in nanodiamonds also show high molar fluorescence brightness, but only if the particles are comparably large (>60 nm) and contain several hundred NV centers. Despite a high quantum yield, single NV centers show very low fluorescence due to their low absorption coefficient. [64] Carbon dots [68,124] and gold clusters [13,125] have absorption coefficients comparable to those of organic fluorophores, but often significantly lower quantum yields. (Finding reliable data for both materials is challenging.) Graphene oxide shows a rather low fluorescence brightness [109,126,127] and usually only fluoresces in the red and into the NIR1. Carbon nanotubes have a very broad absorption spectrum. While the absorption coefficient for a single carbon atom in SWCNTs is on the order of m 1 cm 1, [128,119] the absorption is proportional to the particle length and SWCNTs of about 200 nm length have a molar absorption coefficient of about m 1 cm 1. Quantum yields reported for SWNCTs vary by several orders of magnitude. [129] Upconversion nanoparticles are usually not very efficient light absorbers and also often have low (<1%) and highly excitation intensity dependent fluorescence quantum yields. [86, ] The fluorescence brightness is sometimes dismissed as being of minor importance with reference to the fact that the intensity of the illumination can always be increased to obtain a stronger fluorescence signal. In a biological imaging context this is generally incorrect for two reasons. First of all, autofluorescence from endogenous fluorescent molecules sets a sample dependent lower bound for the fluorescence brightness of a fluorophore/np if it is to create optical contrast in an image. (For some NPs of low brightness, autofluorescence can be avoided for example by time-gating.) Secondly, light illumination can alter biological samples physically (heating) and chemically and should therefore be reduced to a minimum. Phototoxicity in live imaging applications in vivo and in vitro is a particular concern and many mechanisms leading to phototoxic effects even in the absence of an endogenous fluorophore have been identified. [133] The fluorescence lifetime of materials can be exploited in bioimaging and sensing applications in two ways: 1. Via fluorescence lifetime imaging, where the fluorescence decay is recorded for every pixel in an image and structural and chemical information about the sample and materials used as labels inferred from the decay traces; 2. Through time gating, where the fluorescence intensity is recorded with a temporal delay relative to the pulsed light excitation to remove background fluorescence. [134] In both cases the lifetime of the fluorescent label or probe must be significantly longer than the autofluorescence of the biological sample (typically < 8 ns). [134] Rare-earth-based materials both up- and down-converting exhibit long emission lifetimes of several 100 µs up to milliseconds [86,115] and are very well suited for time-gated imaging as well as nanoruby particles, which show phosphorescence on the millisecond timescale. [97,98] Depending on the material used, quantum dots have fluorescence lifetimes from the nanosecond to the microsecond regime, [47,121] similar to metal clusters, most of which have fluorescence lifetimes above 100 ns. [79] The intermittent emission of photons (so-called blinking) of individual molecules or particles is a well-studied, but still not fully understood phenomenon many materials exhibit. [135] It can be exploited in super-resolution imaging techniques such as stochastic optical reconstruction microscopy (STORM), but can also be a disadvantage e.g., in single particle tracking experiments. Quantum dots, fluorescent proteins and organic dyes are known to exhibit blinking, while the natural on and off times are usually not suitable for use in STORM-type imaging experiments and have to be switched by other means. [3] Only recently has a dye with an intrinsic blinking rate suitable for STORM imaging been engineered. [136] Particles that do not exhibit blinking are mainly those containing a large number of dopants or color centers in one particle such as rare-earth-based particles, ruby and diamond. [64,97,115,137] (Although smaller nanodiamonds have been reported to exhibit binking [138] and used for blinking-based super-resolution imaging. [139] ) The photostability of a fluorescent material determines how long the material can be imaged before it bleaches and ceases to emit photons. In most cases a light-induced change in the chemical or electronic structure causes molecules or particles to become nonfluorescent over time. To some degree, photostability is an intrinsic property of the material. However, it also strongly depends on several other factors such as the light excitation intensity and chemical environment of the material. Therefore, photostability in one application, e.g., low magnification wide-field fluorescence imaging, does not imply photostability for confocal microscopy using a 100x magnification objective, where excitation intensities are orders of magnitude higher. Accordingly, the term photostable is used excessively in literature and often merely indicates that a material bleaches slowly or is photostable relative to an organic dye. Quantum dots for example are often said to be highly photostable, while it is well known that the fluorescence of certain quantum dots can increase, decrease and blue shift upon illumination [140] and does photobleach. Nonetheless, for many bioimaging applications, most quantum dots show a high degree of photostabilty and do not bleach significantly during the course of a measurement. The fluorescence emission of NV centers in diamond on the other hand does not change at all for days and weeks even under high intensity illumination. [141] Rare-earth based [115] and ruby particles [97] also show a very high degree of photostability. Carbon dots, [142] gold clusters, [143] polymer dots and carbon nanotubes [144] have all been reported to be more stable than organic dyes. However, unless investigated quantitatively e.g., in terms of the total number of photons emitted by a particle before it photobleaches or under identical conditions, the value of these claims is very limited for practical purposes. A direct comparison of the photobleaching characteristics of some of the materials discussed here has been reported recently by our group. [13] 3.3. Surface Chemistry and Colloidal Stability Molecular fluorophores do not possess a surface as nanoparticles do, even though this distinction is not very clear in the 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com (9 of 26)

10 Figure 5. Generic structure of a functional nanoparticle for bioimaging applications: native nanoparticle surface, surface functionalization and biofunctionalization layers. Abbreviations are: polyethylene glycol (PEG) and mercaptopropionic acid (MPA). Sizes are not drawn to scale. case of metal clusters or small polymeric particles for example, which exhibit molecular as well as particle-like properties. Similarly, the term colloidal stability does not apply to molecules. Therefore, this section will focus on nanoparticles and only briefly discuss purely molecular systems like organic dyes, fluorescent proteins and dendrimers. In the context of bioimaging applications, a nanoparticle can be divided into three parts: a nanoparticle core, a layer of surface groups or molecules (also called ligands) and a biofunctionalization layer (Figure 5). For example silica particles typically exhibit hydroxyl groups on their surface without any functionalization. This native surface can be functionalized with amine groups to enable the covalent bonding of proteins or antibodies (biofunctionalization) to the particle surface. In other cases the distinction between native particle surface and surface functionalization is not meaningful. Semiconductor quantum dots for example are almost exclusively synthesized in the presence of ligands, which already represent a basic functionalization of the particle surface. In a ligand exchange step this functionalization is often changed to modify particle solubility or enable the attachment of biomolecules. We will first discuss native particle surface terminations and basic functionalization strategies. The next section will focus on strategies to attach biologically relevant molecules to particles. A minimum requirement for most bioimaging applications is that fluorescent molecules are water-soluble and particles are colloidally stable in water. Many applications also require stability under physiological conditions or in serum, which involves the presence of millimolar concentrations of salt, amino acids and glucose. Fluorescent proteins are intrinsically stable in water and also under physiological conditions. The chemical structure and functional groups of organic dyes are generally also designed to be highly water-soluble and also stable under physiological conditions. One strategy to use hydrophobic dyes in an aqueous environment is encapsulation e.g., in a polymer particle. Most semiconductor quantum dots and rare-earth based materials (up- and downcoverting) are usually chemically synthesized in organic solvents, are not water soluble, and have to be made hydrophilic through a ligand exchange or similar modification of the surface with hydrophilic molecules. [137,145] For quantum dots thiol-based ligands such as mercaptopropionic acid and thiolated polyethylene glycol molecules are common choices to infer good colloidal stability in water and enable further biofunctionalization. [146] Thin shells of organosilane molecules or polymers are common to make rare earth particles water soluble and graft reactive groups onto their surface. [115] Carbon dots and metal clusters are usually synthesized in aqueous environments and in the presence of stabilizing ligands. In both cases, the stabilizing ligands have a strong influence on the particles fluorescent properties and can be used to tune the fluorescence wavelength. The native carbon dot surface typically consists of COOH and OH groups, while basic functionalization with cysteine, mercaptosuccinic acid and polyethylene glycol (PEG) is common. [147,148] Aqueous gold ions are generally reduced by reducing agents (e.g., NaBH 4 ) in the presence of thiol-based ligands such as glutathione or tiopronin. [79] Similar strategies are employed for silver clusters, while the role of ligands on the optical properties of the clusters is not well understood yet. [82] Silica particles are generally stable in water, their surface functionalization is well established and almost any functional group can be grafted onto their surface, [149] making fluorophore doped silica particles a versatile tool for biological applications. Other particles such as nanodiamonds and nanorubies are milled from bulk samples and generally need to undergo physical (e.g., air oxidation) or chemical (e.g., acid treatment) cleaning processes to become stable colloids in water. Many surface terminations and functional groups including carboxylic acid, amine and hydroxyl can be created directly on diamond particle surfaces, while ligands can be used as well to infer a desired functionality. [150,151] Ruby nanoparticles have been far less explored to date, but have been reported to be stable in water with a strongly positive surface charge. [97] A number of different fabrication processes have been developed for carbon nanotubes. [152] The creation of COOH groups on carbon nanotube surfaces via oxidation is a common first step to infer some water solubility and enable further functionalization. [153] Doped graphene sheets as well as graphene (10 of 26) wileyonlinelibrary.com 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

11 oxide are usually stabilized in water through carboxylic acid and hydroxyl groups. [69,71] Many conjugated polymers used for the synthesis of polymer dots are not water soluble and their hydrophobicity is used in several processes for their fabrication. [34,36] Water soluble conjugated polyelectrolytes or surfactant molecules can be used to stabilize them in water. Other strategies include encapsulation in silica or in other polymers such as the biocompatible poly(lactic-co-glycolic acid) (PLGA). [36] Generally, electrostatic and steric stabilization are the two main mechanisms for stabilizing nanoparticles in solution. In deionized water, charged particles repel each other electrostatically, thereby preventing aggregation and stabilizing them in solution. However, most biological media contain salt concentrations in the millimolar range. The salt ions shield (or screen) the electrostatic charge of particles and lead to aggregation, making purely electrostatically stabilized particles problematic for many applications. Most as synthesized carbon dots and nanodiamonds are charge stabilized in water, usually through the negative charge of carboxylic acid and hydroxyl surface groups, while a variety of charged surface groups can be inferred on nanodiamonds. [151] After a ligand exchange quantum dots are also often charge stabilized by ligands such as mercptocarboxylic acids. [146] Similarly, upconversion nanoparticles can be stabilized in an aqueous environment through ligands with carboxylic acid functional groups. [137] NIR2-emitting DCNPs, stabilized with a phosphonic acid based ligand, have recently been used for nontargeted in vivo imaging. [95] Steric stabilization on the other hand can be achieved by surfactants or polymers adsorbed or chemically bound to the particle surface. When particles approach each other in solution, the stabilizing molecules around the particle essentially act as a buffer layer, which prevents particles from aggregating. (For more insight into the underlying physical mechanisms, the reader is referred to an excellent review on the topic. [154] ) In biological environments this type of stabilization is preferred, as it is not compromised by the presence of salts. PEG is often used for steric stabilization and is very commonly used to stabilize different types of carbon dots, [65,67,155] since it is also used in many synthesis protocols. Recently, PEG-stabilized Ag 2 S quantum dots with tunable emission in the NIR1 optical window have been reported. Nanodiamonds can also be stabilized with PEG. [156] NIR2-emitting PbS quantum dots have been stabilized with PEG as well as with another common polymer polyvinyl alcohol (PVA) since their discovery, [157] while a silica-peg layer around these QDs has been developed more recently specifically for in vivo imaging applications. [158] Singlewalled carbon nanotubes have been functionalized with PEGbased ligands for in vivo imaging. [159,160] Another way to change or improve the colloidal stability or other surface properties of a particle is encapsulation in a shell, which has the desired properties or can easily be further functionalized. Silica is a popular choice as its surface chemistry is well understood and its bioconjugation is established. Red and NIR1-emitting Nanodiamonds, [156,161] semiconductor quantum dots [162,163] and upconversion particles, [ ] as well as NIR2-emitting quantum dots [167] and DCNPs, [166] have been successfully embedded in silica shells to name just a few examples. Depending on the application, none of the functional layers may be required and the as-synthesized particle can be used nonspecifically e.g., to image HeLa cells as done in many reports as a proof-of-concept experiment for new materials or used as a fluorescent contrast agent to image vasculature. Often only a basic surface modification such as a ligand exchange or the attachment of a PEG layer must be carried out to enhance the colloidal stability. However, in order to fluorescently label specific sites or areas to be investigated in a biological system, biological molecules must be attached to the nanoparticle surface, which target this site or enable penetration or uptake into the region of interest. 4. Bioconjugation 4.1. General Strategies A large number of strategies have been developed to attach biomolecules to nanoparticles and fluorescent molecules. These have been reviewed comprehensively by Sapsford et al. [168] and other good reviews on this topic exist. [146,169] They can be divided into two main groups: covalent binding and noncovalent attachment. Covalent bioconjugation strategies generally exploit the functional groups on the nanoparticle surface as well as those on the biomolecule of interest. The two most common functional groups on a nanoparticle surface used for the creation of covalent bonds are carboxylic acids and amines. Carboxylic groups are activated to react with primary amines or N-hydroxysuccinimide (NHS) to form an amide bond. Similarly particles with amine functionality can be reacted with active ester compounds like NHS to form amide bonds. Maleimide coupling reactions are also common. Here thiol groups are coupled to primary amines using maleimide derived linkers such as sulfosuccinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (sulfo-smcc). Another important class of conjugation reactions is commonly referred to as click chemistry, which can be used for the biofunctionalization of nanoparticles [168] as well as the development of biochemical assays [170] and the direct labeling of biomolecules inside a biological system (due to the generally mild reaction conditions). [171] Quantum dots, [ ] polymer dots, [175] nanodiamonds, [176] UCNPs, [177,178] SWCNTs, [179] silica [180,181] and silicon [182] nanoparticles have all been functionalized using click chemistry. Covalent bonds have the advantage of being chemically stable and usually result in the attachment of the biomolecule with a known orientation with respect to the nanoparticle surface, which can be important for the function of the biomolecule and its affinity to other molecules in the biological sample. Several important biomolecules commonly used to functionalize nanoparticles are illustrated in Figure 6, attached to a generic silica coated nanoparticle, with all sizes drawn to scale. Noncovalent attachment methods include electrostatic adsorption of biomolecules to charged particle surfaces and receptor-ligand type interactions between a functionalized nanoparticle and a biomolecule. Electrostatic adsorption has the advantage of not requiring any additional reactants and/ or chemically harsh conditions. Concerns are that the biomolecule may desorb and that its orientation cannot be controlled WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com (11 of 26)

12 Figure 6. Illustration of important biomolecules attached to a generic silica-coated nanoparticle with a total diameter of 10 nm. All sizes are drawn to scale. Proteins are crystal structures from the Protein Data Bank ( and displayed as surfaces. PEG and DNA were modeled from their chemical structure. Illustration adapted with permission. [146] Copyright 2010, Royal Society. A very successful approach is to exploit the strong and specific affinity of natural receptor ligand systems. One of the technologically most established and well-studied is the avidin biotin system. Despite not being a covalent bond, the bond between the small molecule ligand biotin and the receptor protein avidin is very stable. Either biotin or avidin (or one of its derivatives like streptavidin) must be attached to the nanoparticle surface via a covalent bond or otherwise. Avidin has four binding sites for biotin and can be used as a linker to connect biotin moieties. Bitotinylated proteins, nuleic acids and many other molecules are commercially available and the possibilities for the design of ligand-receptor systems to direct nanoparticles to specific molecules inside a biological system are endless. In the following we will discuss recent advances in the bioconjugation of NIR-emitting nanomaterials Bioconjugation of NIR-Emitting Materials The bioconjugation of materials fluorescing in the visible spectrum is not necessarily different from that of NIR-emitting ones. However, much fewer nanomaterials fluorescing at wavelengths longer than 700 nm exist. Many of them have been developed fairly recently and, compared to e.g., organic fluorphores in the the visible, reliable bioconjugation protocols are often not yet well established. Organic fluorophores emitting in the red and NIR1 like Cy 7 or IRDye 800 conjugated to streptavidin, a wide range of immonuglobulins (Ig), DNA and RNA are commercially available. If a molecule of interest is not immediately available, custom bioconjugation services for fluorophores (and sometimes fluorescent beads) are commercially available as well. [183] Most fluorophores are covalently bound to biomolecules via amide or thioether bonds. Beyond 900 nm emission wavelength, most organic fluorophores known to date suffer from water insolubility. These can be incorporated into a polymer matrix and then conjugated to biomolecules based on known polymer nanoparticle conjugation strategies. [35,184] Tao et al. [44] have reported such a system, where the dye doped particle emitting around 1000 nm was conjugated to PEG. For intrinsically fluorescent polymer particles (or p-dots) the bioconjugation is more challenging since their formation is mainly driven by hydrophobic interactions and hence not very rigid and are often encapsulated in other materials. [36,185] Hong et al. have reported a NIR2-emitting p-dot conjugated with PEG, which was further conjugated to the cancer drug Erbitux for targeted imaging of cancer cells (Figure 7). [42] Conjugation of red to NIR1-emitting p-dots to streptavidin and folic acid via carboxylic groups and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) for targeted imaging has been recently reported by Lui et al. [40] Another p-dot emitting in the same spectral region has been encapsulated with a second polymer and functionalized with streptavidin using similar chemistry by the same group. [39] (This particle is actually an organic dye-doped polymer particle and not an intrinsically fluorescent polymer, but a clear separation of these two particle types is challenging.) Another streptavidin functionalized p-dot emitting in the same region was reported by Zhang et al. [122] (12 of 26) wileyonlinelibrary.com 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

13 Figure 7. Targeted cell imaging using NIR2 fluorescing polymer dots. A)A) Schematic showing the structure of a p-dot-erbitux (a drug for metastatic colorectal cancer) bioconjugate, where the Erbitux antibody selectively targets the epidermal growth factor receptor (EFGR) on the cell membrane of a MDA-MB-468 cell. B) Bright-field (b) and NIR2 fluorescence images (c) of EGFR-positive MDA-MB-468 cells incubated with the p-dot-erbitux bioconjugate, showing positive staining of cells. White-light (d) and NIR2 fluorescence images (e) of EGFR-negative U87-MG cells incubated with the p-dot-erbitux particles, without obvious staining of the cells. The scale bar in (e) is 40 µm and applies to all images. Reproduced with permission. [42] Copyright 2014, Macmillan. The bioconjugation of carbon dots has been reviewed. [147] Due to the presence of carboxylic acid groups on the native surface of many c-dots, well established NHS- and EDC-based bioconjugation chemistry can be employed. However, most c-dots fluoresce below 700 nm and no particles emitting in the NIR region and bioconjugated for targeted bioimaging have been reported to our knowledge to date. A wealth of bioconjugation techniques has been developed for semiconductor quantum dots in general [114,146] and NIR-fluorescing ones in particular [47] and many NIR-emitting QDs are already commercially available. Recently, mercaptopropionic acid stabilized Ag 2 S with tunable emission in the NIR1 and 2 spectral windows have been conjugated to peptides using NHS chemistry for targeted cancer imaging (Figure 8). [116] BSA stabilized Ag 2 S (NIR1-emitting) QDs were attached to a growth factor antibody also using NHS/EDC based conjugation for targeted cancer imaging. [186] Schieber et al. have reported coupling of transferrin via azide alkyne cycloaddition ( click chemistry) to azide-modified red fluorescing QDs. [172] A commercially available CdSeTe/ZnS NIR1 emitting (λ em = 800 nm) quantum dot has been successfully conjugated to an epidermal growth factor using thiol maleimide based coupling. [187] Maleimide coupling was also used to functionalize another NIR1 fluorescing QD to arginine glycine aspartic acid to target human glioblastoma tumors. [188] The bioconjugation of UCNPs has been reviewed. [132,137] Once the particles are water soluble through the use of a suitable ligand, established carbodiimide or disulfide-coupling chemistry can be used to bind a range of biomolecules to the particle surface. Another important strategy is the use of silanes either as ligands or to grow a thin silica shell around the particles, which then can be readily coupled to biomolecules. Using a multi-step functionalization based on silanes as well as PEG and biotin, streptavidin functionalized DCNPs have been reported recently and were employed for targeted cancer imaging in the NIR2 spectral region. [189] UCNPs were biotin functionalized using a click reaction as reported by Mader et al. [178] Biofunctionalization strategies for carbon nanotubes for imaging, sensing and drug delivery have been reviewed. [129] SWCNTs have been attached to M13 phages via peptides for targeted in vivo imaging in the NIR2 spectral region. [190,191] Polyethylenimine has been used as a linker to functionalize SWCNTs with small interfering RNA for tumor targeting for Figure 8. Bioconjugation and targeted cancer imaging in vivo using Ag 2 S nanoparticles. A) conjugation of Ag 2 S particles to a cyclic peptide (arginineglycine-aspartic acid-(d)phenylalanine-lysine) using EDC/NHS chemistry to target the α v β 3 integrin receptor (ABIR). B) In vivo fluorescence imaging (λ em = 785 nm, λ em = 820 nm) of the Ag 2 S peptide particles in a tumor-bearing mouse at different time points after intravenous injection of the particles. Circles indicate bilateral subcutaneous tumor locations. Reproduced with permission. [116] Copyright 2015, American Chemical Society WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com (13 of 26)

14 Figure 9. Single molecule imaging and tracking of fluorescent nanodiamonds functionalized with a transforming growth factor (TGF) in live cells. A) Bioconjugation of nanodiamonds to a TGF via PEG-streptavidin-biotin. B) 2D image of the nanodiamond-tgf particles in HCC827 cells at different time points (0, 30, and 60 min) and a control sample of HCC827 cells treated with unconjugated nanodiamonds on the right. White dotted circle represents the cell boundary. Scale bar: 3 µm. C) Representative 3D trajectories of nanodiamond-tgf particles tracked in live cells. Left: immobile particles (Diffusuion coefficient D = 0.01 µm 2 s 1 ); middle: anomalous diffusion (D = 0.2 µm 2 s 1 ); right: brownian diffusion (D = 3 µm 2 s 1 ). Reproduced with permission. [197] Copyright 2015, Wiley-VCH. photothermal therapy. [192] SWCNTs have also been successfully conjugated with β-cyclodextrin via click chemistry. [179] The conjugation of nanodiamonds to biomolecules is relatively well established and has been reviewed. [151] Recently, nanodiamonds were conjugated to a β-lactamase-tag [193] through a hyperbranched polyglycerol linker to target the cell membrane receptor interleukin-18. [194] Zhang et al. recently reported a DNA-biotin-avidin based system for the controlled selfassembly of nanodiamonds. [195] In another recent study, nanodimaonds were conjugated to a transforming growth factor via PEG-streptavidin-biotin and used for 3D particle tracking in cells in vivo (Figure 9). Single stranded DNA molecules were conjugated to nanodiamonds by Akiel et al. [196] using click chemistry. Gold clusters (λ em = 700 nm) have been conjugated to streptavidin via a PEG linker for use in cellular imaging. [143] Zhou et al. have reported ribonuclease-a encapsulated gold clusters (λ em = 680 nm) functionalized with vitamin B12 for targeted imaging of cancer cells. [198] The general progress in the area of gold nanocluster based bioimaging and sensing has been reviewed, [79,199] but not the bioconjugation specifically Toxicity Assessing nanoparticle toxicity is a complex endeavor. In contrast to most macroscopic objects, nanomaterials are able to overcome many natural barriers in living organisms such as cell membranes and target specific biomolecules for visualization or sensing purposes even inside individual cells. This also means that nanomaterials interact with and affect biological systems at the scale and in locations where many fundamental biochemical processes take place. This interaction can occur in many different ways, e.g., affect the proliferation of cells, change gene expression, lead to accumulation of nanomaterials in certain parts of an organism, trigger immune responses or affect the molecular transport in cells. Accordingly, there are numerous approaches to assess this interaction between nanoparticles and biological systems from cellular systems to animal models and from viability essays to complex functional assays and behavioral studies. The effect nanomaterials (or any material for that matter) have on biological systems generally depends on their concentration. Even if all other properties are identical, a change in nanoparticle size on the nanometer scale alone is very likely to strongly effect for example endocytosis and cytotoxicity of the particles and [200] whether they are cleared through the kidneys or the liver of an organism. [201] Often the functional surface or ligand of a particle has a greater effect on the biology than the nanomaterial itself. [ ] Therefore, demonstrating adverse effects of nanomaterials on biological systems is often simple. Proving the general nontoxicity of a material is virtually impossible. Another important and often underestimated aspect of toxicity studies is the thorough characterization of the material (14 of 26) wileyonlinelibrary.com 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

15 that is added to the model system. Unless the physical and chemical properties of a material are precisely known, a toxicological study is meaningless and worse, can potentially hinder the progress in a research area by generating flawed results. Of particular concern are nanomaterials composed of of toxic materials such as selenide, lead or heavy metals. However, if sufficiently protected from their environment, semiconductor quantum dots have been demonstrated to have no adverse effects in a number of studies [206] and their toxicity in various systems is still actively debated in literature. [207] At the other end of the scale are inert carbon-based materials such as diamond, which are generally regarded as nontoxic. [ ] However, a potential embryotoxicity and teratogenicity of nanodiamonds depending on their surface functional groups has been demonstrated in an in vivo study. [211] Most other materials including molecular systems such as organic dyes, are located between intrinsically toxic materials and extremely stable and chemically inert carbonaceous materials in terms of their toxicity. Some recent reports as well as review articles on the toxicity of the nanomaterials discussed here are listed in Table Bioimaging and Sensing Applications 5.1. Imaging Technology Fluorescence microscopes operating in the visible and NIR1 spectral region up to about 900 nm are commonplace and commercially available. In this spectral region light is usually detected using photomultiplier tubes (PMTs), silicon-based photo detectors such as a charge-coupled device (CCD) or an avalanche photodiode (APD) or complementary metal oxide semiconductor (CMOS) detectors (Figure 10). These detectors can be used as point detectors or as two dimensional arrays (except PMTs) depending on the application. Imaging above 900 nm and in the NIR2 requires optical filters, mirrors, objectives and photo detectors tailored for this spectral region and fluorescence microscopes are not yet commercially available. InGaAs point detectors or arrays with quantum efficiencies well above 50% between 1000 nm and 1500 nm [231] are often used in custom-built NIR2 fluorescence imaging systems. The fact that many components of visible to NIR1 microscopes must be replaced or a custom-built, dedicated NIR2 system used, represents a big hurdle for many biology laboratories to enter this research area. For red and NIR1 fluorescence imaging, illumination occurs in the visible and far-red part of the spectrum using LED or laser sources. Both LEDs and lasers operating in the NIR1 are also widely available and are used for imaging in the NIR2. Time resolved fluorescence imaging usually requires pulsed laser excitation in the picosecond time domain or at least intensity-modulated excitation to determine fluorescence lifetimes of fluorescent materials on the nanosecond timescale. Fluorescence lifetime imaging microscopy (FLIM) systems operating in the visible region are commercially available. For the NIR1 and NIR2 regions custom-built setups have to be employed using picosecond laser sources and often employing fast APD or PMT point detectors that operate in the desired spectral range. [232] To date, amongst all the different materials discussed here, only one organic fluorophore emitting in the NIR1, indocyanine green, is approved by the US food and drug administration (FDA) for clinical use. [233] No NIR2-emitting material has FDA approval. Therefore, the majority of applications of near-infrared fluorescence imaging and sensing take place in research laboratories and experiments range from live whole animal imaging to single cell experiments in vitro. The main advantage of imaging in the NIR1 and NIR2 spectral windows is the relatively low absorption, scattering and autofluorescence of biomolecules and tissues in this spectral region compared to the visible. Therefore, the focus in this section will be on applications, which make good use of these advantages Organic Fluorophores and Fluorescent Proteins Indocyanine green (λ em 830 nm) is already used clinically in applications such as minimally invasive guided surgery, [ ] retinal angiography, [237] imaging of vasculature, tumor delineation and imaging of the lymphatic system. [233] It has a comparatively low fluorescence quantum yield of about 1%, which strongly decreases with increasing concentration [238] and is neither chemically stable nor photostable. [235] Nonetheless, it proves to be a very useful tool in many clinical applications and highlights the great potential of novel fluorescent materials for clinical applications. In biological research, red and NIR1-emitting organic fluorophores and fluorescent proteins remain the most important fluorescent materials. Commercially available dyes such as ICG, Cy7 and IR-780 are routinely used for targeted cancer imaging either directly conjugated to tumor targeting ligands or embedded in multifunctional nanoparticle systems. [239,240] Recently, IR-780 was successfully used to specifically target and image prostate cancer cells in vitro and in vivo (mouse model) and even a potential tumor killing ability of the dye was suggested, while no significant impact on physical activity and tissue histology was found. [241] Fluorescence-based imaging of the brain in vivo has been employed for several years, but mostly using minimally invasive endoscopy techniques. [242] Only recently through the development of an NIR2-emitting and water soluble organic fluorophore has it become feasible to image brain vasculature of living animals non-invasively through the scull up to 4 mm deep. [27] The same dye was used to image a tumor (Figure 11) and lymphatic vasculature in a mouse model and showed renal excretion, boding well for further preclinical trials. NIR1 fluorophores have also been used to image cellular proteins in vitro and ex vivo using super-resolution microscopy, [243] while a translation of this approach to an in vivo setting will be challenging. One strength of organic fluorophores is that they can be designed to very specifically interact with analyte molecules or ions in sensing applications. A wide range of red and NIR1 dyes to detect hydrogen peroxide, singlet oxygen, hydroxyl and other reactive oxygen species, Zn 2+, Ca 2+, Cd 2+, Ag +, Cu 2+ and to measure ph in vitro and in vivo exist and have been reviewed. [30] Recently, Fu et al. reported a NIR1-emitting organic fluorophore to detected and localized Amyloid-β 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com (15 of 26)

16 Table 2. Studies assessing the toxicity of fluorescent nanoparticles and molecules. Particle type Intrinsic material toxicity Material Biological system Assessment Reference Carbon dots Low C-dots, PEG stabilized Mice Acute toxicity, subacute toxicity, and genotoxicity Carbon nanotubes Low-medium Many types of CNTs Various in vitro/in vivo [ ] Dendrimers High Various dendrimer types Various in vitro/in vivo [216] Doped graphene QDs Medium N-doped graphene quantum dots [212] Red blood cells Hemolytic activity [217] Fluorescent beads Medium (polymer) Polystyrene nanoparticles Endothelial cells Lysosomal function [218] Low (silica) Silica nanoparticles Epithelial cells and fibroblasts Size, dose, and cell-type dependent cytotoxicity Fluorescent proteins Medium Red fluorescent protein HeLa cells Cytotoxicity [220] Graphene oxide Medium Graphene oxide Various in vitro/in vivo [221] Organic dyes Medium Various organic fluorophores Metal clusters Medium MPA or GSH stabilized Au clusters Graphene oxide Red blood cells Hemolytic activity [217] GSH and BSA stabilized Au 25 clusters [219] Various in vitro/in vivo [222] Colonic epithelial cells Mice Generation of intracellular ROS, cytotoxicity and genotoxicity Renal clearance, biodistribution, and toxicity responses Nanodiamonds Low Detonation nanodiamond Various in vitro/in vivo [208] Various diamond types Detonation nanodiamond P-dots Medium Quinoxaline based polymer, STV conjugated Polybutylcyanoacrylate Quantum dots High CdSe ZnS; PEG, BSA or polymer stabilized Rare earth nanoparticles Medium-high UCNPs, NaYF 4 :Yb,Tm, polyacrylic acid coated Human liver cancer and HeLa cells in vitro Human embryonic kidney cells and Xenopus laevis embryos [205] [223] Cytotoxicity [210] Cytotoxicity, mortality, embryotoxicity and teratogenicity [211] Zebrafish embryo Cytotoxicity [40] HeLa and human embryonic kidney cells/rats Rats Cellular metabolic activity/survival and morphology of neurons Biodistribution, animal survival and mass, hematology, clinical biochemistry, organ histology CdTe Mice Biodistribution, pharma- cokinetics, and toxicity Several types Various in vitro/in vivo [226] Several types Various in vitro/in vivo Meta analysis [227] UCNPs, NaYF 4 :Yb,Tm DCNPs, Gd 2 O 2 S:Tb 3+ Mice HeLa cells, caenorhabditis elegans Humanperipheral blood mononuclear cells, humanderived macrophages, HeLa cells Biodistribution, clearance, cytotoxicity, histology and hematology Cytotoxicity, protein expression, life span, egg production, egg viability, and growth rate Cell viability, apoptosis, cell-cycle progression, and immunological response [224] [206] [225] [228] [229] [230] plaques (pathological manifestation of Alzheimer's disease) in a mouse brain in vivo and ex vivo. [244] 5.3. Fluorescent Beads Cy5.5 doped silica nanoparticles (λ em 700 nm) are currently undergoing clinical trials for FDA approval in the US for NIR1 imaging assisted guided surgery of sentinel lymph nodes for various cancers. [245] The use of silica-based nanoparticles for in vitro and in vivo imaging applications has been reviewed [246] as well as their use as theranostic agents. [247] One advantage of using dye doped particles is that advances in the field of organic fluorophores can often be quickly translated to a nanoparticle system, for which the surface chemistry and resulting interaction with the biological system is already well understood (16 of 26) wileyonlinelibrary.com 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

17 Figure 10. Detection efficiencies for cameras operating in the NIR1 and NIR2 spectral regions based on silicon (Si), indium gallium arsenide (InGaAs) or mercury cadmium telluride (HgCdTe) sensors. Si and InGaAs cameras are sensitive within the first and second near-infrared windows, respectively, whereas HgCdTe is most sensitive at longer wavelengths. Reproduced with permission. [9] Copyright 2009, Macmillan. as in the case of silica particles. For example, methylene blue doped silica nanoparticles have been used for combined NIR1 imaging and photodynamic therapy of xenograft tumors in mice. [248] Similarly indocyanine green doped polymer particles can be used for targeted cancer imaging in the NIR1. [249] The water insoluble dye IR-1061 (λ em 1050 nm) was encapsulated in a PEGylated polymer matrix for in vivo vasculature imaging of mice in the NIR2 (Figure 12). [44] The general approach of encapsulating organic dyes in polymer matrices for tumor imaging applications has been reviewed. [250] Lipid particles doped with NIR fluorophores have been used recently for tumor imaging in living mice. [251] Here, the integrity of the lipid particle was monitored via Förster resonance energy transfer (FRET) between two NIR dyes (Cy5.5 and Cy7.5). NIR fluorescence guided photothermal therapy using fluorophore doped lipid-based particles has been reported by Ng et al., where the particle integrity was also monitored via a FRET-induced shift in the fluorescence emission spectrum of the dye. Recently, Liu and Wu have reviewed the use of micelles, liposomes and exosomes doped with NIR1-fluorescing dyes for in vivo tumor imaging. [252] 5.4. Quantum Dots Semiconductor quantum dots fluorescing from the red to NIR2 are commercially available and have been used extensively for in vivo imaging of animal models. Their application as in vivo markers for NIR tumor imaging has been reviewed [253] and recently Xu et al. reviewed quantum dots for imaging and sensing in the NIR2 spectral region. [254] Despite their excellent Figure 11. A NIR2-emitting fluorophore (λ em 1055 nm) for tumor imaging and guided surgery. A) Simplified reaction scheme illustrating the synthesis of the fluorophore-affibody system used to target the tumor. B,C) In vivo fluorescnce imaging of the tumor 1 h (B) and 6 h (C) after intravenous injection of the fluorophore (60 µg). The upper panel shows the tumor targeting and the bottom panel shows mice injected with a blocking dose consisting of fluorophore-affibody plus free affibody concomitantly intravenously injected. Inset shows the tumor ex vivo after excision in both a targeted and blocked mouse. D) Images of a mouse 24 h post-injection before and after performing NIR2 fluorescence image-guided surgery to excise the tumor. Reproduced with permission. [27] Copyright 2016, Macmillan WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com (17 of 26)

18 Figure 12. In vivo NIR imaging of the hindlimb and abdomen of nude mice with fluorophores circulating in the blood streams after intravenous injection. a) Image of a mouse hindlimb taken after IRDye 800 injection. Fluorescence is collected in the NIR1 at ca 800 nm. b,c) Images recorded after injection of a PEG functionalized polymer nanoparticle doped with the fluorophore IR Excitation occurs at 800 nm and fluorescence is collected in the far NIR2 from 1100 nm 1700 nm (b) and 1300 nm 1700 nm (c). d) The same nanoparticles in the abdomen imaged in the same spectral region, but excited at 980 nm. e) Image of a mouse hindlimb acquired in the 1100 nm 1700 nm region after injection of single-walled carbon nanotubes under 808 nm laser excitation. f) Image of a mouse hindlimb acquired between 1100 nm 1700 nm, but without injection of any fluorophores. Reproduced with permission. [44] Copyright 2013, Wiley-VCH. optical properties and the fact that quantum dot technology is very well established in terms of material processing and functionalization, no clinical trials have been carried out to date due to remaining concerns about the particles toxicity. Nonetheless, quantum dots are and will remain important probes for biological research. Recently, NIR1 to NIR2 tunable Ag 2 S quantum dots have been used to image subcutaneous tumors in mice in vivo by targeting an integrin overexpressed by the tumor cells. [116] Ag 2 S QDs have been used as combined drug delivery vehicles and NIR1 emitting labels for in vitro cancer imaging. [255] Generally, quantum dots are not as sensitive to their environment as organic fluorescent probes are and very few quantum dot based sensors in the NIR exist. [254] Lin et al. have reported an aptamer conjugated CuInS 2 QD (λ em 650 nm) for the sensing of daunorubicin (a cancer drug) and imaging of cancer cells in vivo Single-Walled Carbon Nanotubes The intrinsic and broad NIR2 emission of SWCNTs has been used for in vivo tumor imaging studies, [190,191,256,257] also in combination with photothermal treatment [256,257] and micro CT imaging. [258] Liang et al. report the combined imaging and successful photothermal treatment of primary tumors as well as cancer cells in sentinel lymph nodes using 808 nm laser excitation of PEGylated SWCNTs for both imaging and treatment. [257] SWCNTs stabilized in water with a M13 virus have been used to target and image tumors in vivo using targeting peptides and the concept of using this system for surgical guidance was demonstrated. [191] In vivo imaging of mouse brain vasculature through the scull has been reported up to depths of 2.9 mm using SWCNTs conjugated to the IRDye-800 fluorophore (Figure 13). [259] A SWCNT-based system for the detection (18 of 26) wileyonlinelibrary.com 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

19 Figure 13. Through scull imaging of the cerebral vasculature of a mouse brain in vivo using single-walled carbon nanotubes at a depth of 2.6 mm. The magnification increases from A to C clockwise and the fluorescence emission is collected in the far NIR2 between 1300 nm and 1400 nm. Reproduced with permission. [259] Copyright 2014, Macmillan. of nitric oxide has been developed and sensing experiments in vitro and in vivo (mouse model) have been carried out. [260] The sensing mechanism employed here relies on the nitric oxide concentration dependent decrease of photoluminescence from the SWCNTs Carbon Dots Since most carbon dots fluoresce in the blue and green part of the spectrum, few reports of red and NIR1 imaging applications exist and none in the NIR2. C-dots fluorescing from blue to far-red have recently been reported to selectively target brain tumor cells in vitro and in vivo and were used to image brain gliomas in vivo. [261] Tao et al. demonstrated the feasibility of NIR1 imaging in vitro and in vivo (mouse) of c-dots synthesized from carbon nanotubes and investigated the biodistribution and toxicology of the material (Figure 14). [262] 5.7. Nanodiamonds Despite the fact that many fluorescent optical defects in diamond exist, only the nitrogen vacancy color center has been explored widely in imaging and sensing applications in the NIR1. This is mainly due to a lack of simple and up-scalable production methods for nanoparticles containing high densities of other color centers such as the silicon vacancy center. [263,264] The fluorescence of the NV center (λ em 700 nm) does generally not photobleach and can be used to measure magnetic fields (e.g., of magnetic ions), temperature and electric fields and even membrane potentials inside biological systems often with high (diffraction limited) optical resolution. [64] However, most of these measurements are carried out via optically detected magnetic resonance (ODMR), which requires the presence of microwave fields in the sample volume investigated, which limits possible areas of application in biology. Due to their extreme photostability, nanodiamonds are ideal for single particle tracking and long term imaging applications. [141,265] Simpson et al. have tracked individual 130 nm sized nanodiamonds in drosophila melanogaster embryos and found both freely diffusing particles as well as actively transported particles inside the embryo. [266] Recently, Lui and coworkers targeted 3D single particle tracking of nanodiamonds to monitor the real-time dynamics of a trabsforming growth factor receptor (TGF-β). [197] Nanodiamonds were also used to identify transplanted lung stem cells in vivo and track the cells regenerative capabilities using confocal fluorescence microscopy and FLIM (Figure 15). [267] Nanodiamonds resistance to photobleaching also makes them well-suited for super-resolution [268] and two photon microscopy, [269] where high illumination intensities and long imaging times are often needed. [141] Nanodiamonds have also been used for background-free in vivo imaging of sentinel lymph nodes by exploiting their magnetic field dependent fluorescence. [270] 5.8. Metal Clusters Gold nanoclusters are by far the most common metal clusters used in bioimaging and sensing applications followed 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com (19 of 26)

20 Figure 14. Left: Carbon dots excited at 704 nm and imaged at 770 nm after injection into a mouse. Right: Fluorescence emission spectra of carbon dots and autofluorescence recorded at the positions indicated in the image. Reproduced with permission. [262] Copyright 2012, Wiley-VCH. by silver. Despite the fact that larger (mostly nonfluorescent) gold nanoparticles have been used extensively in preclinical trials and even in clinical trials for photothermal treatment of cancer, [271] only few reports of gold and silver clusters for NIR imaging in vivo exist. Trypsin-stabilized Au NCs (λ em 700 nm) have been used for the in vitro sensing of heparin and folic acid functionalized trypsin Au NCs for targeted in vivo tumor imaging in a mouse model (Figure 16). [272] Wu et al. have reported the use of Au NCs (ligand: BSA, λ em 710 nm) for in vivo imaging of xenograft tumors in mice. [273] Silver clusters (λ em 680 nm) synthesized inside cancer cells using exogenous glutathione as a ligand were used for in vivo imaging of xenograft tumors in mouse models. [274] It is also suggested that the in-situ synthesis of the Ag NCs led to a significant reduction and often to a complete remission of the tumor. Kong and co-workers report ribonuclease-aencapsulated gold nanoclusters (λ em 680 nm) functionalized with vitamin B12 for in vitro targeting and imaging of Caco-2 cells. [198] An Au NC-Herceptin complex (λ em 650 nm) was used for specific targeting and imaging of breast cancer cell nuclei and the anticancer therapeutic efficacy of Au NC-Herceptin complex evaluated. [275] Multimodal optical / CT / MRI in vivo tumor imaging using an Au gadolinium nanocluster was reported by Hu et al. [276] 5.9. Polymer Dots Figure 15. Fluorescence lifetime imaging of nanodiamond labeled lung stem cells in lung tissue sections at different times after intravenous injection of the cells into mice. The images show that the nanodiamond-labeled stem cells (red) are primarily located in the subepithelium of the bronchiolar airways. Scale bar: 50 µm. Reproduced with permission. [267] Copyright 2013, Macmillan. NIR1- and NIR2-emitting polymer dots have been used for imaging in vivo. Small (ca 3 nm) p-dots fluorescing in the NIR2 (λ em 1050 nm) have been used to image arterial blood flow in mice with a temporal resolution of about 40 ms and monitor single cardiac cycles of about 200 ms. [42] The same p-dots were functionalized with Erbitux (a cancer drug) for the targeted imaging of cancer cells in vitro. Chan and coworkers have reported several p-dot particles for imaging and sensing in the NIR1 spectral region. [39,40,277] Recently, four different streptavidin functionalized quinoxaline-based p-dots (λ em nm) were used for targeted cell imaging as well as imaging and a toxicity assessment in zebrafish. [40] Rare-Earth-based Nanoparticles The use of upconverting nanoparticles in biological imaging and sensing applications has been reviewed. [115] In most applications of UCNPs, particles are excited at 980 nm and fluorescence is collected in the visible. However, several NIR1 to NIR1 upconversion particles have been used for in vivo imaging often with multimodal imaging capabilities. A polyphosphoric acid stabilized NaLuF 4 :Yb,Tm, [156] Sm radioactive/upconverting nanoparticle (λ em 800 nm) was used for in vivo blood pool imaging in a mouse model and particle toxicity evaluated (Figure 17). [278] The potential use of a similar particle (not radioactive) in guided surgery applications was explored by in vivo lymphatic imaging in a mouse model. [279] Chen and co-workers used α-naybf 4 :Tm 3+ /CaF 2 core/shell nanoparticles (λ em 800 nm) for deep tissue imaging in vivo and demonstrated the excitation and emission collection from a sample filled cuvette through up to 3.2 cm of pork tissue. [280] Some particles can be used as up- and downconverting particles for in vitro and in vivo whole animal (mouse) imaging. [96] Other particle types are purely downconverting, are (20 of 26) wileyonlinelibrary.com 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

21 Figure 16. Heparin sensing and in vivo tumor imaging using gold nanoclusters (λ em 700 nm). A) Schematic illustration of the heparin sensing process based on surface plasmon enhanced energy transfer between gold nanoparticles and fluorescent gold nanoclusters. B) Schematic illustration of the use of folic acid modified AuNCs for in vivo cancer imaging in the near-ir, which is shown in C) for a mouse model. Reproduced with permission. [272] Copyright 2013, American Chemical Society. also mostly excited at 980 nm and emit in the NIR2, increasing the penetration depth of the emitted light. Wang et al. reported a comparably complex core/shell nanoparticle with far NIR2 emission (λ em 1525 nm) coated with phospholipids for in vivo imaging of mice. [281] Recently, LiYF 4 based downconverting particles doped with Nd 3+ were used for lymphatic and vascular imaging in mice. [95] Hyperspectral whole body and tumor imaging in mice was reported by Naczynski et al. using four different DCNPs based on a NaYF 4 Yb:Ln core emitting throughout the NIR2. [93] In this study the penetration depth of NIR1 and NIR2 light through phantom tissue is also compared. 6. Conclusion The synthesis, characterization, bioconjugation and use of fluorescent nanomaterials particularly in the near-infrared spectral region is an extremely active and growing area of research. Realistically, only very few materials will be able to establish themselves as important fluorophores in biology and even fewer will become clinically relevant in the medium term. For many of the emerging materials, mainly proof-of-principle type studies have been carried out to date in various in vitro, ex vivo and in vivo settings. From a scientific point of view, the next step will be to demonstrate that these materials enable interdisciplinary research teams to answer biological questions, which could so far not be answered with traditional organic fluorophores in the visible spectral region. In this context, the higher penetration depth of near-infrared light through biological matter may be important as well as the superior photostability of materials like nanodiamonds or the background-free imaging capabilities of phosphorescent and upconverting materials. Nanomaterials for multimodal imaging that enable the Figure 17. Polyphosphoric acid-capped NaLuF 4 nanoparticles co-doped with Yb 3+, Tm 3+, and 153 Sm 3+ are used for combined upconversion fluorescence (λ ex 980 nm, λ em 800 nm) and radioactive single-photon emission computed tomography blood pool imaging in vivo. Reproduced with permission. [278] Copyright 2013, Elsevier WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com (21 of 26)