The great amount of solid particles contained in a weighting agent is a major cause of the problems in both rheology properties and damage control mechanism of an oil-based drilling fluid (OBM). Therefore, a proper type of weighting agent can be a solution for the application of OBM. In this study, three weighting agents that have been commonly used with OBM, namely, standard barite, submicron barite and superfine manganese ore, are studied. Rheological properties of OBM and the degree of formation damage are assessed with regard to the three weighting agents. The agents are also studied in aspects of particle size, micromorphology, filtration loss and wall-building property, acid dissolution efficiency of mud cake, lubricity and sedimentation stability to analyse the effects of the agents on rheological properties and the degree of damage as well as to figure out the mechanism of rheology control and damage control. For the OBM, there is a mutual effect between rheological stability and the degree of damage. In consideration of the agents' properties, we can enhance the rheological stability of the OBM and control the degree of formation damage by properly selecting particle size, using acid-soluble materials and forming the mud cake with ultra-low permeability that can easily be cleared away. It is an efficient tool to reduce drilling accidents and raise the drilling speed . In mostStandard barite and a small amount of superfine barite have been the major weighting agents for the oil-based drilling fluid over the years. In an application where the density is high, however, the suspension of the standard barite is not stable and can easily be affected by temperature and pressure, which will seriously affect the drilling speed and also increase the probability of accidents . By far,The usage of a great amount of weighting agent will worsen the rheology, filtration loss and wall-building property, and lubricity of the oil-based drilling fluid . The rheThe oil-based drilling fluid contains a great number of solid-phase particles due to the usage of a weighting agent, and the plugging of these particles is a core factor for the formation damage . In compThe damage control of the oil-based drilling fluid depends partly on its rheological stability. In this article, the rheology of the oil-based drilling fluid and the damage of the steel core are assessed at different amounts of three weighting agents that have been commonly used in oil-based drilling fluid, namely, standard barite, submicron barite and superfine manganese ore. The effects of the weighting agents' particle size and micromorphology have also been analysed. With respect to filtration loss and wall-building property, acid dissolution efficiency of the mud cake, lubricity and sedimentation stability of the oil-based drilling fluid, we have revealed the mechanism of the weighting agents to control the rheological properties and the formation damage, with the intention of providing some useful information for the use of weighting agents in oil-based drilling fluid.2.2.1.Experimental materials: Standard barite ; millimetre–micron barite powder ; superfine manganese ore powder ); agents for treating oil-based drilling fluid, including diesel, organic soil, emulsifying agent, fluid-loss agent and rheology modifier (Jinzhou Jiahua Science and Technology Ltd); steel core (304 steel); the core of matrix strata (dense sandstone with permeability less than 0.1 × 10−3 µm3) (Keshen Block of Tarim Oilfield of CNPC).Experimental apparatus: Laser particle size analyser; electrical stability tester (Qingdao Chuangmeng Apparatus); field emission scanning electron microscope SU-8010 (Hitachi); high-temperature and high-pressure rheometer (FEI); lubrication coefficient tester; centrifugal machine; multifunctional core dynamic damage tester.Formula of the oil-based drilling fluid:2 brine (80/20) + 2.5% fluid-loss agent + 2.5% lime + 0.5% wetting agent + weighting agent. To ensure the correctness of the experimental results, when the experiment did not specify which drilling fluid is to be used, the oil-based drilling fluid is used as the 1# formulation.1#: base oil + 1.0% organic soil + 1.5% primary emulsifier + 2.1% secondary emulsifier + 20% CaCl2 brine (80/20) + 1.0% fluid-loss agent + 1.5% lime + 1.0% wetting agent + weighting agent.2#: base oil + 1.0% organic soil + 3.2% primary emulsifier + 0.8% secondary emulsifier + 20% CaCl2 brine (80/20) + 3.0% fluid-loss agent + 2.0% lime + 1.2% wetting agent + weighting agent.3#: base oil + 2.4% organic soil + 1.2% primary emulsifier + 1.2% secondary emulsifier + 20% CaCl2.2.Rheological properties: The prepared oil-based drilling fluid is weighted by using standard barite, submicron barite and superfine manganese ore, respectively. Different amounts of the same weighting agent, i.e. 200, 400 and 600 g, are added into the oil-based drilling fluid to test the rheological properties of the fluid. The compound of different weighting agents: different amounts of the compound of standard barite and superfine manganese ore that are mixed in the ratio of 1 : 1 are added into the oil-based drilling fluid to test the changes of the rheological properties.Damage assessment: In view of the fact that acidification is a common measure for increasing production, we carry out the assessment in two ways based on the consideration whether the weighting agent is acid-soluble or not. The flow-back pressure and damage rate of both the steel core and the artificially fractured matrix core are tested after they have been damaged by the oil-based drilling fluid. The experiment procedure without acid dissolution is: kerosene displacement—inverse displacement of oil-based drilling fluid—kerosene displacement. The experiment procedure with acid dissolution is: kerosene displacement—inverse displacement of oil-based drilling fluid—inverse displacement of acid solution—kerosene displacement.3.3.1.Rheology reflects the flow and the deformation of a drilling fluid under external forces. It is a basic performance index and also a key property ensuring that the drilling fluid can suspend and carry away the drilling cuttings, and clean the downhole and the borehole, and can drill safely and quickly. In accordance with the rheology-based assessment, we have obtained the relationship between shear rate and apparent viscosity  and the 3.1.1.The experimental data of 3.1.2.3.2.−3 µm3. Two experimental procedures (i.e. with and without acid dissolution) are performed. The degree of damage reflects the effect of the oil-based drilling fluid on the permeable capability of reservoir rocks, and permeability is the most direct reflection of the permeable capability. In consideration of the experimental impact of different cores, we use a steel core and matrix core taken from Keshen Block of Tarim Oilfield to carry out the experiments. Fractures are artificially created to the matrix core because its permeability is very low, and the permeability of the matrix core with artificial fractures is 5–10 × 10As can be seen from the assessment of the damage to both the steel core and the natural core caused by the oil-based drilling fluid, the steel core recovers better than the natural core. The reason is that the fractures of the steel core are artificially created and the steel core is not as coarse as the natural core, which contributes to a higher recovery rate of permeability. In the experiment without acid dissolution, the degree of damage follows the distribution of the particle size. This is because the permeation area becomes decreased due to the damage, and the particle size distribution is greatly related to the amount and depth of the oil-based drilling fluid that permeates into the cores. In the experiment with acid dissolution, the effect of the oil-based drilling fluid weighted by superfine manganese ore on the permeability of the core is much lower than that of the drilling fluid weighted by acid-soluble barites. In general, acid dissolution can greatly help recover the permeability of the core and efficiently protect the reservoir of the oil-based drilling fluid.To test the rheological properties of different oil-based drilling fluids, the evaluation of the core damage of three different oil-based drilling fluids was carried out after adding 600 g of different weighting agents; the results are shown in tables 3.2.1.3.2.2.4.4.1.4.1.1.Analysis of particle size is an effective means to observe microscopic properties of a weighting agent. According to the analysis, we are able to distinguish the distributions of different particle sizes and, to some extent, predict potential damage to permeation channels of the reservoir caused by the weighting agent particles. To observe micromorphology, the three weighting agents are, respectively, added into 0.6% sodium hexametaphosphate solution to perform the ultrasonic dispersion for 5 min. The laser particle size analyser (LA-950A2) is used to measure the size distributions of the three weighting agents. For the purpose of accuracy, each sample is tested three times. As shown in The analysis of particle sizes of the three weighting agents shows that: the distribution intervals of submicron barite and superfine manganese ore particles are much narrower than those of the other two agents. If submicron barite or superfine manganese ore with this size is used, then structural viscosity will be formed in the oil phase. This result is consistent with the changing trend of the rheology, i.e. the oil-based drilling fluid becomes more stable. On the other hand, however, this is easy to result in flocculation if the system is not well controlled, which has great impact on the performance of the drilling fluid and also can cause damage to the reservoir. Standard barite is a proper weighting agent for drilling fluid. When acid dissolution is needed to remove the plug, then a small amount of superfine manganese ore can be added into the fluid.4.1.2.The weighting agent has a great effect on the rheological properties of a drilling fluid as well as on formation damage. The size and morphology of the weighting agent decide its dispersion state in an oil-based drilling fluid and, to some extent, decide the permeability of the mud cake formed on the wall. The overall performance of an oil-based drilling fluid depends partly on distributions of particle size and morphology. The micromorphology of the three weighting agents is scanned by using field emission scanning electron microscopy (Hitachi-SU8010) to observe the morphology and size of the particles, as shown in figures As can be seen from 4.2.Filtration loss and wall-building property is a core function of a drilling fluid and has great effect on the stability of the borehole wall of unstable stratum. For an oil-based drilling fluid, the filtration loss and wall-building property reflects mainly in the filtration loss rate and mud cake quality. If the filtration loss rate is too high, then the friction between fractures in the stratum is small and the stability of the borehole wall will therefore be affected. In addition, the large amount of the fluid filtrated into the stratum can cause formation damage such as emulsion plugging and liquid-phase entrapment. A poor control of filtration loss can also generate a thick mud cake, which may bring about a differential pressure sticking accident and, consequently, affect the accuracy of the downhole test. The solid-phase particle is an important tool to control the filtration loss of an oil-based drilling fluid, while the weighting agent is the major source of solid-phase particles. Therefore, the filtration loss and wall-building property of different amounts of weighting agents has been assessed with respect to high-temperature and high-pressure filtration loss and mud cake permeability, as illustrated in tables The experimental results suggest that: with increasing the amount of the weighting agent, the filtration loss of the drilling fluid also increases and the permeability of the mud cake increases accordingly. Under the condition that the amount of the weighting agent is the same, the mud cake formed by submicron barite and superfine manganese ore is thick and porous, so the effect of reducing filtration loss is not good and the penetration rate is high. Factors that have an impact on the permeability of the mud cake include the size, particle size composition and concentration of solid-phase particles in the drilling fluid. For the drilling fluid containing submicron barite and superfine manganese ore, the quality of the mud cake is poor and the penetration rate is high. This is because although submicron barite and superfine manganese ore particles are small, the size range of submicron barite particles is narrow and the size composition is not good. As a result, pores of the mud cake cannot be well filled during the process of filtration loss. Consequently, the rate of filtration loss is high and the quality of the mud cake is poor. In comparison, the quality of the mud cake formed by standard barite particles is better. The size range of standard barite particles is wide enough for the formation of a multilevel bridge. Compared with submicron barite and superfine manganese ore, standard barite has better mud cake quality and the permeability of the mud cake is also lower. Generally speaking, standard barite has the best filtration loss and wall-building property and, therefore, is applicable for the drilling fluid used in a stratum with high water sensitivity or sandstone formation with high permeability. For submicron barite and superfine manganese ore, the filtration loss and wall-building property is poor, so they should be carefully adjusted and controlled in usage. The high-temperature and high-pressure filtration loss rate and the mud cake permeability of the compound are better than those of a single agent. This is because the multilevel bridge can form a more dense mud cake and the filtration loss and wall-building property is also better.4.3.m1; (ii) put the mud cake into a porcelain bowl and add 15% HCl to soak for 10 h; (iii) filter the soaked pottery bowl with a 100 mesh screen and dry the residue at 50°C, weighing m2; (iv) compare the quality of mud cake before and after the experiment; the acid dissolution rate of the mud cake is (m1 − m2/m1) × 100%. The assessment suggests that: for the barite weighting agents, the acid dissolution efficiency is lower than 4%; for superfine manganese ore, the efficiency is above 97%; and for the compound of standard barite and superfine manganese ore that are mixed at a ratio of 1 : 1, the efficiency is as high as 50%, also showing a good effect of acid dissolution.Acidification is a common technology for increasing the production in mining in dense and unconventional oil and gas reservoirs and is also an important step in protecting the reservoir. It can be used to open the contaminated area around the wall of the borehole in the target reservoir, with a good effect of increasing the production. The weighting agent is a major source of solid-phase particles in an oil-based drilling fluid. The acid dissolution efficiency of the weighting agent is of great importance to help recover the permeability of the reservoir that has been contaminated by the drilling fluid. In this article, the acid dissolution efficiency of mud cakes formed by different weighting agents are assessed . We used4.4.A weighting agent can be used to increase the density of an oil-based drilling fluid. However, high density can cause a series of problems such as high solid content, high viscosity and high friction coefficient. The lubrication performance of an oil-based drilling fluid can be assessed by using a coefficient. In our experiment, the mud cake viscosity coefficients of the three weighting agents and the compound agent are tested, and the results are listed in 4.5.2 brine + 2.5% fluid-loss agent + 2.5% lime + 0.5% wetting agent + 600 g weighting agent. The total amount of the compound agent that is mixed at the ratio of 1 : 1 is 600 g. In the experiment, the fluid to be tested is poured into a 500 ml measuring cylinder. The difference between densities of the upper and the lower oil-based drilling fluid is measured at room temperature after 12 h and 24 h, respectively. The experimental results are listed in Sedimentation stability is a basic index to assess an oil-based drilling fluid system. The weighting agent can disperse and suspend well in an oil-based drilling fluid if the sedimentation is stable. In most cases, however, an oil-based drilling fluid has some problems relating to sedimentation stability, which is one of the two technological problems of an oil-based drilling fluid. In our experiment, the sedimentation of three different weighting agents in an oil-based drilling fluid has been observed to figure out the differences of the three agents, so as to provide some reference for practical application. The formula for the experiment is: oil base + 1.0% organic soil + 1.5% primary emulsion + 2.1% secondary emulsion + 20% CaCl−3 when 600 g of weighting agent is added into the fluid, which is lower than the density of 2.0 g cm−3, which is commonly required in engineering applications. In addition, the measurement is performed at normal temperature and pressure. In fact, basic properties of the oil-based drilling fluid will change greatly at high temperature and pressure. The above result indicates that the sedimentation stability should be carefully investigated. If fast sedimentation appears, it is not good for protecting the reservoir.The experiment of sedimentation stability shows that: the suspension of particles in the oil-based drilling fluid is not as good as that in the water-based drilling fluid. Although the change of the sedimentation stability is acceptable in engineering applications, the measured density is about 1.7 g cm4.6.−3. In comparison with a water-based drilling fluid, the rheology-control mechanism for an oil-based drilling fluid is much different. For drilling fluid with low density, the rheology-control technology has been well developed, but for drilling fluid with high density, the adjustment and control of rheology is still a technological challenge, and the weighting agent is the core factor that has an effect on rheology control of an oil-based drilling fluid [An oil-based drilling fluid is often used in an environment with a complex structure such as a shale formation, deep well or ultra-deep well, where the wall of the borehole is not stable. Generally, the density should be higher than 1.5 g cmng fluid ,28.The solid content in the oil-based drilling fluid with high density is very large, and there is a high probability of the contact of solid-phase particles. So, the major force resistant to the flow of the drilling fluid is the friction between solid-phase particles. The viscosity of the drilling fluid system with high density comprises three parts: non-structural viscosity, structural viscosity and friction between solid-phase particles. As the number of solid-phase particles (coming mainly from the weighting agent) increases, the viscosity is more affected by the friction between the particles. With the interaction between the particles at a certain concentration, a force chain will be formed, which makes the rheology control more difficult. The weighting agent is the major source of solid-phase particles. Therefore, weighting agent is the main content for studying the rheology control of the oil-based drilling fluid.Stribeck conducted a study on friction between particles and developed a curve named the ‘Stribeck curve’, which describes the friction coefficients of particles in different drilling fluids ,30. ThesAccording to both the assessment of the rheology of different weighting agents and the analysis of the particle size and morphology of the weighting agents, the apparent viscosity of the oil-based drilling fluid that is added with the standard API barite falls in a normal range. However, when the fluid is added with a micro-nano weighting agent, i.e. submicron barite and superfine manganese ore, the viscosity increases sharply. If the viscosity increases in a normal range, the drilling fluid can take away more rock debris, but if the viscosity increases too fast, the drilling efficiency will decrease. The rheology can be controlled if the friction between particles becomes smaller, which can be realized by increasing the spacing between the particles through changing the size of the weighting agent. Therefore, the compound made of different sizes of particles is an effective way to control rheology.The surface of the weighting agent is generally hydrophilic. A surface-active agent or surface modification can be purposely used to make sure that the particles of the weighting agent are evenly dispersing in the oil-based drilling fluid, which is a key to controlling the rheology. In addition, different material properties can be properly used to enhance the control of the rheology of the oil-based drilling fluid, for example, the changes of the friction caused by different particle shapes.4.7.For either an oil-based or water-based drilling fluid, the damage to the reservoir comes mainly from both physical and chemical effects of the particles and the fluid that intrude into the reservoir. Therefore, reducing the intrusion of the oil-based drilling fluid is the key for damage control, while the most direct and the most efficient way to reduce the intrusion is to control the quality of the mud cake. There are two patterns to reduce the intrusion of the fluid and particles: extremely short formation time of the mud cake and extremely low permeability of the mud cake .The formation of the mud cake is the result of the sedimentation of solid-phase and other particles captured in the permeation channel in the stratum, which depends on some physical and mechanical effects, such as inertia, diffusional sedimentation, gravitational sedimentation and electrostatic interaction. The mud cake is formed in two stages: the dynamic formation during the drilling process and the static formation after the drilling is stopped. The static filtration loss rate is smaller than the dynamic filtration loss rate, and the static mud cake is thicker than the dynamic mud cake. The fluid loss and wall-building property of the drilling fluid gradually becomes worse as the content of the weighting agent increases. This is because the solid-phase particles break the original particle composition in the fluid. As a result, the mud cake with extremely low permeability will not be formed. Besides, the rheology also becomes worse, which prolongs the formation of the mud cake.In the oil-based drilling fluid without adding bridging particles or a reservoir protection agent, the solid-phase particles come mainly from organic soil, weighting agent and a small amount of fluid loss agent. Generally, the size of organic soil is about 0.2 µm, the size of standard barite is 10–50 µm, and the sizes of submicron barite and superfine manganese ore are 1–3 µm. There are two causes for the poor quality of the mud cake in an oil-based drilling fluid with high density: (i) The difference between the sizes of the standard barite and the organic soil particles. Consequently, the composition of particle sizes is not appropriate. (ii) In the mud cake, the number of large standard barite particles is much higher than the number of organic soil particles.a is the mud cake formed by the matrix of the oil-based drilling fluid without adding any weighting agent. Particles with a size of 0.2 µm can accumulate at superfine pore throats to form a dense mud cake, which is efficient to control the filtration loss rate. But when the pore throat and fracture are larger, then the quality of the formed mud cake is poor. Figure 9b is the mud cake formed by the barite and organic soil, in which the particle size composition is 0.2 µm and 10–50 µm. The spacing between the two different sizes of the particles is large, and, in the drilling fluid with high density, the number of the organic soil particles is not big enough to form the bridge, which, therefore, cannot result in the formation of a dense mud cake. Figure 9c is a dense mud cake formed by 1–3 µm submicron barite and superfine manganese ore particles and 0.2 µm organic soil particles. Likewise, the number of organic soil particles is small, and the drilling fluid containing a superfine weighting agent is affected by many factors. Furthermore, the apparent viscosity is too large, which limits the use of superfine weighting agents in a large amount. Consequently, the quality of the mud cake formed by the particles of the two small sizes is affected. Figure 9d shows the mud cake formed by standard barite, submicron barite and superfine manganese ore, which have three levels of particle size, forming a multilevel particle size composition of ‘bridging by large particles—bridging and filling by medium particles—filling by small particles’. For each level, the particle size follows the normal distribution, which makes the particle size composition more reasonable. In addition, if the particles of the three sizes are mixed in a reasonable ratio, the properly increased viscosity is more favourable to take away solid-phase particles and improve the rheology of the oil-based drilling fluid. Of course, a reservoir protection agent, bridging particle and other treating agents can be used in accordance with practical conditions to refine the particle size composition, so as to further reduce the intrusion of the drilling fluid and particles and also protect the reservoir.To describe the quality of the mud cake, the weighing agent particles and the organic soil particles in the oil-based drilling fluid are simply seen as round particles, as shown in Figure 9Seen from the perspective of reservoir protection, the reservoir shall be plugged quickly and tightly and the permeability of the reservoir shall be efficiently recovered after completion of the well. Measures to recover permeability of the reservoir include natural flow-back of solid-phase particles under the formation pressure, perforation and acid pickling, etc. . NaturalGenerally speaking, the weighting agent for an oil-based drilling fluid shall be selected in accordance with practical geological conditions. Standard barite has a cost advantage. Submicron barite is suitable for reservoirs that have higher requirements on viscosity and can reduce the damage by using less treating agents. Superfine manganese ore is more protective for reservoirs, which is why it has been used in some blocks. The assessment of the weighting agents has focused largely on studying the effects of the agents on the oil-based drilling fluid, by which we have figured out the damage caused by the agents, i.e. the damage to the reservoir is influenced by material, particle size distribution and surface properties of the weighting agents. In applications, one or several compounds can be used in accordance with practical conditions to meet engineering requirements and also protect the reservoir.5.(1) The rheological and suspension stability are two technical problems that cannot be solved well in an oil-based drilling fluid. The weighting agent is the core factor affecting the two properties. The rational selection of the weighting agent can improve the stability of the oil-based drilling fluid.(2) The particle size and morphology of the weighting agent greatly affect the rheology of the oil-based drilling fluid. The particle size of most standard barite particles is above 10 µm, and submicron barite and superfine manganese ore are basically distributed in the range of 1–3 µm. The morphology of standard barite is irregular; submicron barite particles are quasi-circular structures; and superfine manganese ore particles are longer. The differences in both particle size and morphology have brought about changes in filtration loss and wall-building property, and the lubricity and sedimentation stability of the oil-based drilling fluid. A proper utilization of these differences can improve the rheological stability of the oil-based drilling fluid.(3) The particle size, morphology, surface wettability and acid solubility of the weighting agent affect the reservoir protection performance of the oil-based drilling fluid. The degrees of the damage to permeability caused by the three weighting agents are higher than 50%, but when the acid-soluble superfine manganese ore particles are added into the oil-based drilling fluid after the acid dissolution, the damage degree can be controlled below 10%.(4) For the oil-based drilling fluid, rheology control and damage control are complementary to each other. To improve the rheological stability of the oil-based drilling fluid and protect the reservoir, we should take full consideration of the properties of both the drilling fluid and the weighting agents, rationally design the particle granular composition, make use of acid dissolution, quickly form the mud cake with extremely low permeability and efficiently remove the mud cake.


Imaging has an essential role in the planning and delivery of radiotherapy. Recent advances in imaging have led to the development of advanced radiotherapy techniques—including image-guided radiotherapy, intensity-modulated radiotherapy, stereotactic body radiotherapy and proton beam therapy. The optimal use of imaging might enable higher doses of radiation to be delivered to the tumour, while sparing normal surrounding tissues. In this article, we review how the integration of existing and novel forms of computed tomography, magnetic resonance imaging and positron emission tomography have transformed tumour delineation in the radiotherapy planning process, and how these advances have the potential to allow a more individualised approach to the cancer therapy. Recent data suggest that imaging biomarkers that assess underlying tumour heterogeneity can identify areas within a tumour that are at higher risk of radio-resistance, and therefore potentially allow for biologically focussed dose escalation. The rapidly evolving concept of adaptive radiotherapy, including artificial intelligence, requires imaging during treatment to be used to modify radiotherapy on a daily basis. These advances have the potential to improve clinical outcomes and reduce radiation-related long-term toxicities. We outline how recent technological advances in both imaging and radiotherapy delivery can be combined to shape the future of precision radiation oncology. The integration of imaging technology into modern radiotherapy treatment machines has led to an increase in the precision and accuracy of radiation delivery.10 Imaging throughout a course of radiation treatment enables anatomical changes to be detected, and radiation plans to be adapted accordingly. Indeed, adaptive radiotherapy, whereby an individual plan can be created for each treatment fraction depending on the precise positioning, and shape, of the tumour at the time of imaging, is a new goal for radiation oncology. For this goal to become a realistic possibility, advanced imaging and the integration of automated segmentation processes for the delineation of target volumes and surrounding structures need to be developed.Over the past decade, a number of significant technical developments, such as image-guided radiotherapy (IGRT), intensity-modulated radiotherapy (IMRT), stereotactic body radiotherapy (SBRT) and proton beam therapy (PBT), have enabled the concept of ‘precision radiotherapy’ to be advanced rapidly.14 The development and application of new functional imaging techniques might offer a greater understanding of individual tumour biology and could help to identify potential areas of radio-resistance within a tumour that require radiation dose escalation.The first step in being able to deliver precision radiotherapy is accurate target delineation during the radiotherapy planning process. Advanced multi-modality diagnostic imaging with computed tomography (CT), high-resolution magnetic resonance imaging (MRI) and 18-F-fluorodeoxyglucose positron emission tomography (FDG-PET)/CT imaging now forms part of the routine staging process for a number of tumour types.In this review, we begin by outlining the target delineation process before providing an overview of recent advances in technology relevant to biological target volume definition, IGRT, adaptive radiotherapy and the technologies under development that will define the future of precision radiation oncology.15 Errors in delineation can ultimately lead to an insufficient dose being delivered to the tumour, leading to reduced tumour control, or too high a dose being delivered to nearby OARs, resulting in increased toxicity for the patient. Imaging thus serves a vital role in defining the targets for radiation therapy.The more conformal and precise radiation delivery becomes, the more vital it is to accurately identify the tumour, its local extension and the exact location of surrounding critical structures: organs at risk (OARs). Inaccuracies in tumour and OAR delineation are recognised as a common source of radiation therapy error.16 The macroscopic, radiologically measurable tumour is delineated as the gross tumour volume (GTV). Tumour delineation from CT imaging can be challenging, especially for soft tissues. To improve the accuracy of GTV delineation, algorithms have been developed that allow co-registration of diagnostic images, particularly those derived from MRI and FDG-PET, with the primary simulation CT scan.18 MRI provides higher resolution and greater soft-tissue contrast in pelvic tumours when compared to CT imaging, while FDG-PET provides more accurate tumour delineation in head and neck, lung, lymphoma and oesophageal cancers.22 Registration can be relatively simple (rigid), where the transformation preserves the distance between all points, or more complex (non-rigid or deformable), which involves a significantly greater number of degrees of freedom and allows for distortion between two image sets. Such co-registration or ‘fusion’ of images allows for the GTV to be contoured on to the planning CT dataset, while using information from the diagnostic images to ensure accurate tumour delineation. The development of MR compatible devices—devices that are safe and unaffected by the MR environment—also means that MRI can now be performed with the patient in the radiotherapy planning position.23 This approach avoids the need for complex registration algorithms, and helps to minimise inaccuracy that may occur during the image fusion process.The radiotherapy planning process typically starts with the acquisition of a CT scan with the patient immobilised in the optimal radiotherapy treatment position. CT scans are the standard imaging modality for radiotherapy planning, as they provide a three-dimensional (3D) view of the tumour as well as data regarding electron density, which are required for dose calculations.26 Delineation of the CTV is based on anatomical/spatial information gained from cross-sectional imaging. However, this information might be an oversimplification, as it does not reflect the underlying complexity of the tumour; instead, current practice is to deliver a relatively homogeneous radiation dose to the entire CTV.27 Information on the risk and extent of tumour spread in expanding GTV to CTV is based on historical practice rather than the extent of tumour quantified on imaging for an individual patient.28A second volume, the clinical target volume (CTV), adds a margin to the GTV to cover neighbouring sites that are at risk of harbouring microscopic disease, and that are important to target to achieve cure.26The planning target volume (PTV) is a geometrical concept that allows for uncertainty in planning or treatment delivery. It takes into account the internal motion of the tumour during treatment ), in addition to random and systematic variations that occur in patient set-up on a daily basis. These variations can include changes in the tumour position and shape due to tumour regression or growth, bladder filling or rectal distension, as well as unpredictable changes arising from a change in the patient position or set-up methodology between each delivered fraction. The PTV is therefore the recommended parameter to ensure that an adequate dose of radiotherapy will actually be delivered to all parts of the CTV Fig. .26Fig. 129 FDG-PET imaging is currently the most widely used functional imaging technique that could facilitate BTV delineation, although a number of other novel functional imaging techniques are in development  Fig. . DelineaCT is the most widely used imaging modality for cancer staging and response assessment. A standard diagnostic CT scanner comprises of an X-ray source and a detector, mounted on a rotational gantry that moves around the patient. Computing algorithms are then used to create cross-sectional images from the mathematical reconstruction of the measured X-ray intensities from the detector. New reconstruction algorithms, particularly iterative reconstruction, and increased detector sensitivity have facilitated a significant reduction in exposure to ionising radiation, and continuous improvements in data processing (e.g. noise and artefact correction) have enabled high-quality CT imaging for cancer. These developments have not only driven an improvement in anatomical image quality but have spawned a number of quantitative CT techniques that are better able to characterise tissue and describe function. Imaging biomarkers derived from techniques such as perfusion imaging and dual-energy CT could improve target definition and form the basis for more individualised cancer therapy.32 Qualitative and semi-quantitative evaluation and mapping of these data can improve tumour detection and differentiation of tumour tissue from healthy or treated tissues.33 More complex kinetic perfusion models can derive functional physiological parameters such as blood flow, blood volume, mean transit time, extracellular-extravascular volume fraction, and permeability surface area product. Blood flow reflects the delivery of oxygen and other nutrients to tumour tissue, and is an indirect measure of hypoxia and angiogenesis, whereas the permeability surface area product reflects the leakiness of tissue vasculature, and interstitial pressure. Neovascularisation, hypoxia and reduced contrast extraction are parameters associated with tumour aggressiveness and treatment resistance, and could be specifically targeted.Perfusion CT, or dynamic contrast-enhanced CT (DCE-CT), imaging employs standard low molecular weight iodinated contrast agents. When administered as an intravenous bolus, tissue concentration is dependent on vascular flow and interstitial accumulation, resulting in differential attenuation on CT imaging, before recirculation and clearance of the agent by the kidneys. Neovascularisation and hyperpermeability within the tumour vasculature results in a concentration curve that differs from that of healthy tissue, with a more rapid initial uptake of contrast, high peak enhancement and rapid washout.34 Studies have also investigated changes in tumour perfusion following treatment with systemic therapy.36 The identification of such biomarkers, that represent areas at risk of radio-resistance, may therefore highlight areas within the tumour that may require focal dose escalation.Tumour perfusion rate has been assessed as an imaging biomarker in patients with head-and-neck squamous cell carcinoma (HNSCC) treated with radiotherapy and found to be an independent predictor of local control of the tumour—patients with lower perfusion rates had a significantly higher local failure rates.37 and hypovascular pancreatic tumours,38 and could aid with tumour delineation in the future.Contrast resolution, and therefore sensitivity to abnormal tissue (hypo-enhancing or hyper-enhancing), can be improved by using dual-energy CT techniques to produce low-tube-voltage imaging. Dual-energy CT imaging is a technique that uses attenuation data acquired at two different tube voltage settings. Using these data, images at any tube voltage can be reconstructed. At low-tube-voltages the X-ray absorption of iodine is increased relative to water and soft tissues, resulting in increased image contrast. This technique has been shown to increase the conspicuity of hypervascular liverStandard MRI relies on the magnetic properties of hydrogen protons within a tissue to generate image contrast. The past decade has seen the development of several new quantitative MRI techniques that have expanded the use of these properties to start to probe the underlying tissue microstructure.Diffusion-weighted MRI has already been widely adopted in cancer imaging due to its ability to enhance soft-tissue contrast. Diffusion-weighted imaging (DWI) adds further sensitivity to the detection of the intrinsic random thermal movement of hydrogen protons within water molecules that occurs as part of the natural (Brownian) diffusion process of water. Sensitisation to this motion is achieved with a pair of equal and opposing magnetic gradients, applied in succession, such that any net displacement of water dipoles can be attributed to diffusion along the orientation of the field gradients. Measured DWI signals from water movement within tissues thus reflect properties such as cellular density, intracellular structure, membrane permeability, interstitial volume and blood flow.40 ADC values have been assessed in patients with HNSCC treated with chemoradiation. Significantly lower pre-treatment ADC values were found in complete responders when compared to partial responders, suggesting that this biomarker could be used to predict response to treatment and may even allow for dose-modification.41 Technological advances have allowed quantitative functional assessment with MRI to be broadened into whole body acquisitions (WB-MR-DWI) with accurate staging of cancers including lymphoma42 and myeloma.43Tumour growth, resulting in areas of increased cellularity, tends to cause restriction of water motion, which can be quantified by the apparent diffusion coefficient (ADC). A change in tumour cellularity, perfusion and cellular necrosis following treatment is reflected in a change in the ADC value, and the potential utility of DWI in tumour detection, grading and assessment of treatment response has been demonstrated in a range of tumour types.44 This approach allows separation of the diffusion and perfusion signal component of the water molecules, and could improve tissue characterisation such that tumours might be distinguished by pathological grade.46 However, these existing DWI models lack specificity for various elements of the tissue microstructure, such as cell size, shape, intracellular structure, and perfusion effects. An improved framework for describing diffusion behaviour was proposed by Panagiotaki et al.47 This Vascular, Extracellular, and Restricted Diffusion for Cytometry in Tumours (VERDICT) method uses a three-compartment model to specifically account for intracellular, interstitial and intravascular water and map descriptors, which form key components of histopathological tumour assessment  model to the DWI-MRI. This model is based on the approximation of the diffusion of water molecules within a tissue. More complex models that divide the diffusion signal into pseudo-diffusion (like perfusion) and true molecular diffusion can also be applied .ent Fig. . VERDICT52 The technique has become the backbone of some MRI protocols for breast examinations and a sensitive imaging test for breast cancer detection53 with qualitative analysis of contrast kinetics. DCE-MRI is, however, complicated by the non-linear relationship between contrast agent concentration and signal intensity, and its dependence on scan parameters such as flip angle, repetition time and pre-contrast signal. Furthermore, quantitation can be complex, and measurement variation across centres remains an issue, which has limited the clinical implementation of DCE-MRI in cancer imaging.54 Some of these limitations could potentially be overcome by combining more than one type of MRI imaging in each patient to personalise treatment planning and assessment.56Dynamic contrast-enhanced-MRI (DCE-MRI) is a quantitative MRI technique that allows investigation of tissue microvascular structure and function by evaluating the pharmacokinetics of an extracellular low molecular weight contrast agent (e.g. gadolinum-based agents) as it passes through the tumour vasculature. The lack of ionising radiation allows temporal sampling frequency to be limited only by sequence acquisition time, and the technique is therefore attractive for repeated interval assessments. DCE-MRI is sensitive to alterations in vascular volume, blood flow and vascular permeability, and its role in tumour assessment has been extensively investigated1H, 31P, 13C and 19F). MRS allows the analysis and quantification of metabolites present in tissue, such as choline, creatine and myoinositol, which could act as tumour biomarkers. MRS has shown promise in the diagnosis and monitoring of tumours in the brain, prostate and breast.59 The technique is, however, technically complex, offering lower signal strength and sensitivity in comparison to other MRI techniques, which has limited its clinical applicability.Magnetic resonance spectroscopy (MRS) is a technique based on the detection of radiofrequency signals generated by specific endogenous nuclei , and causes increased signal loss on T2*-weighted imaging compared with diamagnetic oxyhaemaglobin (in which only paired electrons are present). This fundamental difference in the paramagnetic properties of oxygenated and deoxygenated blood means that BOLD MR imaging is able to detect changes in oxygenation within a tissue. Although BOLD MR methods have mainly been used to map functional activity in the brain, they have also been used to help evaluate tumours61 and interventions designed to disrupt tumour oxygenation.62 In the context of radiotherapy planning, BOLD-MRI may therefore enhance treatment plans by improving the delineation of critical neural structures that must be spared from high radiation doses. Furthermore, BOLD-MRI may provide a non-invasive method of imaging tumour hypoxia, which could identify tumours (or even areas within a tumour) at risk of radio-resistance, and could predict response to treatment.Blood oxygen level-dependent (BOLD) MRI is a non-invasive functional imaging method that exploits endogenous contrast.63Molecular information can be extracted by MRI methods that exploit the difference in magnetic properties between tissues of different composition. Signal can be modulated by cross interaction between mobile water and relatively immobilised protons within macromolecules. By using selective radiofrequency pulses to excite the magnetisation of exchangeable protons  and decrease the associated water MRI signal because of proton magnetic interaction, Chemical Exchange Saturation Transfer MRI is able to sensitise the acquisition to smaller macromolecules. Signal from even low concentrations of metabolites is magnified and each can be mapped separately.31 Other PET tracers have since been developed, including fluorine-18 fluoromisonidazole (F-MISO),64 which binds to hypoxic areas of the tumour, and 3′-deoxy-3′-fluorothymidine (FLT), which is a measure of tumour proliferation. PET-CT and PET-MRI with 18F-fluoroethylcholine have also been shown to be of value in the diagnosis and response assessment of paediatric brain tumours.66Positron emission tomography (PET) is a functional imaging technique involving the use of radioactive tracers—positron emitters attached to targeted biologically active molecules. 3D images of tracer concentration within the body are then constructed to assess the distribution of the targeted molecule in vivo. FDG, an analogue of glucose, is the most frequently used tracer, as FDG avidity reflects a number of key biological processes likely to be relevant to radiotherapy response, including receptor-mediated glucose uptake, cellular respiration, hypoxia and perfusion.124I is a positron-emitting isotope of iodine, and 124I-labelled metaiodobenzylguanidine (mIBG) can be used in the assessment of neuroblastoma by combined PET-CT or PET-MRI, in place of 123I-mIBG scintigraphy and single photon emission computed tomography (SPECT)-CT, to identify diseased areas with greater resolution. The longer half-life of 124I-mIBG allows serial scans to be performed over several days to track the biokinetics of the radiopharmaceutical. This might have predictive value for dosimetry prior to 131I-mIBG molecular radiotherapy.67Although functional imaging techniques have the potential to provide important prognostic information on a tumour prior to radiotherapy treatment, translating this information into improving precision radiotherapy remains a challenge. The aim is that functional imaging techniques would identify subvolumes of a tumour representing areas of radio-resistance–the biological target volume—and that these areas would then receive an extra dose of radiation with high precision without increasing the dose to the whole tumour.69 Early clinical trials have also used tumour burden, based on FDG-PET and DCE-MRI imaging, to enable focused escalation.72 These trials show that focused dose escalation using functional imaging is feasible, but large prospective clinical trials are required to establish the benefit for tumour control and toxicity. Although moving from a GTV to a BTV is the ultimate aim in being able to deliver precision biologically-directed radiotherapy, a number of challenges still need to be overcome before this approach becomes standard clinical practice. At present, there is no single imaging modality that reflects all underlying mechanisms of radio-resistance, and studies have shown that it may be difficult to establish the spatial relationships between imaging techniques.73 Imaging biomarker prioritisation will therefore need to be established before functional imaging can be reliably used for dose escalation.A number of radiotherapy planning studies have assessed the role of hypoxia to target biological dose escalation. These have mainly involved PET-based imaging, including F-MISO, and have focused on head and neck cancers.16 While traditional radiation treatments deliver a consistent intensity across the treatment field, IMRT uses multiple beams to deliver the dose, with a high degree of intensity modulation across each beam.75 Advanced computation, including inverse planning—an optimisation process which begins with specification of the desired dose to the tumour targets and dose limits to critical structures—is required to decide how to alter the intensity of the beam in order to make the treatment most conformal. IMRT can therefore allocate different dose targets for treatment to achieve optimal dose distributions.75 This technique enables dose escalation to the tumour target or, potentially, to defined ‘high risk areas’ within the target (creating an inhomogeneous dose across the tumour), while sparing dose to the surrounding normal tissue.The delivery of radiotherapy has changed significantly over the past two decades. The introduction of 3D imaging has moved radiation delivery away from simple treatment fields to a more complex approach using 3D conformal radiotherapy (CRT). This was followed by the introduction of IMRT, an advanced form of CRT that optimises the delivery of radiation to irregularly shaped objects.79 Intensity-modulated arc therapy (IMAT) is a form of IMRT, but in this approach the linear accelerator gantry head rotates in an arc around the patient;;80 it shows improved conformity and reduced treatment times.81 Optimal IMRT or IMAT delivery does, however, require more advanced image guidance than 3D CRT does. For 3D CRT, patients are aligned for daily treatment with the use of external skin marks (tattoos) and correct positioning is verified on radiographs by matching to anatomical landmarks.82 With the greater dose conformality provided by IMRT or IMAT, however, there is a greater need to reliably localise the target at the time of treatment.Dose escalation using IMRT has been shown to improve outcomes at a number of tumour sites, without increasing toxicity.75 As such, CBCT imaging prior to treatment enables the safe delivery of radiotherapy by IMRT and IMAT approaches.The development of on-board imaging systems means that linear accelerators are now capable of performing cone beam CT (CBCT) scans prior to treatment, which increases the certainty of tumour location at the time of treatment. Imaging with CBCT can identify daily changes in tumour position due, for example, in the case of pelvic tumours, to changes in bladder and bowel filling. Localising the tumour can be enhanced further by the implantation of fiducial markers, either within or near to the tumour, prior to the start of treatment.84 The doses delivered in SBRT are therefore ‘ablative’ and, accordingly, the safe delivery of such large doses per fraction necessitates effective patient immobilisation, precise target localisation, accurate treatment delivery and daily image guidance to allow very steep isodose gradients between target volumes and surrounding normal tissues.85 The excellent results achieved with ablative doses suggest that the anti-tumour effects of SBRT are different to those induced by conventional fractionation. Endothelial cell damage, enhanced anti-tumour immunity and a lack of time for reoxygenation and repopulation are possible underlying mechanisms for the increased efficacy of SBRT.86Traditionally, radiation treatments are fractionated into daily doses of 1.8–2.0 Gy to maximise the therapeutic ratio between the dose to the tumour and to normal tissue, and this approach applies to CRT and IMRT techniques. SBRT is an alternative method that accurately delivers a high dose over one to five fractions to an extracranial target.85 These advances include ‘motion-encompassing methods’ (in which motion will be present during radiotherapy), ‘forced shallow breathing’ techniques, ‘breath hold’ techniques, ‘respiratory gating’ techniques and ‘respiration synchronised’ techniques.87 The advent of four-dimensional CT (4D-CT) imaging, or respiratory-correlated CT imaging, has allowed high-quality CT data to be obtained at the radiotherapy planning phase in the presence of respiratory motion. 4D-CT data can then be analysed to assess the mean tumour position and tumour range for treatment planning, in addition to the relationship of the tumour position to phases of respiration.87 Tumour motion throughout the breathing cycle can then be accounted for when the radiation plan is created, delivered and verified. Abdominal compression devices can restrict respiratory motion and enforce shallow breathing patterns. Alternatively, motion can be arrested through voluntary or device-assisted schemes and radiation delivered only when the patient holds his or her breath (using ‘breath hold’ techniques). These schemes include deep-inspiration breath hold and active-breathing control systems. Treatment machines that interrupt radiation delivery when the tumour is out of position (gating techniques) can also be used. This approach involves verification of the patient’s anatomy with in-room imaging, and the use of either an external marker of respiration or implanted fiducial markers that move with the tumour.87 Finally, ‘respiratory synchronised’ techniques enable the radiation beam to ‘track’ the tumour during treatment, by either movement of the linear accelerator, or movement of the patient. Dedicated stereotactic radiotherapy machines capable of compensating for tumour motion in this way are available. Cyberknife®  is a dedicated robotic delivery system that can deliver SBRT with real-time tracking. This technique can allow radiation delivery with an accuracy of 0.5 mm.75Recent advances in IGRT allow for patient movement due to respiration to be measured and accounted for during treatment.89 One potential limitation of PBT is that the range of a proton is subject to considerable uncertainties caused by imaging, patient set-up, beam delivery and dose calculation.90 Proton ranges are highly sensitive to the composition of the media through which the beam traverses, and so are highly sensitive to variations in patient positioning.92 As the range of a proton beam varies as a function of the relative stopping power (RSP) of the absorbing material, there is a degree of range uncertainty due to the conversion of Hounsfield units (HU) in the planning CT scan to RSP. This uncertainty is ~2% for soft tissue, and as high as 5% for lung, fat, and bone; an average value of 3.5% is often assumed for clinical practice.95 To reduce this range uncertainty, there is a developing interest in using dual-energy CT imaging as opposed to standard single-energy CT imaging. Dual-energy CT imaging provides greater information on the elemental composition of the absorbing material, as the attenuation of the material is known at two energies; recent studies have suggested that dual-energy CT imaging could improve the accuracy of the predicted RSP, and therefore the estimation of proton range, in the order of 0.1–2.1%.97All of the treatment techniques discussed so far have utilised megavoltage photons to deliver radiotherapy. Charged particle therapy, such as proton beam radiotherapy (PBT), has gained particular interest as an alternative to photons. Protons deposit little energy as they interact with matter, until they reach the end of their range . This gives rise to a low dose on entering the tissue, which reaches a relatively flat dose plateau before rising sharply to a maximum, the Bragg peak, and then falling to zero at the end of the beam range. This unique characteristic dose release pattern has led to the use of PBT in paediatric cancers , and in adults to provide an effective dose to tumours that are localised adjacent to structures sensitive to the effects of radiation, such as the brainstem and spinal cord.131I-sodium iodide for thyroid remnant ablation and treatment of metastatic differentiated thyroid carcinoma.98 Even patients with extensive metastatic disease can be cured using this form of molecular radiotherapy.99 Other commonly used treatments are 131I-mIBG for childhood neuroblastoma100 and 177Lu-DOTATATE in the management of neuroendocrine cancers.101 A range of studies over the past 30 years have shown that about one in three patients has an objective response to 131I-mIBG102 and there is level one evidence of a survival benefit of 177Lu-DOTATATE.101Molecular radiotherapy, sometimes called radionuclide therapy or therapeutic nuclear medicine, is the administration of biologically targeted radiopharmaceuticals which are concentrated and retained in tumour tissue to a significantly greater extent than in normal tissues or OARs, resulting in the sustained delivery of radiation to the cancer at both the primary site and in metastatic deposits. One advantage over external beam therapy is that it is anatomically unconstrained—all tumour cells expressing the physiological uptake mechanism for the radiopharmaceutical in question will be targeted no matter where they are located anatomically. The oldest example is the use of 123I-mIBG scintigraphy is used for the staging and response assessment of neuroblastoma,103 and avid uptake into known sites of disease is an essential prerequisite for 131I-mIBG therapy. Similarly in neuroendocrine cancers, the demonstration by PET-CT of the uptake of 68Ga-DOTATATE into tumours with a greater standardised uptake value (SUV) than in liver is required before proceeding to 177Lu-DOTATATE therapy.104Accurate imaging is essential for the planning, delivery and evaluation of molecular radiotherapy. Selection of the targeted radiopharmaceutical for treatment is typically based on the use of imaging biomarkers. Semi-quantitative assessment of the uptake of mIBG using Imaging is essential for tumour dosimetry, which is now mandatory under the European Union Council Directive 2013/59/EURATOM. In relation to all radiotherapeutic exposures, practitioners must ensure that exposures of target volumes are individually planned and their delivery is appropriately verified, taking into account that doses to non-target volumes and tissues must be as low as reasonably practicable and consistent with the intended radiotherapeutic purpose of the exposure. This requires a series  of SPECT-CT scans to allow for quantification of the absorbed dose to the tumour and critical normal organs. Although the administration of a greater activity, proportional to body weight, will result in a higher dose to the whole body, tumour and organs, the precise doses vary depending on the biokinetics of the radiopharmaceutical in individual patients. Personalised molecular radiotherapy can therefore be based on modification of the activity of second and subsequent administrations based on the measured doses achieved following an initial therapy administration, or on measurements taken following the administration of a tracer.105 Indeed, recent advances in IGRT, particularly the use of CBCT, have shown that there can be significant volumetric changes to the tumour and OARs during a course of treatment.107 As radiation can now be delivered with advanced techniques that can create sharp dose gradients at the margins of tumours, even a slight change in anatomy could result in significant dosimetric changes. Imaging throughout the course of a treatment should therefore detect these anatomical changes, and allow for treatment plans to be modified accordingly. At present, imaging is most commonly performed using CBCT (carried out on the treatment unit) either on a daily or weekly basis (depending on the duration and type of treatment schedule and the likelihood of significant movement of the target volume). If significant anatomical changes are detected, then a new radiation plan can be created; this is termed ‘Adaptive Radiotherapy,’ but it is time consuming and can lead to delays in treatment. CBCT imaging, however, does not allow for optimal imaging of tumours and OARs when they are surrounded by soft tissues.108 Furthermore, it only allows for the assessment of anatomical, and not biological, changes in response to radiation.Radiotherapy practice traditionally involves delivering the original radiotherapy treatment plan to completion, without accounting for anatomical or biological changes that can occur over the course of treatment, which can last up to 7 weeks. However, anatomical changes could occur due to patient weight loss, tumour shrinkage, oedema, inflammation, and normal tissue volume alterations over this time period.112 Although FDG-PET is the most widely studied imaging modality, the roles of FLT-PET, DCE-MRI and DWI-MRI have also been assessed.116 Imaging the response of the BTV in this way could be then be used to adapt the radiation plan, and allow for dose escalation. There are, however, several issues in using functional imaging to guide adaptive radiotherapy. First, there will be differences in patient positioning between the planning CT and functional images taken during treatment; as such, a reliance on accurate co-registration algorithms is required. In addition, the optimal timing for assessing functional imaging during a course of radiation is unknown, as is the effect of concurrent chemotherapy or immunotherapy agents on functional imaging (Box 117 This is because the SUV of a tumour decreases in response to radiation (indicating a decrease in the number of tumour cells), while no change occurs in the normal background tissue. This results in the automated software not being sensitive enough to distinguish tumour from background and define a volume.118A number of studies have therefore evaluated the role of functional and molecular imaging techniques in evaluating the response of the tumour to radiation.In order to deliver optimal adaptive radiotherapy, images need to be taken throughout the course of the radiation treatment (not just at one time point), anatomical and functional changes need to be assessed, and rapid changes in the radiotherapy plan need to be implemented. These requirements have led to substantial interest in the role of MRI in real time, and online therapy guidance, whereby daily images are used to adapt the radiotherapy treatment plan before each fraction of radiotherapy is delivered.Box 1 Imaging techniques to assess immunotherapy126 As changes in tumour burden are used as a measure of response to treatment, typically using the Response Evaluation Criteria in Solid Tumours (RECIST) criteria, questions have been raised about how to assess these changes following treatment with novel immunotherapeutic agents,127 which has led to the development of a modified set of RECIST criteria for immune-based therapeutics (termed iRECIST).128 This approach allows for pseudo-progression to be taken into account when assessing initial response, but tumour measurements are still based on CT and MRI imaging methods.Tumours have been shown, by imaging, to respond differently to immunotherapies compared to chemotherapeutic agents. The novel mechanism of action of these agents, with immune and T-cell activation, can lead to unusual patterns of response that even resemble tumour flare (pseudo-progression).18F-FDG is widely used to assess the response of tumours to traditional therapy, its use is limited in assessing response to immunotherapy agents.130 This is due to the complex tumour microenvironment, in which metabolically active immune cell infiltrates can cause an expansion in tumour volume with increased glucose uptake.130 As the generation of an immune response leads to T-cell activation, detecting T-cell subsets (CD8+ and CD4+) within tumours might enable enhanced imaging of this response. For example, Granzyme B is a serine protease released by CD8+ T cells and natural killer cells during the cellular immune response, and represents one of the two dominant mechanisms by which T cells mediate cancer cell death. Early studies suggest that granzyme B might represent a biomarker for active cytotoxic immune response by PET imaging, with a potential predictive capability in immuno-oncology.130Although PET-CT with 119 Diagnostic quality MRI yields excellent soft-tissue visualisation, and therefore offers better soft-tissue images of both the tumour and the surrounding OARs when compared to CBCT images.120 MRI also provides several imaging modalities for the identification of movement, function and physiology, which could potentially include DWI and DCE-MRI. As such, it offers scope to combine the delineation of biological indices along with standard anatomical delineation. Integrating MRI functionality with a linear accelerator could potentially make these capacities available for high precision, real-time IGRT.121 However, at present DCE is not reproducible across multiple sites, and DWI, while reproducible, is biologically non-specific.73 As such, these limitations would need to be overcome before these imaging biomarkers can be integrated into clinical practice.121The MR Linac is a hybrid linear accelerator (linac) combined with an MRI scanner that can generate daily images of a patient during treatment.108 This would involve delivering an additional dose of radiation (or ‘boost’) to areas likely to show a poor response to radiation, while keeping the dose as planned to areas showing a good response. Research is ongoing as to how best to implement this new approach in clinical care.Improvements in soft-tissue imaging could allow for dose escalation to the tumour, resulting in improved tumour control. The integrated system allows for daily fast adaptive re-planning in addition to gated/tracked radiotherapy treatments, which can allow a reduction in the margins for uncertainty and tumour motion, thus decreasing the dose to normal surrounding tissue, and ultimately leading to a reduction in toxicity. Furthermore, functional imaging with MRI during a course of treatment could allow for adaptive focal boosting and personalised inhomogeneous target dosage based on response.To achieve real-time adaptive radiotherapy planning, including the use of the MR Linac, images need to be interpreted at the time of treatment. This involves delineation of the tumour and surrounding OARs immediately after the images have been taken, followed by registration with the treatment planning data. Once the images have been segmented, a new plan can be created. An alternative approach is to create a library of plans for each treatment course, and to then select the ‘optimal’ plan based on the current imaging. The creation of a new plan ensures that the dose from previous fractions can be taken into account in order to optimise the overall dose distribution. However, this approach can be time consuming.123 Currently available segmentation tools include those based on image greyscale interrogation, statistical shape modelling and body atlas methods. Machine learning and integration of artificial intelligence into the planning process might also offer a solution. Machine learning has already been employed in the context of image segmentation; it can be used to automatically detect and classify different types of tissue, and can also aid segmentation by learning image context and tissue appearance and by learning appropriate priors (such as reference images or atlases) for structures and organs.122The advent of auto-contouring systems integrated within the planning system offers one solution to this, by reducing the burden of re-delineating normal tissues and target volumes.124 Radiogenomics involves the integration of radiomic quantitative features  with underlying molecular features of the tumour at the genomic level. Radiomic and radiogenomic data can be integrated with other clinical data, such as outcomes for individual patients, to develop models that might be able to predict how radiotherapy should be adapted in clinical decision making.125The availability of large cohorts of images is essential to the development of auto-contouring software, and to the development of radiomics and radiogenomics, which are emerging as potential means of personalising radiotherapy. Radiomics involves the extraction of quantitative information on tumour pathophysiology from medical imaging , and the transformation of these data using pattern recognition tools into mineable high-dimensional data.Rapid advances in imaging and radiotherapy delivery techniques have occurred over the past decade, allowing radiotherapy to be delivered at higher doses and in a more highly conformal manner. However, the future of ‘precision radiation oncology’ needs to encompass more than an ability to deliver precise technical radiotherapy Fig. . It needA particularly timely research interest is the prospect of combining precision radiotherapy with novel immune modulators. The success of novel immune modulators, such as inhibitors of cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death ligand-1 (PD-L1), in treating certain types of cancers, has recently led to the emergence of ‘immuno radio-oncology’ as a new field of research. Combining radiation with immune modulators is expected to lead to synergistic effects, due to the complex interaction between radiation and the immune system. However, in order to treat, and monitor response to treatment, in this new cohort of cancer patients, new imaging biomarkers will need to be developed systematically and incorporated into the radiotherapy planning process (Box


The tumour switches on the NIR-AZA emission for fluorescence guided surgery. off to on bio-responsive NIR-fluorophores are utilised to switch-on first within the tumour. Bio-responsive cRGD, iRGD and PEG conjugates have been synthesised using activated ester/amine or maleimide/thiol couplings to link targeting and fluorophore components. Their off to on emission responses were measured and compared with an always-on non-responsive control with each bio-responsive derivative showing large fluorescence enhancement values. Live cell imaging experiments using metastatic breast cancer cells confirmed in vitro bio-responsive capabilities. An in vivo assessment of MDA-MB 231 tumour imaging performance for bio-responsive and always-on fluorophores was conducted with monitoring of fluorescence distributions over 96 h. As anticipated, the always-on fluorophore gave an immediate, non-specific and very strong emission throughout whereas the bio-responsive derivatives initially displayed very low fluorescence. All three bio-responsive derivatives switched on within tumours at time points consistent with their conjugated targeting groups. cRGD and iRGD conjugates both had effective tumour turn-on in the first hour, though the cRGD derivative had superior specificity for tumour over the iRGD conjugate. The pegylated derivative had similar switch-on characteristics but over a much longer period, taking 9 h before a significant emission was observable from the tumour. Evidence for in vivo active tumour targeting was obtained for the best performing cRGD bio-responsive NIR-AZA derivative from competitive binding studies. Overall, this cRGD-conjugate has the potential to overcome the inherent drawback of targeted always-on fluorophores requiring prolonged clearance times and shows excellent potential for clinical translation for intraoperative use in fluorescence guided tumour resections.The use of NIR-fluorescence imaging to demarcate tumour boundaries for real-time guidance of their surgical resection has a huge untapped potential. However, fluorescence imaging using molecular fluorophores, even with a targeting biomolecule attached, has a major shortcoming of signal interference from non-specific background fluorescence outside the region of interest. This poor selectivity necessitates prolonged time delays to allow clearance of background fluorophore and retention within the tumour prior to image acquisition. In this report, an innovative approach to overcome this issue is described in which cancer targeted  Advantages lie in its ease of use, enhanced safety profile over radiolabelling and the ability to acquire image data in real-time during surgical procedures.in vivo fluorescence imaging using molecular fluorophores exists. Following intravenous administration, fluorophore distributes to all vascularised regions within seconds, resulting in a strong non-specific fluorescence. This necessitates an unpredictable time delay to allow background fluorophore clearance, following which imaging is achievable if sufficient fluorophore is retained in the region of interest (ROI) (e.g. antibody), as an initial broad distribution will still occur. The time between administration and imaging depends on several parameters such as rates of accumulation and clearance from both the ROI and surrounding tissues and elimination from the body via metabolic and excretion pathways. Each of these factors can be influenced by the structure of fluorophore itself and by groups conjugated to it, but a time lag before imaging is unavoidable. To provide sufficient contrast for imaging, it is necessary to identify an optimal time point at which a maximum quantity of fluorophore is retained in the ROI with a minimum remaining in the surrounding tissues. For example, antibody conjugated fluorophores have been adopted in recent clinical trials for visualising breast and colorectal cancers utilising labelled bevacizumab and carcinoembryonic antigen (CEA) respectively.As a result, new classes of NIR-fluorophore have recently emerged, several of which are bio-conjugated to enhance their affinity for specific cancer types.st (ROI) . This liThus, for rapid and accurate intraoperative imaging innovative alternative approaches are needed to enhance target-to-background signal ratio at early stages following fluorophore introduction. One plausible solution is to exploit a mechanism of selective fluorescence quenching in the background areas, whilst first establishing the emitting potential of the fluorophore in the ROI . This ov1 performs as an excellent probe capable of real-time continuous imaging of fundamental cellular processes such as endocytosis, lysosomal trafficking and efflux  interactions. They act as transmembrane linkers between extracellular ligands such as ECM proteins, growth factors, matrix degrading proteins and the cytoskeleton, which serves to modulate various essential signalling pathways in most cells.For this study we have chosen one cRGD (c[RGDfK(PEG-PEG)]) and one iRGD (cCRGDKGPDC) peptide sequence for conjugation to the bio-responsive NIR-AZA imaging platform. It was hoped that these low molecular weight peptides would promote rapid uptake and switch-on of emission preferentially within tumours allowing a high tumour to background ratio (TBR) to be established without waiting for prolonged clearance times. In practice, it is envisaged that they would be administered and visualised intraoperatively, thereby not impeding the normal surgical or hospital workflow.in vitro cellular emission profiles and tested their in vivo tumour imaging performance using a human breast tumour model in mice.Incomplete tumour removal during surgical resection is closely related to cancer reoccurrence and patient survival rates. A major challenge in achieving cancer free margins is to fully distinguish between all of the cancerous growth and normal tissue during surgery. While high definition images obtained by PET, CT or MRI scans identify and diagnose tumour growths prior to surgery, such images are not overly useful to guide surgical resection during the operation. Currently, tumour margins are typically assessed by visual assessment and palpation of the tumour intraoperatively. However, the possibility of micro-invasion of the surrounding tissues can make it difficult to determine an adequate tumour-free excision margin. In this report, we have developed synthetic routes to RGD conjugated bio-responsive fluorophores, examined their photophysical and 2 and 3 were selected for synthesis using activated ester/amine coupling to conjugate the cRGD sequence and cysteine to maleimide addition for the covalent linkage of the iRGD peptide sequence ]) was selected as it is known to be an ideal construct for housing the integrin recognising tripeptide sequence  which was coupled with N-acetyl protected cysteine, through the amine of the lysine residue, to provide the final thiol substituted peptide.via cysteine to maleimide addition was efficiently achieved by reaction of 7 with iRGD 8 in DMSO at rt for 30 min to produce the required derivative 3  containing 10% fetal bovine serum (FBS). Absorption and emission wavelengths are listed in always-on10 was 17- and 15-fold greater than 2 and 3 respectively  was 23 and 18 respectively  and time-lapse NIR-fluorescence and differential interference contrast (DIC) images were acquired over 120 min. Image data showed that for always-on10, a fluorescence specific to the plasma membrane was rapidly observed within 15 min, which could be attributed to its strong association with the cell membrane  and later (due to retention of switched on probe) stage discrimination of tumour from background.The different cell staining patterns between 1, 2 and 3 were tested using subcutaneous tumours grown in nude mice. Fluorophore 10 was also included as a positive control in the study to demonstrate the advantage of using off to on responsive fluorescence over a constant emission. It was anticipated that 1, 2 and 3 would remain predominately fluorescent silent until tumour uptake occurred causing a fluorescence signal modulation to on. Experimental measurement of changes in tumor-to-background ratio (TBR) over time was the preferred method to quantify differences between bio-responsive and always-on fluorophores. Pegylated bio-responsive 1 was included to compare the turn-on time differences between passive PEG and the active integrin targeting of 2 and 3. The expectation being that the larger EPR dependent PEG conjugate would be slower.For this study, the human breast cancer cell line MDA-MB 231 was selected for its relevance to clinical forms of aggressive breast cancers for which the first line of treatment is often surgical resection. The ability of bio-responsive conjugates in vivo analysis using a standard dosing set at 2 mg kg–1 delivered by i.v. tail vein injection. Post injection, images were acquired initially at regular intervals between 10 min and 9 h and thereafter less frequently at 24, 48 and 96 h. The method used for image analysis was consistent across all experiments with TBR values calculated by measuring tumour ROI fluorescence against an average of three equally sized background ROI regions, two of which were close to and one distant from the tumour  for the first 60 min then rose to 1.6 and 1.8 at 6 and 9 h respectively. It took 9 h to reach maximum tumour fluorescence intensity with this level remaining relatively unchanged at 24 h. Pleasingly, the TBR threshold of 2 was reached at 24 h and this was maintained for a further 48 h. Comparing the changing TBRs of pegylated 1 and RGD 2 over time illustrates that substituting with the peptide sequence provides a considerably faster tumour accumulation. If clinically adopted, the slower time frame of 1 would most likely require its administration 24 h before surgery, though its long retention within the tumour may provide a prolonged window in which it could be imaged.Finally, the pegylated bio-responsive in vivo tests were performed on the most promising derivative RGD NIR-AZA 2. To gain insight into the initial rate of tumour fluorescence turn on, images were acquired every 10 min for 3 h immediately after introduction of 2. To establish that the RGD substituent was influencing the rate of tumour uptake, competitive binding or blocking experiments were also carried out. Experimentally this was achieved by first administering an i.v. tail injection of the RGD peptide 5  and following a short time period (5 min) 2 was then administrated. It would be expected that the first administration of RGD 5 would result in the integrin receptors being bound by the free peptide such that when NIR-AZA 2 was next introduced there would be a reduced fluorophore uptake and as such a lower switch-on of fluorescence. In order to make direct comparisons, pairs of mice with closely matching sized tumours were selected for each experiment (n = 4 pairs). One animal was first given RGD 5 then both animals were administered with 2, following which fluorescence images of both animals were taken every 10 min for the following 3 h. Averaged results from four experiment showed a 1.75 fold reduction in the total tumour fluorescence intensity after three hours for the mice which were first treated with the peptide 5 prior to receiving the NIR-AZA conjugate 2versus the mice which only received 2  or passive (PEG) tumour targeting groups and their photochemical, cellular and in vivo assessment of tumour imaging performance for bio-responsive probes 1, 2, 3 and always-on derivative 10 was conducted with monitoring of the fluorescence distributions over 96 h following administration. As anticipated, the always-on10 gave an immediate, non-specific and very strong emission profile throughout animals whereas the bio-responsive 1, 2 and 3 displayed relatively very low initial fluorescence. In the case of 10, clearance from normal tissue with accumulation and retention in tumour, allowed for a TBR above 2 to be reached between 9 and 24 h. All three bio-responsive derivatives switched on within tumours at time points consistent with their conjugated targeting groups. cRGD 2 and iRGD 3 both had effective switch-on in the first hour though 2 had superior specificity for tumour than 3. Probe 2 achieved the threshold TBR value of above 2 within 3 h and this was maintained for a further 24 h. Relatively, the PEGylated 1 had slower similar turn on characteristics taking 9 h to reach maximum fluorescence from the tumour. Despite the slower accumulation, its retention was biased to the tumour tissue with the threshold TBR value being reached at 24 h and maintained out to 96 h. The side-by-side imaging comparison of 1 and 2 is an important and unique illustration of the dynamic differences between passive EPR and active targeting in action.A comprehensive 2 has been identified as showing excellent potential for clinical translation for intraoperative fluorescence guided tumour margin identification. Its bio-responsive nature with early accumulation at the tumour periphery may overcome the inherent drawback of always-on fluorophores requiring prolonged clearance times. The PEGylated derivative 1 does also offer potential for clinical translation though its slow switch-on rate may ultimately limit its clinical scope.Overall, the cRGD-conjugate The next ongoing stage of this research is to record continuous NIR-fluorescence video of the bio-responsive turn on at tumour margins to gather more kinetic data on the tissue dependent rates of emission increase over the first 90 min. This real-time data will be utilised in conjunction with specifically developed algorithms for dynamic image analysis that could provide the surgical team with an augmented reality (AR) heat map representation of the tissue to be excised during the operation. An intraoperative use of dynamic fluorescence tissue imagery combined with AI analysis and a clinical AR interface has the potential to transform surgical practice.Detailed experimental procedures and characterisations are provided in the ESI.3, 7, 9, 10, carried out in vitro cell imaging and in vivo image analysis. H. D. synthesised, measured photophysical properties and analysed compounds 1, 2. M. G. developed the synthetic route to compounds 1, 2, 4 and determined their photophysical properties. E. C. performed in vivo imaging studies, preliminary analysis and ethical approval application with assistances from B. L. and W. M. G., R. E. synthesised and analysed compound 8. D. F. O. conceived the project, designed experiments and wrote the manuscript with input from all the co-authors.D. W. synthesised, measured photophysical properties and analysed compounds 2-azadipyrromethene based NIR fluorophores (EP2493898 and US8907107) in which he has a financial interest.DOS declares the following competing financial interest. Patents have been filed on BFSupplementary informationClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.