Many macroscopic imaging technologies (shown above the timeline) are in routine clinical use, and there have been huge advances in their capabilities to obtain anatomical and physiological information since the beginning of the twentieth century. Shown are some examples of bones (X-rays), soft tissue (ultrasound, MRI and CT rows), three-dimensional organs (CT and MRI rows) and physiological imaging (MRI and PET rows). Microscopic and other intravital optical techniques (shown below the timeline) have developed over the past decade and now allow studies of genetic, molecular and cellular events in vivo. Shown are surface-weighted, whole-mouse, two-dimensional techniques (macroscopic reflectance row); tomographic three-dimensional techniques, often in combination with other anatomical modalities (tomography row); and intravital microscopy techniques (microscopy row). The timeline is approximate and is not to scale. BLI, bioluminescence imaging; CT, computed tomography; DOT, diffuse optical tomography; FMT, fluorescence-mediated tomography; FPT, fluorescence protein tomography; FRI, fluorescence reflectance imaging; HR-FRI, high-resolution FRI; LN-MRI, lymphotropic nanoparticle-enhanced MRI; MPM, multiphoton microscopy; MRI, magnetic resonance imaging; MSCT, multislice CT; OCT, optical coherence tomography; OFDI, optical frequency-domain imaging; PET, positron-emission tomography. (PET image reproduced, with permission, from ref. 4. Diffusion MRI image courtesy of B. Ross and A. Rehemtulla, Univ. Michigan Medical School, Ann Arbor. X-ray image (left) reproduced, with permission, from ref. 86. Diaphanoscopy image reproduced, with permission, from ref. 87. Fibre-optic image reproduced, with permission, from ref. 88. BLI image courtesy of K. Shah, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts. Lifetime FRI image courtesy of U. Mahmood and C. Salthouse, Massachusetts General Hospital. DOT image reproduced, with permission, from ref. 89. FPT image courtesy of G. Zacharakis and V. Ntziachristos, Massachusetts General Hospital. FMT-MRI image courtesy of J. Chen, Massachusetts General Hospital. Bright-field image (left) reproduced, with permission, from ref. 14. Bright-field image (right) courtesy of T. Mempel, Massachusetts General Hospital. Epifluorescence image courtesy of F. Jaffer, Massachusetts General Hospital. OCT, OFDI image reproduced, with permission, from ref. 22. MPM image reproduced, with permission, from ref. 51. Microendoscopy image reproduced, with permission, from ref. 17.)

Different strategies have been pursued to develop agents for molecular imaging, and the various types of agent available and examples of these are indicated. Small molecules are shown in the top row, and macromolecular agents and nanotechnology-derived agents are shown in the bottom row. Four main types of small molecule are used. Small ligands refers to imageable small molecules (for example, ¹⁸F-labelled drugs and fluorescent peptides), whereas active-site binders (green) attach to specific protein pockets in enzymes (blue) either covalently or non-covalently. Site-specific protein tags (pink) achieve a similar function but at sites of interest in engineered proteins (white). Environmentally sensitive probes (for example, 4-N,N,-dimethylaminophthalimidoalanine (4-DAPA); yellow) change their physicochemical properties on interaction with the target (in this case, Tyr and T90β). Two main types of macromolecule are used. Supramolecular structures are synthetic agents that have been useful as enzyme substrates for the imaging of cathepsins and proteases or for delineating new microvasculature: shown is part of a poly-l-lysine backbone (blue) derivatized with protease-cleavable side chains (red) and polyethylene glycol (grey). Engineered proteins (with optimized pharmacokinetics) refers to other macromolecules that have been used for some targets. Finally, a host of nanomaterials, including inorganic nanoparticles (grey) and bionanoparticles, that can be used for imaging phagocytic cells or cell-surface targets (green) is emerging. (Images adapted, with permission, from ref. 90 (active-site binders), ref. 91 (site-specific protein tags), ref. 92 (environmentally sensitive probes), Mimetibody.com (engineered proteins) and ref. 93 (inorganic nanoparticles.)

Intravital microscopy can be used to derive quantitative parameters on intravascular and interstitial cell migration, interaction and viability in the tumour environment and the lymphatic organs. A, In vivo microscopic recordings in anaesthetized mice can be used to obtain data at cellular resolution. Some of the types of process that can be observed are illustrated. a, Leukocytes (green) trafficking in blood microvessels (red). b, B cells (pink) and T cells (green) inside and outside high-endothelial venules (diffuse red), respectively, in a tumour-draining lymph node. Collagen fibres are also visualized (blue). c, A cytotoxic T cell (CTL) (green) recognizing and killing a target cell (pink). Yellow dots track the position of the centre of the CTL at 15 s intervals. Collagen fibres are also visualized (blue). d, Simultaneous tracking of multiple cell types: a CTL (green) is firmly bound to an antigen-presenting cell (blue), and another CTL is transiently interacting with a FOXP3⁺ regulatory T (T_reg) cell (red). (Panels b-d reproduced, with permission, from ref. 51.) B, A typical analysis of cellular behaviour from data retrieved from time-lapse intravital imaging can assess four properties: the motility of cell populations (based on mean displacement over the square root of time); the instantaneous velocity of a single cell over time; the average cell-cell interaction time (in this case, A shows CTL-T_reg-cell interactions, and B shows CTL-target cell); and the viability of a single cell over time (as measured by the ratio of cytoplasmic fluorescence to nuclear fluorescence, F_c:F_n)15,51. C, A model for the in vivo role of tumour stromal cells in tumour growth and metastasis, based on intravital imaging studies, is shown. Immune cells such as monocytes (which differentiate into tumour-associated macrophages, TAMs), CTLs and T_reg cells are recruited into the tumour stroma from the blood vessels (dashed lines indicate direction) and can be visualized by intravital microscopy. Imaging studies can be used to investigate how these and other cells (such as carcinoma-associated fibroblasts, CAFs) interact with each other or with tumour cells and how they participate in tumorigenesis. In particular, in vivo studies have examined how CTLs recognize tumour-associated antigens at the surface of tumour cells41,48,49 and exert cytotoxic activity51 (a), how T_reg cells suppress the function of CTLs51 (b), how TAMs and CAFs remodel the extracellular matrix (ECM) and promote invasion (c) and angiogenesis54,56 (d), and how TAMs also participate in tumour-cell intravasation54 (e). Macroscopic imaging techniques are complementary and allow bulk cell migration to be measured. Objects that can be imaged in vivo include the cell types described here, as well as molecular targets (or parameters) for which specific probes have been designed (indicated in red). bFGF, basic fibroblast growth factor; CSF1, colony-stimulating factor 1 (also known as M-CSF); CXC-L12, CXCchemokine ligand 12; EGF, epidermal growth factor; EGFR, EGF receptor; IFN-γ, interferon-γ; IL, interleukin; MMP, matrix metalloproteinase; mTOR, mammalian target of rapamycin; PI(3)K, phosphatidylinositol-3-OH kinase; ROS, reactive oxygen species; TGF-β, transforming growth factor-β; TIE2, endothelial tyrosine kinase (also known as TEK); TNF, tumour-necrosis factor; UPA, urokinase-type plasminogen activator; VCAM1, vascular cell-adhesion molecule 1; VEGF, vascular endothelial growth factor; VVF, vascular volume fraction.

With a common computing platform, data obtained by different imaging techniques can be seamlessly assembled (and fused) for the screening, detection, characterization (in vivo pathology) and real-time treatment of early-stage cancers. In the example shown, a multislice CT image of the abdomen is acquired, and the slices are assembled in three dimensions. A suspicious colonic lesion is identified (at the intersection of the dashed lines) and reconstructed in three dimensions to reveal a polyp-like growth (arrow). Post-processing algorithms that explore differences in tissue and air-barium attenuation (insets) can be used to suggest, but not confirm, the presence of a malignancy. Near-infrared endoscopy using protease beacons then allows the detection of small and otherwise difficult-to-identify lesions. The bright signal in the near-infrared fluorescence channel indicates large amounts of proteases in a tumour (lower right) but not in a normal colon (upper right). White-light views of the same images are shown on the left. Microendoscopy shows a malignant signature in vivo, which can be used to make treatment decisions on the spot in real time. The microscopy image shows a polyp-like lesion, which contains mucosal cells (green), proteases (red) and new microvasculature (blue). Real-time local treatment can be achieved by lesion removal or light-activated therapy. 3D, three-dimensional.