Table 2.

Summary of imaging methods discussed including resolution, depth of imaging, contrast used & safety, quantitation accuracy, clinical translation, and the tumor microenvironment factors the imaging methods have been used to investigate.

Imaging method		Resolution	Imaging depth	Contrast agents & safety	TME factors	Accuracy in quantitation	Clinical translation
Magnetic resonance	Magnetic resonance imaging[375,376]	25-100 mm	Whole body	Gd- or iron-oxide-based probes and dendrimer-based macromolecules; conventional MRI is safe for imaging for patients without embedded metals in their body while GD-based contrast may cause some adverse health concerns	ECM proteins, matrix metalloproteinase, mesenchymal stromal cells, cancer associated fibroblasts, immune cells, tumor vasculature, metabolic-choline-phospholipid metabolism, hypoxia, pH, and tumor stroma interactions	Only semi-quantitative, relying on regional differences in signal intensities, and primarily used to reveal gross morphological abnormalities	Currently clinically used technique, making future TME studies feasible; however, some preclinical studies using higher strength magnetic fields may pose challenges
Nuclear	SPECT[375,376]	1-2 mm	Whole body	Radiolabeled antibodies, antibody fragments, and antigens; SPECT requires exposure to radiation	Matrix metalloproteinase, mesenchymal stromal cells, and immune cells	Although clinically only semiquantitative, quantitation is still a key benefit of nuclear medicine and can be improved with quantitative SPECT (only tested preclinically)	Currently a clinically available technique; however, clinical translation suffers from increased attenuation and decreased resolution
	PET[375,376]	1-2 mm	Whole body	Radiolabeled antibodies, antibody fragments, and nutrients, as well as activatable probes; PET requires exposure to radiation	Matrix metalloproteinase, mesenchymal stromal cells, immune cells, tumor vasculature, glycolysis, hypoxia, pH, and tumor stroma interactions	Similar to SPECT, quantitative accuracy can be improved with advanced algorithms, but these have only been applied in preclinical imaging	Currently clinically used for evaluation of therapy response, making clinical translation feasible
X-Rays	Computed tomography[375,376]	50-200 mm	Whole body	Water-soluble iodinated probes; requires exposure to radiation	Immune cells, tumor vasculature, hypoxia, and pH	Accurate quantitation is a benefit of CT	Clinical translation will be limited by approval of appropriate contrast agents
Ultrasound	Ultrasound[289,375]	50-500 mm	mm-cm	Endogenous, targeted microbubbles; no safety concerns with conventional ultrasound and microbubble contrast is FDA approved for cardiac[377] and liver[378] imaging	Immune cells and tumor vasculature	Generally qualitative but use of contrast and mathematical algorithms have improved ability for quantitation	Clinically available technique where microbubble contrast is FDA approved for other types of US imaging, making translation of preclinical techniques feasible
Optical	Photoacoustic imaging[379]	5-300 mm	0.7-40 mm	Fluorophores, nanoparticles, and quantum dots; no safety concerns, can be used repeatedly on tissue	ECM proteins, immune cells, tumor vasculature, and pH	Generally qualitative but several studies have shown realization of quantitative information	Size and cost of laser sources, building a prototype, and clinical trials limit translation
	Intravital microscopy[376,380]	100 nm - 1 mm	100-300 mm	Endogenous; requires a surgically implanted window	Immune cells, tumor vasculature, and tumor stroma interactions	Quantitation is difficult; however, methods are being developed to improve ability for quantitation	Only feasible for introperative guidance
	Bioluminescence imaging[375,376]	3-5 mm	1-2 cm	Reporter genes; requires use of lentiviral vectors, although toxicity is low	Matrix metalloproteinase, mesenchymal stromal cells, immune cells, tumor vasculature, and pH	Quantitation is difficult, preclinical methods are being developed for improvements	Only ideally used as a preclinical technique, human tumors do not express luciferases
	Fluorescence imaging[375,376]	2-3 mm	< 1 cm	Fluorophores, fluorescent nanoparticles; fluorescent imaging in the near infrared is biologically safe and fluorescent particles show little to no toxicity	Matrix metalloproteinase, mesenchymal stromal cells, immune cells, tumor vasculature, pH, and tumor stroma interactions	Quantitation is difficult and is only relative to other regions in the tissue	Translation feasibility lies in fluorescence guided surgery, in which clinical trials have been completed
	Fluorescence molecular tomagraphy[352]	< 1 mm	1-2 mm	Near infrared dyes, quantum dots, and reporter genes; imaging with near infrared light is biologically safe; however, quantum dot composite material is toxic in elemental forms	Matrix metalloproteinase	Improves quantitation over fluorescence imaging but is still challenging	Clinical translation limited by the development of scanners for smaller and more superficial structures
	Optical coherence tomography[359]	1-15 mm	2-3 mm	Endogenous; no biological safety concerns	ECM proteins, immune cells, and tumor vasculature	Typically, quantitation requires specialized proprietary software, but methods are being developed to overcome this	Currently clinically used for ophthalmic imaging, clinical translation for TME imaging depends on development of compact probes