Optical Microscopy and Coherence Tomography of Cancer in Living Subjects

Intravital microscopy (IVM) and optical coherency tomography (OCT) are high-resolution optical imaging tools for in vivo cancer studies. Both techniques allow visualization of cancers in living subjects at subcellular-scale resolutions. They are complementary in terms of spatial and temporal resolutions, fields of view, and depth of penetration, thus providing different perspectives in basic cancer research and clinical oncology investigation.By leveraging advanced labeling and label free approaches, IVM and OCT have been extensively implemented preclinically and clinically in recent years for various aspects of cancer imaging, including tumor anatomy, physiology, intratumor cell migrations, and molecular dynamics.Label-free IVM is enabled by the development of nonlinear optical microscopy techniques, which empowers IVM to image the extracellular matrix in cancers. Label-free OCT techniques have been advanced by novel optical designs and algorithms, which have allowed OCT to more accurately detect the tumor margin and vasculature.Novel fluorescent labeling techniques have advanced applications of IVM for tracking cancer stem cells, visualizing the intratumoral genetic diversity during tumor progression and tracking the migrations of various immunocytes during cancer therapies. Emerging OCT contrast agents have enhanced the sensitivity of OCT angiography and allowed OCT to image the physiology, molecular expression, and cellular behaviors in and surrounding tumors.Research advancements that combine IVM and OCT in a dual modal endoscope have permitted more efficient and thorough cancer screening in endoluminal locations, such as the gastrointestinal tract and urinary bladder, and greatly improved morphological and molecular characterizations of endoluminal tumors in preclinical and clinical settings. Intravital microscopy (IVM) and optical coherency tomography (OCT) are two powerful optical imaging tools that allow visualization of dynamic biological activities in living subjects with subcellular resolutions. Recent advances in labeling and label-free techniques empower IVM and OCT for a wide range of preclinical and clinical cancer imaging, providing profound insights into the complex physiological, cellular, and molecular behaviors of tumors. Preclinical IVM and OCT have elucidated many otherwise inscrutable aspects of cancer biology, while clinical applications of IVM and OCT are revolutionizing cancer diagnosis and therapies. We review important progress in the fields of IVM and OCT for cancer imaging in living subjects, highlighting key technological developments and their emerging applications in fundamental cancer biology research and clinical oncology investigation. Intravital microscopy (IVM) and optical coherency tomography (OCT) are two powerful optical imaging tools that allow visualization of dynamic biological activities in living subjects with subcellular resolutions. Recent advances in labeling and label-free techniques empower IVM and OCT for a wide range of preclinical and clinical cancer imaging, providing profound insights into the complex physiological, cellular, and molecular behaviors of tumors. Preclinical IVM and OCT have elucidated many otherwise inscrutable aspects of cancer biology, while clinical applications of IVM and OCT are revolutionizing cancer diagnosis and therapies. We review important progress in the fields of IVM and OCT for cancer imaging in living subjects, highlighting key technological developments and their emerging applications in fundamental cancer biology research and clinical oncology investigation. Imaging is an indispensable tool for preclinical investigation of clinical management of tumors. Clinical imaging techniques such as magnetic resonance imaging and ultrasound permit macroscale measurement of the location and anatomy of cancers and their change over time, but lack cellular- and molecular-scale details of the lesions [1.Ntziachristos V. Going deeper than microscopy: the optical imaging frontier in biology.Nat. Methods. 2010; 7: 603-614Crossref PubMed Scopus (976) Google Scholar]. In contrast, intravital microscopy (IVM) and optical coherence tomography (OCT), employing distinct imaging mechanisms, provide subcellular-scale resolutions that enable a broad range of preclinical and clinical cancer investigations (Figure 1). IVM relies on the scanning of tissues in living subjects with single- or multiphoton fluorescent microscopy (MPM) (see Glossary) and can be performed acutely at a single time-point or chronically with longitudinal observation of a tissue site over a period of days to months. OCT employs dual-beam interferometry to capture the patterns of light scattering through tissue. By levering advanced fluorescent labeling techniques, IVM is a very powerful tool to spatiotemporally characterize the complex tumor microenvironment (TME) in preclinical cancer biology studies, including tracking tumor progression [2.Ricard C. et al.Dynamic quantitative intravital imaging of glioblastoma progression reveals a lack of correlation between tumor growth and blood vessel density.PLoS One. 2013; 8e72655Crossref PubMed Scopus (14) Google Scholar], tumor vasculature growth and regression [3.Fisher D.T. et al.Intraoperative intravital microscopy permits the study of human tumour vessels.Nat. Commun. 2016; 7: 1-9Crossref Scopus (30) Google Scholar], metastasis of cancer cells [4.Zomer A. et al.In vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior.Cell. 2015; 161: 1046-1057Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar], propagation of cancer stem cells [5.Zomer A. et al.Brief report: intravital imaging of cancer stem cell plasticity in mammary tumors.Stem Cells. 2013; 31: 602-606Crossref PubMed Scopus (69) Google Scholar], migration of tumor-associated immunocytes [6.Qi S. et al.Long-term intravital imaging of the multicolor-coded tumor microenvironment during combination immunotherapy.eLife. 2016; 5e14756Crossref PubMed Scopus (16) Google Scholar], interaction of cancer therapeutic agents with cancer and immune cells [7.Arlauckas S.P. et al.In vivo imaging reveals a tumor-associated macrophage–mediated resistance pathway in anti–PD-1 therapy.Sci. Transl. Med. 2017; 9eaal3604Crossref PubMed Scopus (0) Google Scholar], etc. Recent advances in nonlinear optical microscopy have allowed IVM to visualize the extracellular matrix in major and metastatic tumors without any fluorescent labeling [8.Burke K. Brown E. The use of second harmonic generation to image the extracellular matrix during tumor progression.IntraVital. 2014; 3e984509Crossref PubMed Google Scholar]. Compared with preclinical cancer research, clinical applications of IVM are less prevalent, mainly focusing on endoscopic evaluation of gastrointestinal cancers [9.Sturm M.B. et al.Targeted imaging of esophageal neoplasia with a fluorescently labeled peptide: first-in-human results.Sci. Transl. Med. 2013; 5: 184ra61Crossref PubMed Scopus (109) Google Scholar] and cystoscopic evaluation of bladder tumors [10.Pan Y. et al.Endoscopic molecular imaging of human bladder cancer using a CD47 antibody.Sci. Transl. Med. 2014; 6: 260ra148Crossref PubMed Scopus (80) Google Scholar]. OCT is commonly used as a label-free technique to noninvasively characterize the anatomies of superficial skin cancers [11.De Carvalho N. et al.The vascular morphology of melanoma is related to Breslow index: an in vivo study with dynamic optical coherence tomography.Exp. Dermatol. 2018; 27: 1280-1286Crossref PubMed Scopus (4) Google Scholar] and endoluminal tumors [12.Gora M.J. et al.Endoscopic optical coherence tomography: technologies and clinical applications.Biomed. Opt. Express. 2017; 8: 2405Crossref PubMed Scopus (11) Google Scholar]. Compared with IVM, OCT has greater tissue penetration, wider field of view, and higher imaging speed (Table 1), which makes it a promising tool for clinical oncology investigation, such as detecting tumor margins for intraoperative surgical guidance [13.Yecies D. et al.Speckle modulation enables high-resolution wide-field human brain tumor margin detection and in vivo murine neuroimaging.Sci. Rep. 2019; 910388Crossref PubMed Scopus (0) Google Scholar,14.Kut C. et al.Detection of human brain cancer infiltration ex vivo and in vivo using quantitative optical coherence tomography.Sci. Transl. Med. 2015; 7: 292ra100Crossref PubMed Scopus (0) Google Scholar]. The recent emergence of OCT contrast agents has extended the capabilities of OCT for cellular and molecular imaging of cancers, such as near real-time tracking of tumor-associated leukocytes [15.SoRelle E.D. et al.Spatiotemporal tracking of brain-tumor-associated myeloid cells in vivo through optical coherence tomography with plasmonic labeling and speckle modulation.ACS Nano. 2019; 13: 7985-7995Crossref PubMed Scopus (0) Google Scholar] and imaging overexpressed cancer biomarkers [16.John R. et al.In vivo magnetomotive optical molecular imaging using targeted magnetic nanoprobes.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 8085-8090Crossref PubMed Scopus (74) Google Scholar] in preclinical animal models. The preclinical cancer imaging research conducted by IVM and OCT have provided powerful insights to answer the basic cancer biology questions, which can be difficult to elucidate by other approaches. For example, Karagiannis et al. used IVM to study the dynamics of resident macrophages in response to neoadjuvant chemotherapy and found that treatment increased the rate at which macrophages facilitated the metastatic dissemination of breast cancer [17.Karagiannis G.S. et al.Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism.Sci. Transl. Med. 2017; 9: eaan0026Crossref PubMed Scopus (104) Google Scholar]. Using OCT, SoRelle et al. were able to plot the migration trajectories of tumor-associated macrophages (TAMs) in glioblastoma multiforme after exposure to gold nanoparticles [15.SoRelle E.D. et al.Spatiotemporal tracking of brain-tumor-associated myeloid cells in vivo through optical coherence tomography with plasmonic labeling and speckle modulation.ACS Nano. 2019; 13: 7985-7995Crossref PubMed Scopus (0) Google Scholar]. The clinical imaging conducted by combined IVM and OCT approaches has enabled more efficient and accurate cancer screening and allowed for better morphological and molecular characterization of tumor lesions [18.Tang Q. et al.Depth-resolved imaging of colon tumor using optical coherence tomography and fluorescence laminar optical tomography.Biomed. Opt. Express. 2016; 7: 5218Crossref PubMed Scopus (15) Google Scholar,19.Li Y. et al.Multimodal endoscopy for colorectal cancer detection by optical coherence tomography and near-infrared fluorescence imaging.Biomed. Opt. Express. 2019; 10: 2419Crossref PubMed Scopus (1) Google Scholar]. Although many cancer imaging studies can be conducted by either IVM or OCT, each of these two techniques has its own advantages and limitations in terms of spatial and temporal resolutions, imaging depth and field of view, labeling and multiplexability, etc. (Table 1). This review aims to elucidate the strengths and deficiencies of each technique for cancer imaging. We focus on the major progress of IVM and OCT technologies over the past 5 years and their applications to fundamental and clinical oncology studies in living subjects that have greatly advanced our understanding of cancer biology and facilitated the clinical management of cancer through anatomical, physiological, cellular, and molecular imaging.Table 1The Imaging Performance Characteristics of IVM and OCTaAbbreviations: ICG, indocyanine green; SHG, second harmonic generation; THG, third harmonic generation.IVM techniquesOCT techniquesSpatial resolution<0.5 μm<10 μmDepth of penetration<600 μm2–3 mmTemporal resolution<7 million pixels/second>50 million pixels/secondMultiplexabilityHigh (5–10 fluorescent signals)Low (up to three OCT spectral signals)Contrast agentsFluorochromes (Rhodamine-B, FITC, Alexa®, ICG, etc.); genetically encoded fluorescent proteins; nanoparticles (quantum dots, upconversion nanoparticles)Gold nanoparticles, magnetic nanoparticles, microbeads, intralipid, fluorescent dyes (i.e., ICG)Photobleaching of contrast agentsHigh (small molecule fluorophores)Low (fluorescent proteins and nanoparticles)LowToxicity of contrast agentsLow (ICG has been FDA approved)LowLabel-free techniquesSHG and THGOCT, angiography, and lymphangiographya Abbreviations: ICG, indocyanine green; SHG, second harmonic generation; THG, third harmonic generation. Open table in a new tab IVM extends optical microscopy to the interrogation of tissues within living animals. IVM can be performed label-free by using either brightfield confocal microscopy, as had been done since the 19th century, to image the tissue reflective signals [20.Groner W. et al.Orthogonal polarization spectral imaging: a new method for study of the microcirculation.Nat. Med. 1999; 5: 1209-1212Crossref PubMed Scopus (574) Google Scholar,21.Nyvad J. et al.Intravital investigation of rat mesenteric small artery tone and blood flow.J. Physiol. 2017; 595: 5037-5053Crossref PubMed Scopus (15) Google Scholar], or using MPM to image specific tissue structures that emit photons at the second- or third-harmonic wavelength of the illumination source (second/third harmonic generation) [8.Burke K. Brown E. The use of second harmonic generation to image the extracellular matrix during tumor progression.IntraVital. 2014; 3e984509Crossref PubMed Google Scholar,22.Beerling E. et al.Intravital characterization of tumor cell migration in pancreatic cancer.IntraVital. 2016; 5e1261773Crossref PubMed Google Scholar]. However, brightfield confocal IVM works poorly outside of very specific tissue sites with high translucency, such as inner ear or eye, and multiharmonic generation is limited to imaging certain specific tissue structures, such as collagen and cell lipids with strong harmonic emission spectra. To image the dynamics of specific cells and molecules in living subjects, IVM is usually conducted using fluorescent labeling strategies. Both 1-photon fluorescent microscopy (1PM) and MPM can be used to excite fluorophore labels. 1PM allows simultaneous imaging of many different fluorophores with distinct emission spectra, whereas MPM enables deeper tissue penetration despite somewhat reduced multiplexability. Fluorophores can be designed to circulate for a long time in the vasculature [2.Ricard C. et al.Dynamic quantitative intravital imaging of glioblastoma progression reveals a lack of correlation between tumor growth and blood vessel density.PLoS One. 2013; 8e72655Crossref PubMed Scopus (14) Google Scholar,23.Smith B.R. et al.Selective uptake of single-walled carbon nanotubes by circulating monocytes for enhanced tumour delivery.Nat. Nanotechnol. 2014; 9: 481-487Crossref PubMed Scopus (132) Google Scholar], molecularly target specific cell surface markers [24.Cuccarese M.F. et al.Heterogeneity of macrophage infiltration and therapeutic response in lung carcinoma revealed by 3D organ imaging.Nat. Commun. 2017; 814293Crossref PubMed Scopus (57) Google Scholar,25.Junankar S. et al.Real-time intravital imaging establishes tumor-associated macrophages as the extraskeletal target of bisphosphonate action in cancer.Cancer Discov. 2015; 5: 35-42Crossref PubMed Scopus (58) Google Scholar], penetrate the cell membrane and be retained in a cell [6.Qi S. et al.Long-term intravital imaging of the multicolor-coded tumor microenvironment during combination immunotherapy.eLife. 2016; 5e14756Crossref PubMed Scopus (16) Google Scholar], or be embedded as reporter genes to continually and nondilutively generate fluorescent proteins in cells of interest wherever they traffic [7.Arlauckas S.P. et al.In vivo imaging reveals a tumor-associated macrophage–mediated resistance pathway in anti–PD-1 therapy.Sci. Transl. Med. 2017; 9eaal3604Crossref PubMed Scopus (0) Google Scholar,26.Milo I. et al.The immune system profoundly restricts intratumor genetic heterogeneity.Sci. Immunol. 2018; 3eaat1435Crossref PubMed Scopus (15) Google Scholar]. In some cases, novel 'smart' nanotechnologies that deploy fluorophores or quenchers in response to changes in microenvironmental conditions may also be used [27.Smith B.R. et al.Shape matters: intravital microscopy reveals surprising geometrical dependence for nanoparticles in tumor models of extravasation.Nano Lett. 2012; 12: 3369-3377Crossref PubMed Scopus (125) Google Scholar]. Careful consideration of fluorophore excitation and emission spectra is necessary not only for use with a particular laser light source and microscope filter set, but also for minimizing bleed-through between related fluorophores; background autofluorescence is also a consistent issue that may influence imaging results. Advanced IVM techniques, such as fluorescence-lifetime imaging microscopy, can be used to reduce this issue by selecting fluorophores with very different fluorescence lifetime profiles than surrounding tissue [28.Winfree S. et al.Intravital microscopy of biosensor activities and intrinsic metabolic states.Methods. 2017; 128: 95-104Crossref PubMed Scopus (3) Google Scholar]. This increases imaging contrast through a sort of 'time gating' that, in combination with the standard wavelength filtering used with single-photon and multiphoton microscopy, provides unprecedented fluorophore discrimination and insight into the formation and dynamics of the TME [29.Niesner R. et al.The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging.Biophys. J. 2007; 93: 2519-2529Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar,30.You S. et al.Intravital imaging by simultaneous label-free autofluorescence-multiharmonic microscopy.Nat. Commun. 2018; 92125Crossref PubMed Scopus (15) Google Scholar]. IVM is often conducted with a window or dorsal skinfold chamber installed on the animal to image superficial tumors, such as skin tumors. However, most cancers (e.g., colon cancer, liver cancer, pancreatic cancer) are not directly optically accessible by IVM, which usually has a depth of tissue penetration up to ~600 μm, depending on the tissue [1.Ntziachristos V. Going deeper than microscopy: the optical imaging frontier in biology.Nat. Methods. 2010; 7: 603-614Crossref PubMed Scopus (976) Google Scholar,31.Vakoc B.J. et al.Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging.Nat. Med. 2009; 15: 1219-1223Crossref PubMed Scopus (491) Google Scholar]. Therefore, specific surgical procedures must be performed to expose the specific organ of imaging interest in order to visualize orthotopic tumors. Careful consideration of appropriate animal preparation and imaging procedures are crucial for successful IVM studies (Box 1).Box 1Animal Preparation and Imaging Procedures for IVMIn acute IVM, surgical exposure or surface visualization of a site of interest in an anesthetized animal model permits visualization of labeled events within the field of view where imaging may be performed for several hours, even a day, to watch dynamic processes unfold. Other imaging sites, particularly soft tissues, can be acutely imaged using glass slides between the tissue and the microscope objective. At the conclusion of imaging, the animal may be sacrificed and ex vivo characterization is often done via traditional molecular biology means, including immunofluorescence histology, flow cytometry, and molecular assays such as western blots to obtain deeper insight into the factors driving the observed cell behavior. In chronic IVM, the goal is to image the same tissue site multiple times over an interval of several days, weeks, or even months, necessitating the use of microsurgical techniques and implanted optical scaffolds to isolate the site of interest. For example, mice may be fitted with a dorsal skin chamber, which resembles a flanged backpack with an optical window in which a fold of dorsal skin is immobilized for repeated imaging. This chamber provides direct optical access to the inside of the mouse and enables motion stabilization under the microscope objective via use of customized stages. Alternatively, mice may be fitted with cranial windows to provide an optically clear view into the meninges and brain, or with a small, cylindrical washer or window placed around the inguinal lymph node for lymph node imaging [75.Ito K. et al.Unexpected dissemination patterns in lymphoma progression revealed by serial imaging within a murine lymph node.Cancer Res. 2012; 72: 6111-6118Crossref PubMed Scopus (16) Google Scholar,117.Kotsuma M. et al.Nondestructive, serial in vivo imaging of a tissue-flap using a tissue adhesion barrier.IntraVital. 2012; 1: 69-76Crossref Google Scholar]. Such approaches are well-suited to test the function, pharmacokinetics, and cell type specificity of novel drugs. Ongoing developments in surgical technique and optical window development to repeatedly image soft tissue sites are now permitting longitudinal studies to be performed in an increasing range of tissues of interest, including lung [118.Fiole D. Tournier J.-N. Intravital microscopy of the lung: minimizing invasiveness.J. Biophotonics. 2016; 9: 868-878Crossref PubMed Scopus (7) Google Scholar], mammary fat pad [119.Bonapace L. et al.If you don't look, you won't see: intravital multiphoton imaging of primary and metastatic breast cancer.J. Mammary Gland Biol. Neoplasia. 2012; 17: 125-129Crossref PubMed Scopus (12) Google Scholar], and bone marrow [120.Nwajei F. Konopleva M. The bone marrow microenvironment as niche retreats for hematopoietic and leukemic stem cells.Adv. Hematol. 2013; 2013: 1-8Crossref Scopus (47) Google Scholar]. In acute IVM, surgical exposure or surface visualization of a site of interest in an anesthetized animal model permits visualization of labeled events within the field of view where imaging may be performed for several hours, even a day, to watch dynamic processes unfold. Other imaging sites, particularly soft tissues, can be acutely imaged using glass slides between the tissue and the microscope objective. At the conclusion of imaging, the animal may be sacrificed and ex vivo characterization is often done via traditional molecular biology means, including immunofluorescence histology, flow cytometry, and molecular assays such as western blots to obtain deeper insight into the factors driving the observed cell behavior. In chronic IVM, the goal is to image the same tissue site multiple times over an interval of several days, weeks, or even months, necessitating the use of microsurgical techniques and implanted optical scaffolds to isolate the site of interest. For example, mice may be fitted with a dorsal skin chamber, which resembles a flanged backpack with an optical window in which a fold of dorsal skin is immobilized for repeated imaging. This chamber provides direct optical access to the inside of the mouse and enables motion stabilization under the microscope objective via use of customized stages. Alternatively, mice may be fitted with cranial windows to provide an optically clear view into the meninges and brain, or with a small, cylindrical washer or window placed around the inguinal lymph node for lymph node imaging [75.Ito K. et al.Unexpected dissemination patterns in lymphoma progression revealed by serial imaging within a murine lymph node.Cancer Res. 2012; 72: 6111-6118Crossref PubMed Scopus (16) Google Scholar,117.Kotsuma M. et al.Nondestructive, serial in vivo imaging of a tissue-flap using a tissue adhesion barrier.IntraVital. 2012; 1: 69-76Crossref Google Scholar]. Such approaches are well-suited to test the function, pharmacokinetics, and cell type specificity of novel drugs. Ongoing developments in surgical technique and optical window development to repeatedly image soft tissue sites are now permitting longitudinal studies to be performed in an increasing range of tissues of interest, including lung [118.Fiole D. Tournier J.-N. Intravital microscopy of the lung: minimizing invasiveness.J. Biophotonics. 2016; 9: 868-878Crossref PubMed Scopus (7) Google Scholar], mammary fat pad [119.Bonapace L. et al.If you don't look, you won't see: intravital multiphoton imaging of primary and metastatic breast cancer.J. Mammary Gland Biol. Neoplasia. 2012; 17: 125-129Crossref PubMed Scopus (12) Google Scholar], and bone marrow [120.Nwajei F. Konopleva M. The bone marrow microenvironment as niche retreats for hematopoietic and leukemic stem cells.Adv. Hematol. 2013; 2013: 1-8Crossref Scopus (47) Google Scholar]. In contrast to the relatively straightforward optics of IVM described above, OCT is an interferometry based technique that illuminates tissues with near-infrared (NIR) low-coherence light and detects the back-scattered photons from tissue. The reflective light interferes with a reference beam to record the time delay of back-scattered photons at different depths of tissue in an interferogram, which is then reconstructed to an OCT image using Fourier transforms [32.Kim J. et al.Functional optical coherence tomography: principles and progress.Phys. Med. Biol. 2015; 60: R211-R237Crossref PubMed Scopus (39) Google Scholar]. The scanning philosophy of OCT is similar to that of ultrasound, which captures consecutive line images in the z-plane of tissue (A-scan). By changing the angle of the scanner, the OCT A-scan beam is moved along the x-direction to complete the cross-section scan (B-scan). OCT completes the 3D volume scan by conducting consecutive B-scans along the y-direction and thus builds up a detailed subcellular image of the underlying tissue. OCT can be, and often is, used as a label-free technique, which enables anatomical and physiological imaging of live tissues without contrast agents. However, the image quality and level of information obtained from OCT scanning are highly dependent on the scanning protocols and postimage processing algorithms used [33.Zhu J. et al.Can OCT angiography be made a quantitative blood measurement tool?.Appl. Sci. 2017; 7: 687Crossref Scopus (11) Google Scholar]. For example, to detect blood vessels, repeated A-scan or B-scan have to be conducted in the same imaging location over time and the obtained OCT signals need to be processed by specific algorithms to calculate the signal difference over the repeated scans [34.Kashani A.H. et al.Optical coherence tomography angiography: a comprehensive review of current methods and clinical applications.Prog. Retin. Eye Res. 2017; 60: 66-100Crossref PubMed Scopus (148) Google Scholar]. By combining OCT with contrast agents, the discriminatory power of OCT can be significantly enhanced and the functions of OCT can be extended for cellular and molecular imaging [35.Smith B.R. Gambhir S.S. Nanomaterials for in vivo imaging.Chem. Rev. 2017; 117: 901-986Crossref PubMed Scopus (87) Google Scholar]. For example, by injecting large gold nanorods (LGNR) [36.Liba O. et al.Contrast-enhanced optical coherence tomography with picomolar sensitivity for functional in vivo imaging.Sci. Rep. 2016; 623337Crossref PubMed Scopus (39) Google Scholar] or gold nanoprisms (GNPR) [37.Si P. et al.Gold nanoprisms as optical coherence tomography contrast agents in the second near-infrared window for enhanced angiography in live animals.ACS Nano. 2018; 12: 11986-11994Crossref PubMed Scopus (9) Google Scholar] as intravascular contrast agents, OCT can visualize microvessels located at greater depth in the tumor (i.e., deeper than 1 mm below skin surface), which cannot be detected by conventional label-free OCT. By using microbeads (MBs) as contrast agents, Si et al. were able to monitor the dynamic expression of lymphatic vessel endothelial hyaluronan receptors (LYVE-1) [38.Si P. et al.In vivo molecular optical coherence tomography of lymphatic vessel endothelial hyaluronan receptors.Sci. Rep. 2017; 7: 1-11Crossref PubMed Scopus (3) Google Scholar]. Additional modification of OCT instrumentation further enhances the discriminatory power of OCT. Because OCT image quality can be compromised by speckle noise [39.Schmitt J.M. et al.Speckle in optical coherence tomography.J. Biomed. Opt. 2002; 4: 95-105Crossref Scopus (511) Google Scholar], an optically modified OCT system [speckle-modulation OCT (SM-OCT)] can greatly improve the image contrast and resolution by effectively eliminating the speckles [40.Liba O. et al.Speckle-modulating optical coherence tomography in living mice and humans.Nat. Commun. 2017; 8: 1-12PubMed Google Scholar]. Combining SM-OCT and spectral OCT contrast agents (i.e., LGNR), the de la Zerda group has greatly extended the capability of OCT for cellular imaging (e.g., in vivo imaging of circulating tumor cells [41.Dutta R. et al.Real-time detection of circulating tumor cells in living animals using functionalized large gold nanorods.Nano Lett. 2019; 19: 2334-2342Crossref PubMed Scopus (2) Google Scholar], in vivo imaging of TAMs in a brain tumor mouse model [15.SoRelle E.D. et al.Spatiotemporal tracking of brain-tumor-associated myeloid cells in vivo through optical coherence tomography with plasmonic labeling and speckle modulation.ACS Nano. 2019; 13: 7985-7995Crossref PubMed Scopus (0) Google Scholar]). By extending OCT with a water-cooling electromagnet and using ferromagnetic particles as contrast agents, John and colleagues achieved molecularly targeted imaging of human epidermal growth factor receptor 2 (HER2) biomarkers overexpressed in a rat breast cancer model [16.John R. et al.In vivo magnetomotive optical molecular imaging using targeted magnetic nanoprobes.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 8085-8090Crossref PubMed Scopus (74) Google Scholar]. Tumor anatomy, including shape,