Cells are basic structural and functional units of living organisms. The functions of cells and their interactions with other cells and biological components mediate many crucial biological processes
One of those efforts is the development of whole-body imaging, such as magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). These techniques can show the whole-body distribution of specific probes, such as fluorescence, radioactive tracers, or contrast enhancements in live animals (6, 7). Accordingly, whole-body imaging is currently being used for the diagnosis of various diseases. However, most whole-body imaging has limited resolution (MRI, 10-100 μm; SPECT, 1-2 mm; PET, 1-2 mm, in general) that hinders visualizing objects at cellular level (Table 1) (7).
On the other hand, intravital microscopy (IVM) has been developed as an alternative modality that overcomes the limitation of whole-body imaging. IVM, as a microscopic imaging system, has high spatial (∼1 μm) and temporal resolution (sub-seconds) (8-11). In this regard, IVM has been applied to visualizing and monitoring single-cell biological processes, different from other
IVM incorporates all the microscopy techniques, which can have various resolutions and frame rates, for visualizing and analyzing biological events in living animals. The concept of observing living animals with microscopy was first introduced by the early pioneers of microscopy in the 17th and 18th centuries (19). In the 19th century, Julius Cohnheim used light microscopy to observe the migration of leukocytes in blood vessels toward injury sites in the transparent tongue of a living frog and discovered that their magnetism is a crucial process of inflammation (20). Even though this early conventional optical IVM contributed to discovering new aspects of biology, it had many limitations: the difficult reduction of background signals, dependence in the eyesight of the observer, and no controlling depth of field.
After significant development of microscopy and image processing, microscopic imaging techniques, originally built for
Another adopted approach is multi-photon microscopy (8-10). The essential mechanism of multi-photon (e.g., two-photon) microscopy is to induce fluorescence in the laser-focused site with the summed energy of the photons in the laser. The basic structures of confocal and two-photon microscopy -- most conventional multi-photon microscopy -- are compared in Fig. 1B and 1C. In confocal (single-photon) microscopy, the exciting laser shoots a photon that has enough energy to induce the emission of fluorescence, which occurs in all the areas that the laser hits, thus creating the necessity for the pinhole that rejects the signals from out-of-focus areas. In contrast, in two-photon microscopy, the fluorescence is generated in a small region where the laser is tightly focused, since only the sites which two photons excite receive enough energy to emit the fluorescence. As a result, the out-of-focus signal can be rejected in multi-photon microscopy without needing another element like a pinhole. Also, the use of long-wavelength excitation has the advantage in imaging deep tissues because it can minimize scattering and absorption by certain proteins in tissues (11). Thus, the two-photon laser allows a deep tissue penetration of 300 μm, which is superior to that of confocal imaging (18), and causes negligible photodamage or photobleaching (7). Furthermore, the second-harmonic generation (SHG) can be observed in two-photon microscopy. SHG is a second-order non-linear optical process in which two photons interacting with non-linear optical material (e.g., collagen) combine to form a new photon with twice the frequency and half the wavelength of the initial photons (21). With these properties, multi-photon microscopy is adapted to intravital imaging and used for discovering novel aspects of biology.
IVM has enabled researchers to observe a variety of biological constructs in live animals and to follow the dynamic behavior of single cells over a long period of time. With these properties, researchers have used IVM for visualizing and analyzing biological processes in various research fields, such as vascular biology, immunology, stem cell biology, and oncology.
In the field of cancer immunology, for instance, Jung
IVM has been also used for quantitative evaluation of the organization, structure, and function of blood vasculature. Especially, IVM was used to view irregular hyperpermeability and heterogeneous blood flow in tumor microvessels, resulting from an imbalance of pro- and anti-angiogenic factors. These abnormalities of tumor blood vessels hinder the efficacy of radio-/chemotherapy and promote the growth of more aggressive and metastatic cancer cells (22). Early examination of tumor vessels with fluorescent agents and IVM revealed the difference in static values, such as vessel diameter, length, surface area volume, and branching patterns, between the normal and tumor vessels (23, 24). With the development of IVM, dynamic parameters, such as blood flow velocity, changing vessel diameter, and vascular permeability, are quantified and analyzed for a better understanding and classification of blood vessels (25-27). Microscopic observation of these characteristics of vessels in a living animal with IVM can be used to analyze the restoration of vessel functionality and structure after vessel normalization (28). Also, the development of various types of imaging windows permits the longitudinal monitoring of vasculature development over a long period of time (29-34).
Cell trafficking with IVM reveals novel aspects of cellular immunity by its ability to follow and observe the dynamic behavior of immune cells. The high spatial and temporal resolution of IVM enables researchers to track the movement and behavior of immune cells in desired sites (35). Cell-trafficking parameters, such as the population, migration, clustering, 2D/3D movement pattern, velocity, and morphology of immune cells, can be used to understand the states and functions of leukocytes (36). For example, immature T and B lymphocytes migrate to the secondary lymphoid organs and become immunocompetent cells in these sites (37). For detailed comprehension of mechanisms in those processes, early pioneer researchers produced fluorescence-labeled T and B lymphocytes and transferred them to recipient animals to observe their dynamic behavior, such as velocity, moving pattern, T cell interaction with dendritic cells, morphology changes, and activation in the lymph node (38-40). These studies visualized the dynamic behavior of immune cells and deepened the understanding of adaptive and innate immunity (41). Also, researchers analyzed and quantitatively evaluated the motility of immune cells in the specific condition with IVM. Some studies showed the change in the motility of immune cells by specific molecules. For example, researchers used IVM to reveal how integrins LFA-1 and MAC-1 regulate neutrophil extravasation in the endothelial basement membrane and pericyte sheath (42), and how reactive oxygen species recruit neutrophils to muscle tissue after exercise (43). In addition, IVM was used to show that chemokine CCL22 expression of dendritic cells affects the contact and interaction of dendritic cells with Tregs in the lymph nodes (44). Other studies showed that specific states affect the dynamic patterns and velocity of immune cells. For example, the population and velocity of eosinophils in different organs and their changes in the normal and ovalbumin sensitization state can be seen with IVM (45). Also, IVM was applied to images to analyze how HIV infection changes T-cell motility and migration patterns by disrupting host-cell cytoskeletons (46) and how pathogen-specific T cells interact with intracellular parasite-infected cells (47). In addition,
IVM has been also used for viewing the movement and location of stem cells to understand their functions. Some studies used IVM to view hematopoietic stem cells in the bone marrow niche and analyzed their interaction, which is crucial for proper stem-cell functions. For example, some researchers developed IVM-guided transplantation of a hematopoietic stem cell in the bone marrow of mice to study the functional characteristics of a single stem cell (50). Other researchers used IVM to examine the relationship of hematopoietic stem cells and progenitor cells to blood vessels, osteoblasts, and the endosteal surface in the niche (51, 52), and others used IVM to view the dynamic responses of the hematopoietic stem cells in the niche upon diverse stresses (53). Also, researchers used IVM to discover novel facts of stem cell function. For example, IVM was used to view the interaction of the hematopoietic stem cells with Tregs, which results in Treg accumulation in the niche that prevents immune attacks (54). In another study, IVM was used to analyze the function of mammary stem cells, which regulates the duct-branching morphogenesis (55).
In the field of oncology, IVM is applied to view the single-cell behavior of cancer and immune cells during the progression and metastasis of the tumor, which can reveal new findings in the tumor microenvironment. Imaging the single-cell behavior enables researchers to understand the relationships between the dynamic behavior of cancer cells and the features of the tumor (56). Also, cancer-cell invasion and metastasis, which are the primary reason for the death of cancer patients (57), are made visible and explored by IVM. For instance, IVM was used to view circulating tumor cells, which are known to be highly associated with metastasis and defined their higher metastatic subsets (58). Also, some studies observe the pre-metastasis stage in a liver metastasis model with an abdominal imaging window (59). Another study developed an intravital imaging model of cancer cells in bone marrow to study systemic metastasis (60). In addition, some researchers have used IVM to view and analyze the interaction of cancer cells with immune cells. IVM is used in studies that imaged and analyzed the interaction between tumor-infiltrating T lymphocytes with cancer cells (61) and how adopted cytotoxic CD8+ T lymphocytes induce tumor-cell death (62). Also, other researchers used IVM to reveal the mechanism of how tumor-associated macrophages are related to poor prognosis by viewing macrophage-assisted cancer-cell intravasation (63) and tumor-associated macrophage-mediated drug resistance (64). The increasing number of cancer studies now use IVM to discover and render unexpected findings in the tumor microenvironment.
As mentioned above, IVM has a limited penetration depth, which is an obstacle in imaging deep tissues, such as the brain, colon, and heart (65). Endomicroscopy based on the optical probe is a method that overcomes this obstacle and can observe the deep organs. Micro-diameter probes using graded refractive index (GRIN) lenses enable conventional intravital microscopes to view the internal deep organs of animals with minimal invasion. Some organs can even be viewed without incision or additional components. For example, colon vessels and tumorigenesis are observed by endomicroscopy that enters through the rectum without invasion (35, 66, 67). Also, mesenchymal stem-cell therapy in the bladder-pain syndrome model can be analyzed by endomicroscopy that enters the bladder without an incision (68). On the other hand, viewing the brain with endomicroscopy needs a small incision to implant the probe. To minimize the damage and inflammation, some researchers fabricated a side-view probe with the small GRIN rod lenses with a micro-diameter and viewed the murine brain from the cortex to the hypothalamus (69) and the hippocampus (70). With similar methods, intravital endomicroscopy can make the glioma progression in the brain visible (71). In addition, viewing the heart or airway needs additional caution, because of the vigorous movement of the heartbeat and breathing. To view the heart, researchers used a suction tube to locally stabilize the movement of the heart and examined the flowing and rolling monocytes in the beating heart (2). In airway imaging, the challenge by breathing was overcome by the side-view design, which results in proximity between the epithelium of the airway and the imaging window of the probe (72).
With the development of IVM and endomicroscopy, the clinical adaptation of IVM starts in various studies (16). Fluorescein is the most widely used in the clinical adaptation of IVM, because fluorescein is FDA-approved for angiography of the retina (73). Diagnostic criteria for benign and neoplastic or tumor conditions has been developed by probe-based IVM and fluorescein in the colon (74), esophagus (75), and urinary tract (76). Also, one study emphasizes another important aspect of intravital imaging, as IVM-imaged human tumor vessel diameters in melanoma patients are larger than predicted from immunohistochemistry. Thus, the measurement of vessel diameters with IVM enables to establish more precise strategies for drug delivery and immunotherapies (77).
Although conventional IVM has proved to be an excellent tool for
Fig. 2 compares conventional (non-video-rate scanning) and real-time (video-rate scanning) IVM. The limited frame rate of conventional IVM cannot track the movement of flowing cells. On the other hand, the rapid movement of the flowing cell can be tracked with real-time IVM (Fig. 2A). Also, the different movements of cells such as flowing (Fig. 2B) and rolling (Fig. 2C), can be analyzed by real-time IVM, which is related to their function and population at the site of imaging. This high-temporal resolution of real-time IVM expands the visual perception of researchers for understanding the dynamic cellular behavior, which leads to novel discovery.
Fig. 3 is a schematic of real-time confocal and two-photon switchable IVM with two different options for beam scanner systems. In the upper panel, a schematic shows an example of real-time IVM that has two different modes of intravital imaging. The backscattered light or fluorescent light from the tissue of a living animal was collected by the objective lens and delivered to either one of the two independent PMTs, depending on the mode of imaging (79). A photomultiplier tube with a confocal pinhole is built to detect confocal fluorescence and reflectance imaging. Another one (two-photon PMT) is built to detect two-photon fluorescence and second-harmonic generation imaging. In confocal microscopy mode, the emitted light from the tissue of the animal returns along the same path as the excitation laser until it is reflected at the dichroic beamsplitter, then the confocal pinhole selectively transmits only fluorescence generated at the focal region. In contrast, because the fluorescence originates only at a point on the focal region in two-photon microscopy mode, it does not require pinhole for blocking background signal. Thus, the emitted fluorescence or SHG signals can be sent directly to an optical dector, which can be placed as close to the tissue of the animal as possible to minimize the loss of the emitted light (80). In the lower panel, the scanning by two different scanners is described. The frame rate of IVM is determined by the speed of vertical and horizontal scan mirrors that move the laser across the
The researchers used real-time IVM to view swift biological processes, such as the significant motion of tracheal epithelial cells caused by breathing and heartbeat (72), tumor development in gastrointestinal tracts (83), and the rapid movements of immune cells in cancers (1) and cardiovascular system (2). In addition, this real-time IVM can analyze and evaluate the delivery and effects of nanotherapeutics in living animals (84, 85). In short, real-time IVM will serve as a sophisticated tool for visualizing rapid biological events, which cannot be achieved by non-video-rate scanning IVM.
IVM incorporates diverse optical systems for direct viewing and analyzing the biological structure and dynamic behavior of cells in living animals. Unlike
Nontheless, compared to other
On the other hand, whole-body imaging systems, which has been widely used in the clinic, are suitable for viewing large parts of tissues and analyzing the function of whole tissues, although these methods in general have low spatial resolution, unlike IVM. Thus, the use of IVM and whole-body imaging systems can be well combined to obtain more comprehensive biological information that cannot be obtained by a single method (Fig. 4). IVM and whole-body imaging modalities compensate for the weakness of each other’s imaging system. Also, the constant development of IVM will surely broaden our knowledges on the field of biological and biomedical sciences by providing novel insights, which cannot be achieved by conventional experimental techniques.
This study was supported by the Creative-Pioneering Researchers Program through Seoul National University (SNU) (KJ) and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1C1C10 15062), by the Cooperative Research Program of Basic Medical Science and Clinical Science from the Seoul National University College of Medicine (800-20190261) (KJ), by grant no. 16-2019-007 from the Seoul National University Bundang Hospital research fund (KJ), and by the SNUH Research Fund 03-2018-0290 (KJ).
The authors have no conflicting interests.