In vivo studies are the only way to accurately assess the interactions of multiple cell types in the context of complex anatomical features. Cancer and inflammatory disease, for example, represent the combined processes of cell migration and recruitment, remodeling of extracellular matrices, flow and shear forces, and complex modulation of multiple cytokines and chemokines. New optics, coupled with the use of near-infrared (650 to 900 nm) fluorescent probes, present exciting opportunities for in vivo imaging of multiplexed fluorescent reporters in small-animal models. This enables the simultaneous imaging of multiple cellular parameters and the identification of mechanisms involved in complex biologic processes. Scan head design Confocal and multiphoton microscopy are used for imaging within intact tissue, but even with the increased penetration of the infrared wavelengths of multiphoton lasers, the scatter of photons emitted from fluorophores limits the depth at which these technologies can achieve sufficient signal-to-noise ratios. Developed in collaboration with Ralph Weissleder and his colleagues at the Center for Molecular Imaging Research at Massachusetts General Hospital in Charlestown, the Olympus IV100 intravital laser-scanning microscope has a light path that is optimized for collecting light from deep within living tissue. High-resolution imaging over a wide range of wavelengths also presents challenges. The optics used in the intravital microscope combine glass and coating technologies that optimize the microscope for use from the blue through the far-red wavelength range — and well into the near-infrared. The advantages of imaging in the near-infrared window are twofold: This region not only benefits from the reduced scatter of longer wavelengths, but also avoids the wavelengths confounded by tissue autofluorescence and by hemoglobin absorption. The intrinsic fluorescence of biological molecules, such as those in blood vessels, can be useful, however, in providing anatomic reference points. The lasers used for the intravital microscope are similar to those used in conventional laser scanning, with somewhat higher intensity and an emphasis on the longer wavelengths. Based on the platform of the Olympus FV1000 confocal laser-scanning microscope, the emission path of the intravital system employs high-sensitivity photomultiplier tubes, along with ion-sputtered emission beamsplitters and barrier filters for simultaneous acquisition of multiple wavelength ranges. The small diameter of the optical fiber capturing emission from the scan head, together with the relatively high numerical aperture of the objective lenses, provides a degree of optical sectioning without a pinhole. Available for the system are 10-mW, 488-nm argon-ion; 13-mW, 531-nm diode-pumped solid-state; 10-mW, 633-nm helium-neon; and 18-mW, 748-nm diode lasers. Even when an animal is surgically opened, the orientations and uneven surfaces of organs can cause difficulties when performing conventional imaging with an upright microscope. Therefore, the system’s compact scan head can be tilted for orthogonal imaging of internal organs. This enables optimal imaging with minimal repositioning of the animal (Figure 1). With the instrument’s enclosed system for optimal isoflurane anesthesia and temperature control, some procedures are amenable to observation of up to several hours. Figure 1. The scan head of an intravital laser-scanning microscope tilts from 210° to 70° for optimal imaging of the internal organs of live animal subjects. Elias Gounaris and Rainer Kohler, also of the Center for Molecular Imaging Research, have used the intravital laser-scanning microscope to image protease activity along with neovascularization to investigate tumor progression in a mouse prone to develop adenomatous polyps and colon carcinoma. In this mouse model, adenoma formation was directly imaged using fluorescent probes from VisEn Medical Inc. of Woburn, Mass.: one to detect cathepsin B activity; the other, to delineate the microvasculature. Following surgical opening of the abdomen, a loop of gut was positioned for imaging using a 10x dry objective. Images were acquired from a Z-stack over a 150-μm range (Figure 2). In some studies, two excitation channels were used; in others, a third channel was acquired simultaneously to provide anatomic morphology.1 Figure 2. These representative images are from a Z-stack imaged through the gut wall using the intravital microscope and a specialized 10x objective lens. Courtesy of Elias Gounaris and Rainer Kohler, Massachusetts General Hospital Center for Molecular Imaging Research. The use of long-wavelength excitation (633 and 748 nm), along with the long-wavelength emission of the dyes, enabled excellent image contrast and resolution, even with direct imaging through the intact wall of the gut. The system allowed the researchers to conduct their studies with the organs in situ rather than having to work with exteriorized organs. Specialized objectives For minimally invasive imaging, the intravital microscope’s platform can be coupled with Olympus’ MicroProbe objective lens, which provides a sharp contrast to conventional high-resolution objective lenses. The large barrels of conventional lenses require extensive surgery to expose enough of the subject animal’s internal organs, and many such studies must be performed with the organs completely exteriorized. Exposing an animal to this level of surgical trauma reduces the possibility of longitudinal studies in a single animal and can affect the biological relevance of data, especially in investigations of the inflammatory response. The ability to follow disease progression or response to treatment in a single animal is attractive not only because of the potential to intervene, but also because of the smaller number of animals required for preclinical studies. The slim barrels of the MicroProbe lenses allow close apposition of the front lens element to the tissue without fear of affecting the organ or causing undue trauma to the animal. The lenses have an outer diameter of 1.3 mm and extend up to 2 cm in length, permitting insertion through a tiny incision into the body cavity for imaging organs in situ. Importantly, longitudinal studies often can be enabled by the use of minimally invasive techniques such as keyhole surgery and subsequent suture. The water-immersible objectives can yield Z-stacks that extend 200 μm into an intact organ. There are three lenses in the series (Figure 3), all designed to satisfy in vivo imaging applications: a 27x, 0.7-NA, 3.5-mm-diameter lens with a 220-μm field of view; a 20x, 0.5-NA, 1.3-mm-diameter lens with a 200-μm field of view; and a 6x, 0.14-NA, 1.3-mm-diameter lens with a 670-μm field of view. Figure 3. MicroProbe objectives have especially thin barrels, facilitating in vivo imaging deep within tissue. Each of these lenses can be sealed with an O-ring cap for immersion in sterilization fluid, using a dedicated protective frame. The spring loading is contained within the scan head. Each objective adheres to Royal Microscopical Society thread standards and is transmissive and corrected for a wide range of wavelengths. The transmission at 1000 nm is at least 80 percent as great as transmission at 500 nm. Vascular leakage is an indicator of tissue damage due to ischemia. The increased permeability of small vessels occurs because of intercellular gaps and necrosis of endothelial cells lining the vessel. Research into these areas can help determine the processes underlying this inflammatory response, which often results in life-threatening events. In a mouse model of induced intestinal reperfusion injury, Herlen Alencar and Umar Mahmood of the imaging research center used the intravital laser scanning microscope to image leakage from microvessels in the villi of the jejunum.2 After surgically opening the abdominal wall, they inserted the objective through a small incision in the wall of the jejunum, into the lumen and directly against — but not penetrating — the mucosal surface. Comparing experimental, control and sham-treated mice, the investigators examined the role of the inflammatory response and the possibility of mitigating it in cases of reperfusion injury. In experimental mice, significant leakage of the vascular probe was observed as early as 20 minutes after reperfusion. They then used the imaging system to view the effects of an experimental treatment to prevent damage resulting from reperfusion injury (Figure 4). Figure 4. Leakage of a fluorescent-labeled vascular probe from microvessels within the villi of the jejunum was imaged using intravital laser-scanning microscopy. To image circulation within the villi, a 1.3-mm-diameter objective lens with a 2.5-cm-long barrel was inserted into a small incision in the wall of the jejunum and placed in direct contact with the mucosal surface. The blood vessels within the lamina propria of microvilli are identified by the circulating vascular probe (red), and the enterocytes are stained by rhodamine 6G (green). Both channels are acquired simultaneously but also are displayed separately for clarity. In the treated animal (top), the vascular probe is retained within vessels; in the untreated control (bottom), there is leakage into the lumen. Courtesy of Herlen Alencar, Massachusetts General Hospital Center for Molecular Imaging Research. Imaging with the intravital microscope showed that the agent is retained in the blood vessels inside the colonic villus. In contrast, unmitigated leakage was observed in the saline-treated control. By quantitatively imaging vascular leakage in this way, researchers can better assess compounds selected for preclinical trials. When looking at complex interactions of cells with regard to tissue architecture, imaging in vivo and in situ is beneficial. Cancer and inflammatory disease are significant human health problems, and their pathophysiology is one such area of research. In vivo imaging has the potential to elucidate the underlying mechanisms of disease and to offer direct observation of the effects of therapeutic intervention. Only recently, with the development of new in vivo imaging systems, has such minimally invasive surgery imaging been possible with minimal trauma to the animal. The acceptance of these methodologies will enable better imaging of reporters of molecular activity, which, in turn, can be immensely valuable in target and pathway validation. References 1. M. Zhang et al (Jan. 3, 2006). Identification of the target self-antigens in reperfusion injury. J EXP MED, pp. 141-152. 2. H. Alencar et al (November 2005). Novel multiwavelength microscopic scanner for mouse imaging. NEOPLASIA, pp. 977-983. Meet the author Angela Goodacre is group manager for applications systems, Bio Business Development Div., Life Science Group, Olympus America Inc., Center Valley, Pa.; e-mail: angela.goodacre@olympus.com.
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