by Angela Goodacre

The ability to stimulate accurate axon re-growth is the ultimate goal in spinal cord injury research, but another key area of study comes from the fact that patients who have suffered such injuries are also at risk for potentially fatal vascular events such as deep vein thrombosis and thromboembolism. In the Department of Neurosciences at the University of California at San Diego (UCSD), significant efforts are under way to determine the processes underlying the chronic life-threatening events that patients face after acute trauma to the spinal cord. In vivo monitoring of the vascular changes in animals with spinal cord injuries can lead to a better understanding of the mechanisms of thrombus formation and identify targets for therapeutic intervention.

Real-time imaging of living, intact organisms builds on the last decade of breakthrough discoveries made through fluorescent imaging in live cells. But doing the most humane and successful imaging requires special consideration. Having excitation light penetrate into organs, and even more, collecting photons from fluorescent emission, all deep within an intact animal, present significant challenges for both optical design and for fluorescent probe chemistry.

Collecting light from deep within tissue
Olympus has combined core technologies in endoscopy and microscopy to address the needs of small animal imaging in a family of complementary imaging platforms. The Olympus IV100 laser scanning system has a light path optimized for collecting light from deep within living tissue. The orientation and uneven surfaces of organs in a live animal cause difficulties when doing conventional imaging with an upright microscope, so the system has a compact scan head that can be tilted for orthogonal imaging of internal organs. This enables optimal imaging with minimal animal repositioning (Figure 1).
Figure 1. The Olympus IV100 scan head tilts from -10 degrees to +70 degrees for imaging internal organs.

In vivo imaging benefits greatly from the ability to multiplex fluorescent reporters. This allows multiple cellular parameters to be imaged simultaneously, identifying mechanisms involved in a complex biologic process. Even the intrinsic fluorescence of biological molecules such as those found in blood vessels can be useful in giving anatomic reference points. The lasers used for the IV100 are similar to those used in conventional laser scanning. They include an Argon laser at 488 nm with the addition of a diode pumped solid-state (DPSS) laser at 561 nm, a red Helium-Neon laser at 633 nm and a diode laser emitting at 748 nm. Based on the platform of the Olympus FV1000 confocal laser scanning microscope, the emission path of the IV100 system employs high-sensitivity photomultiplier tubes, along with ion-sputtered emission beam splitters 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.

High resolution imaging over a wide range of wavelengths also presents its own challenges. The UIS2 microscope objectives used with the system combine new technologies in lens coatings and glass development. As a result, the optical elements of the IV100 are optimized for use from blue through far red and well into the near infrared (NIR) range.

Advantages over conventional optical methodologies
Conventional objective lenses are large and thus can require extensive surgery to expose or even exteriorize organs. But exposing the animal to such trauma reduces the possibility of longitudinal studies in a single animal and can also affect the biological relevance of data.

For minimally invasive imaging, the IV100 platform can be coupled with Olympus MicroProbe objective lens technology. These high numerical aperture (NA) optics have been designed with a narrow diameter and extend up to 2 cm in length for insertion through a small incision in the body wall into the body cavity for imaging of organs in situ. The long, extremely narrow MicroProbe optics can help avoid unnecessary trauma to the animal and enhance the possibility of conducting studies over the period of recovery or disease progression in the same animal. Such longitudinal studies not only have advantages in terms of following the progress of events in an animal, they can help avoid the sacrifice of animals at different time points and thus can markedly reduce the number of experimental animals needed for a study.

High numerical aperture MicroProbe objectives can yield Z stacks that extend 200 micrometers into an intact organ. There are three versions designed to satisfy in vivo imaging applications:

• high resolution 27 magnification (NA 0.7) 3.5 mm-diameter lens with 220 m field of view (FOV).
• high-resolution 20 magnification (NA 0.5) 1.3 mm-diameter lens with 20 m FOV.
• wide-angle 6 magnification (NA 0.14) 1.3 mm-diameter lens with a large FOV of 670 m

The lenses can be completely sealed with an O-ring cap for immersion in sterilization fluid, in a dedicated protective frame. The spring loading is contained within the actual scan head. Each objective adheres to RMS thread standards and is transmissive and corrected for a wide range of wavelengths.

New optics coupled with the use of near infrared (NIR) fluorescent probes present opportunities for in vivo imaging of multiplexed fluorescent reporters in small animal models. Confocal and multiphoton microscopy have been used for imaging in intact tissue, but even with the increased penetration of the IR wavelengths of multiphoton lasers, the scatter of emission photons from fluorophores limits the depth at which these technologies can achieve sufficient signal-to-noise ratios. Using NIR dyes, high-resolution images can be obtained with the IV100 up to 500 m deep. In addition, fluorophores in the NIR window (650-900 nm) offer an advantage in their spectral distance from autofluorescence and absorption due to other molecules such as hemoglobin.

Imaging in vivo often requires an enclosed system for optimal anesthesia and temperature control, as some procedures are amenable to long term observation. Isoflurane anesthesia has been shown to be reliable and with rapid recovery once the inhalant supply is removed.
Figure 2. Each image represents a view from a 3-dimensional rendering of a thrombus that formed in an externalized mesenteric vessel. Red and green channels were acquired simultaneously using the IV100, with green representing platelet aggregation and red delineating the lumen of the blood vessel. The platelets are labeled with an antibody conjugated to fluorescein isothiocyanate (FITC) (BD Biosciences, San Jose, CA) and the blood pool with AngioSense 680 (VisEn Medical, Woburn, MA), and an XYZ image stack was acquired using the Olympus MicroProbe 273, NA 0.7 objective. The stack was imaged over time, for up to 45 minutes.

The study
Researchers in the Neuroscience department of UCSD have an extensive program in the study of spinal cord injury. Brendan Brinkman has been using the Olympus IV100 intravital laser scanning microscope to image the vascular changes that occur after spinal cord injury in experimental animals. The work involves creating three-dimensional renderings of multi-focal stacks of images to show the location and morphology of thrombus formation inside the blood vessel over time (Figure 2).

In a separate study in vivo imaging examined ex vivo gene therapy, utilizing modified cells that deliver the growth factor neurotrophin-3 (NT-3) to the acutely injured spinal cord. Such grafting has been shown to elicit regeneration and recovery of function in the adult rat. A small laminectomy window was surgically prepared in a rat some 2-3 days after spinal cord injury and transplant of NT-3 producing bone marrow stromal cells. The animal had been injected one week prior to imaging with a lentiviral green fluorescent protein (GFP) expression system via the dorsal root ganglion, resulting in GFP labeled axons. The small diameter of the MicroProbe lens allowed positioning of the optics within the window to accurately image the site of the lesion and track regeneration over multiple fields of view, a significant advantage over larger objectives (Figure 3).
Figure 3. Axonal re-growth imaged in a small scale laminectomy window, showing the fine structure including the border of the lesion and terminized GFP labeled axons, along with reddish autofluorescence of infiltrating macrophages.

Spinal cord injury in experimental animals can serve as a model to study cardiovascular mechanisms following traumatic injury and has the potential for mitigating long-term chronic effects of such injuries. Moreover, the inflammatory processes that impact the ability of axons to re-grow and the potential for promoting repair of the spinal cord offers possibilities for therapeutic intervention not only in spinal cord injury, but also in neurodegenerative disorders such as Parkinson's, Multiple Sclerosis and Alzheimer's disease. Experimental investigations requiring careful and humane experimental design are aided by technologies that employ minimally invasive procedures.

About the author
Angela Goodacre is Group Manager for Applications Systems, Bio Business Development, Olympus America Inc., Life Science Group. Brendan Brinkman is Managing Director, Neuroscience Microscopy Shared Facility, University of California at San Diego. More information about the systems and procedures discussed in this article is available from:

Olympus America Corp.

Figures 2 and 3 courtesy of Brendan Brinkman, Department of Neurosciences, UCSD. Thanks to Ephron Rosenzweig, Ph.D., laboratory of Mark Tuszynski, Department of Neurosciences, UCSD, and to Ana Kasirer-Friede and Hisashi Kato, laboratory of Sanford Shattil, Division of Hematology, Department of Medicine, UCSD.