February 1, 2013 (Vol. 33, No. 3)

Historically, optical imaging in small animal models was primarily used to evaluate drug efficacy—to answer the question, is a tumor growing or regressing in response to therapy? “We have moved beyond that,” says Alexandra De Lille, Ph.D., director of technical applications for in vivo imaging at Perkin­Elmer.

Dr. De Lille describes two major trends driving the in vivo imaging industry: multiplexing, or the use of two or more probes or reporters in one animal; and a move away from two-dimensional imaging (based on molecular imaging methods including bioluminescence, fluorescence, and radioisotopes, possibly combined with x-ray, for example, to identify the location of a major organ as an anatomical frame of reference) to 3D reconstructive multimodal imaging, such as co-registering optical imaging with micro-computed tomographic (microCT) imaging, magnetic resonance imaging (MRI), positron emission tomography (PET), or single-photo emission CT (SPECT).

The biological questions being asked will determine the anatomical imaging modality used, notes Dr. De Lille, and these tomographic imaging modalities may include the use of contrast agents to highlight specific anatomical features as well.

PerkinElmer’s IVIS® SpectrumCT instrument combines fluorescence, bioluminescence, radioisotopic Cerenkov, and microCT imaging; it is part of the IVIS product line that includes the Kinetic, Lumina II, XR, and Spectrum.

New to the IVIS family is the IVIS Lumina Series III, offering users a benchtop system with greater versatility in setup and more custom options; it incorporates features from the company’s other in vivo technologies into one platform. The IVIS platform is the workhorse of the PerkinElmer in vivo imaging product line, and the company’s FMT system is designed to streamline fluorescence 3D tomography and quantification for drug discovery campaigns.

Tracking Labeled Targets In Vivo

In vivo imaging is increasingly being used to track the biodistribution of labeled cells and compounds and for preclinical toxicity testing, notes Dr. De Lille. For example, PerkinElmer is developing kidney toxicity probes to be used for fast kinetic imaging with quantitative, functional outputs. The probes can be used with the company’s current imaging systems to assess toxicity of a drug in a live animal without having to do multiple blood extractions. The same type of technology could be used for detecting liver and other types of toxicity in small animal models.

In the first quarter of this year, PerkinElmer also plans to introduce a new Bombesin RSense 680, a fluorescent agent to target and quantify upregulation of bombesin receptors in vivo associated with tumor proliferation, as well as a new in vivo cell tracking dye.

When customers began modifying their standard imaging systems to do fluorescent in vivo imaging in plants and small animals, UVP decided it was time to develop its own dedicated in vivo imaging systems.

The company’s iBox® line of small animal imaging systems ranges from the Spectra introductory level instrument; to the Scientia™, designed for automated, repeatable imaging using a cooled CCD camera (either the BioChemi HR 500 or OptiChemi HR 610) and including a darkroom, warming plate, and the BioLite MultiSpectral Source; and UVP’s newest model, the Explorer™2, which is capable of macro to micro, whole animal to individual cell imaging by transitioning through a magnification range of 0.17x to 16.5x.

UVP’s imaging systems are designed for fluorescence imaging and in animal applications used primarily in cancer research. Sean Gallagher, Ph.D., vp and CTO at UVP, describes how the ability to differentially label healthy and cancerous cells makes it possible to study metastasis.

Using a long working distance and macro imaging, the researcher can fluorescently image a whole animal, identify tumor margins, study blood vessel formation, and observe tumor growth. By zooming into the micro scale in the same animal, these processes can then be imaged at the level of individual cells.

The Explorer imaging system is compatible with conventional fluorescent probes, as well as Dylight Quantum Dot and Alexa dye technology, and can capture signals from 400 nm into the near-IR range of 700–900 nm, enabling the use of fluorescently tagged antibodies or drugs for biodistribution and pharmacokinetics studies.

The industry is increasingly moving toward “visualization of fluorescence in the red and near-infrared (NIR) wavelengths,” says Dr. Gallagher, which allow for deeper penetration into the animal and more light able to emerge from the area being imaged.

As new genetically encoded and dye labeling technologies emerge in the 700–900 nm range, “we need to match that with improved detection and hardware; they have to work together,” he says. The imaging systems need to become more efficient at these wavelengths, and “so for NIR applications we changed to a highly cooled scientific camera that has a higher quantum efficiency in the NIR,” he adds.

Quantitation remains one of the biggest challenges in imaging, notes Dr. Gallagher. For example, when imaging a tumor, estimating the size of a tumor versus determining the intensity of the fluorescent signal it generates may require the use of two different imaging techniques. To determine how large a tumor is, one can visualize its margins and use that information to estimate its volume.

Measuring the intensity of a tumor may not always be a good indicator of its size, however. Some melanomas, for example, produce so much melanin, that it will quench the fluorescent signal. Specialized software and deconvolution techniques can sometimes help in generating quantitative results from imaging studies.

High magnification with iBox® Explorer™ Imaging Microscope (16.5x) of two HT-1080 cells migrating within the vasculature, each with a GFP-labeled nucleus and an RFP-labeled cytoplasm. The field of view is 900 x 900 microns. [UVP]

Multimodal and Deep Tissue Imaging

In September, Bruker acquired the preclinical in vivo imaging business of Carestream Health. Bruker also acquired Belgian microCT instrument manufacturer SkyScan last year, renaming the company Bruker microCT N.V. The company produces high-resolution microCT instruments for in vivo and ex vivo applications.

These acquisitions, and the addition of these new product lines to Bruker’s existing offerings in the preclinical imaging market, are a direct response to a developing market trend described by Mat Brevard, vp, preclinical imaging for North America, Bruker Biospin: “Researchers are using an expanding array of different technologies to get answers to biological questions,” he says. They want multiple data points—reliable and simple data acquired using more diverse methods.

Bruker offers the In-Vivo line of optical imaging systems for small animal imaging applications. The In-Vivo F PRO is an entry-level optical molecular imaging system, and the In-Vivo MS FX PRO is a multispectral optical imaging instrument. Both offer either a 10x zoom or f/0.95 fixed lens option. The MS FX PRO features an x-ray head capable of capturing images in small animals in 3 seconds.

Bruker’s In-Vivo Xtreme optical and x-ray small animal imaging system can perform fluorescence, luminescence, radioisotopic, and radiographic imaging and offers the choice of a front-illuminated 16 MP or back-illuminated 4 MP camera. The Xtreme features a 400W xenon illuminator, a 410–760 nm excitation wavelength range, and a 535–830 nm emission filter wavelength range.

The company is currently developing a “magnetic particle-based imaging modality for fast and sensitive detection of iron oxide particles,” expected to launch in the first half of this year, according to Brevard.

Bruker’s corporate roots are in the MRI field, and it focuses its expertise on developing MRI systems for animal imaging. Its benchtop 1 Tesla cryogen-free MRI system, the ICON™, with a compact-shielded permanent magnet from Aspect Imaging and a negligible magnetic fringe field, made lower-cost, on-site magnetic resonance imaging technology more readily available to small animal research laboratories, Brevard notes.

The LI-COR Pearl® Impulse Small Animal Imaging System facilitates dynamic visualization of imaging agents and deep target imaging using laser excitation and near-infrared fluorescent detection. Rapid screening of small animals can be performed through the acquisition of a white light overlay image of the mouse and simultaneous detection of near-infrared probes at 700 nm and 800 nm wavelengths.

The system includes FieldBrite™ Xi Optical Technology, BrightSite™ targeting agents are available, and environmental control options include HEPA-filtered imaging beds for immunocompromised animals, docking stations, and anesthesia systems.

LI-COR customers use the Pearl system mainly for tumor imaging in mice, monitoring drug response, in the development of new imaging agents and targeting compounds such as antibodies and peptides, and for studying the pharmacokinetic and biodistribution properties of drugs and imaging agents as they move through the body, according to Jeff Harford, senior product marketing manager.

High-resolution in vivo MRI image of a mouse brain with 29 micron in-plane resolution acquired in just over 20 minutes: (A) full field coronal view, (B) expanded view of the hippocampal area, and (C) a corres-ponding Nissl stained plate. [Bruker]

Fluorescence Shines Brightly

During the past five years, the biggest trend has been the gradual shift in interest toward fluorescence imaging versus bioluminescence, in Harford’s view. Whereas “bioluminescence was the optical imaging modality of choice, it is not feasible for clinical imaging,” and people are starting to think more about translational technology and developing clinical applications of in vivo imaging technology.

Furthermore, he notes, fluorescence has gained in usage since the introduction of 800 nm dyes. For example, the LI-COR IRDye® 800CW infrared dye is useful for translational research applications and preclinical studies of disease detection and progression and monitoring of drug efficacy. Light absorption and light scattering by endogenous chromophores present in living tissues, such as hemoglobin, melanin, and lipids, decrease above 700 nm; and tissue autofluorescence is minimal at 800 nm. Thus background noise will be minimized in this spectral region, yielding high signal-to-noise ratios.

Berthold Technologies’ NightOWL II molecular imaging system can be used to study in vivo gene expression by detecting luciferase- and fluorescence-labeled proteins and for localization studies using fluorescence-labeled antibodies. It features a deep-cooled CCD camera and provides height-corrected signal intensity.

The company’s NightOWL LB 983 in vivo imaging system includes a deeply cooled back-illuminated slow scan CCD camera. The NightOWL systems are controlled by Berthold’s IndiGO in vivo imaging software that includes image evaluation tools.

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