Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging

European Radiology, Jan 2003

A recent development in biomedical imaging is the non-invasive mapping of molecular events in intact tissues using fluorescence. Underpinning to this development is the discovery of bio-compatible, specific fluorescent probes and proteins and the development of highly sensitive imaging technologies for in vivo fluorescent detection. Of particular interest are fluorochromes that emit in the near infrared (NIR), a spectral window, whereas hemoglobin and water absorb minimally so as to allow photons to penetrate for several centimetres in tissue. In this review article we concentrate on optical imaging technologies used for non-invasive imaging of the distribution of such probes. We illuminate the advantages and limitations of simple photographic methods and turn our attention to fluorescence-mediated molecular tomography (FMT), a technique that can three-dimensionally image gene expression by resolving fluorescence activation in deep tissues. We describe theoretical specifics, and we provide insight into its in vivo capacity and the sensitivity achieved. Finally, we discuss its clinical feasibility.

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Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging

A recent development in biomedical imaging is the non-invasive mapping of molecular events in intact tissues using fluorescence. Underpinning to this development is the discovery of bio-compatible, specific fluorescent probes and proteins and the development of highly sensitive imaging technologies for in vivo fluorescent detection. Of particular interest are fluorochromes that emit in the near infrared (NIR), a spectral window, whereas hemoglobin and water absorb minimally so as to allow photons to penetrate for several centimetres in tissue. In this review article we concentrate on optical imaging technologies used for non- - invasive imaging of the distribution of such probes. We illuminate the advantages and limitations of simple photographic methods and turn our attention to fluorescence-mediated molecular tomography (FMT), a technique that can three-dimensionally image gene expression by resolving fluorescence activation in deep tissues. We describe theoretical specifics, and we provide insight into its in vivo capacity and the sensitivity achieved. Finally, we discuss its clinical feasibility. Tissue observation with light is probably the most common imaging practice in medicine and biomedical research ranging from the simple visual inspection of a patient to advanced in vivo and in vitro spectroscopic and microscopy techniques [1]. While intrinsic tissue absorption and scattering yields significant information on functional and anatomical tissue characteristics, significant attention has also been given to fluorescence investigations of tissue since many biochemical markers can be retrieved due to fluorescence contrast, and many more can be targeted using appropriate fluorescent markers [2, 3]. Numerous different optical imaging approaches can be used for imaging fluorescence in vivo. Traditionally, optical methods have been used to look at surface and subsurface fluorescent events using confocal imaging [4, 5, 6], multiphoton imaging [7, 8, 9, 10] microscopic imaging by intravital microscopy [11, 12] or total internal reflection fluorescence microscopy [13]. Recently however, light has been used for in vivo interrogations deeper into tissue using photographic systems with continuous light [14, 15, 16, 17] or with intensity-modulated light [18] and tomographic systems [19, 20]. Potentially, phased-array detection [21] can also be applied. This recent focus in macroscopic observations of fluorescence in tissues has evolved due to the potential of transferring this technology to imaging through animals and humans [2]. This technology has become feasible mainly due to the development of fluorescence probes emitting in the near-infrared spectrum where tissue offers low absorption, highly sensitive detectors and monochomatic light sources (lasers) with higher but nevertheless safely delivered power per wavelength compared with white-light illuminators. While macroscopic fluorescence imaging in the visible has also been attempted using fluorescent proteins, the penetration depth is limited to only 12 mm [16], whereas it has been predicted that NIR fluorescent light can penetrate for several centimeters [22]. Such an approach could enable investigations available currently only for in vitro studies to propagate in in vivo human disease diagnosis and imaging of treatment response and significantly enhance the field of molecular imaging [2]. In this article we discuss imaging techniques that use the diffuse component of light for probing molecular events deep in tissue. Specifically, we focus on reflectance fluorescence imaging and fluorescence-mediated molecular tomography (FMT), which are the two most common approaches currently used for imaging fluorescent probes in deep tissues. We further discuss recent findings that predict the capacity of near-infrared fluorescent signals to propagate through human tissue for non-invasive medical imaging and address feasibility issues for clinical studies. Reflectance imaging Simple photographic methods, in which the light source and the detector reside on the same side of the animal imaged, are generally referred to as reflectance imaging. Reflectance imaging is currently the typical method of choice for accessing the distribution of fluorescent probes in vivo, but the method can be applied more generally to imaging fluorescent proteins or even bioluminescence even if in the latter case no excitation light is used [23]. Near-infrared fluorescence reflectance imaging in particular operates on light with a defined bandwidth as a source of photons that encounters a fluorescent molecule (optical contrast agent or molecular probe), which emits a signal with different spectral characteristics, that can be resolved with an emission filter and captured by a high-sensitivity CCD camera. A typical reflectance imaging system is shown in Fig. 1. The light source can be either a laser at an appropriate wavelength for the fluorochrome targeted or white light sources using (...truncated)


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Vasilis Ntziachristos, Christoph Bremer, Ralph Weissleder. Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging, European Radiology, 2003, pp. 195-208, Volume 13, Issue 1, DOI: 10.1007/s00330-002-1524-x