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)