Fluorescent proteins (FPs) have revolutionized biological discovery by allowing visualization of cellular and sub-cellular molecules, structures and processes [1
]. The ability to image and quantitate such processes at the whole body level would enable functional ‘-omics’ and/or allow the detailed tracking of cell populations over time. As we move to systematically explore the function and regulation of protein networks - either normally functioning ones, alterations in disease or through therapeutic manipulation - it is increasingly important to visualize various events in situ
in the appropriate organ and within complex living environments. In this role, the use of mammalian animal models has been invaluable to bridge the divide between in vitro
research and the clinical application of therapeutic strategies.
Intravital microscopy [3
] has paved the way for the utilization of FPs in the understanding of cellular and sub-cellular function in vivo.
The use of this technique, however, is significantly limited by the inability to non-invasively visualize activity deeper than a few hundred microns, for example from entire solid tumors, or within organs and systems. Epi-illumination imaging, which uses single projection imaging, has likewise been employed for imaging surface activity at depths of a few millimeters [5
]. In this approach, the fluorescence intensity has a strong, non-linear dependence on both the depth of the activity and the underlying tissue optical properties. Consequently, this impedes the ability to accurately quantify the underlying fluorescence activity. Fluorescence tomography (FMT) [7
], a model-based reconstruction method, has been developed as an alternative, quantitative, three-dimensional imaging technique to overcome the limitations of epi-illumination and to visualize the distribution of fluorescence probes in vivo
. Through several technical developments recent systems utilize CCD cameras, which allow high spatial sampling of photon propagating through tissues at multiple projections, i.e. at multiple illumination detection paths [8
] and wavelengths [9
]. Fluorescence tomography has been previously shown to resolve GFP and dsRed reporter protein expression in-vivo [10
], however limitations associated with operation in the visible spectrum (<600 nm) were noted, such long acquisition times and thus limitation to single view projections.
The ability to resolve fluorescent reporter proteins operating in the near-infrared could significantly improve the applicability of these imaging techniques in the study of biological function. Seminal ongoing research on FP development has resulted in a variety of adept red-shifted constructs that operate in the far-red (RFPs) [13
], and recently, in the near-infrared (IFP) [20
]. It has been predicted that the signals emitted from RFPs can be detected for several millimeters to centimeters in tissues [21
], which would allow visualization through entire small animals, such as mice or rats. This feature is due to a peculiarity in the hemoglobin absorption spectrum that yields a steep drop in the extinction coefficient at wavelengths that are longer than 600-610 nm. As a result, light attenuation in tissues is significantly lower in the far-red and near-infrared (630 – 900 nm) compared to the visible. Yet, the excitation of most RFPs remains in the visible range (<630 nm), where light experiences strong attenuations when traveling through tissue [13
]. This imposes several methodological difficulties on the development of strategies that can accurately illuminate, capture and reconstruct the fluorescence biodistribution of fluorochromes that operate at a highly varying attenuation background.
For this reason, in the present study, we explored a method that can optimally reconstruct the three dimensional distribution of FP activity through the entire volume of a mouse. To achieve optimal performance, the imaging method developed herein incorporates optimum excitation and emission wavelengths, efficient tissue autofluorescence subtraction techniques, and forward light propagation models that work seamlessly at both sides of the steep light attenuation change, as well as, a multispectral reconstruction scheme that is based on normalized Born (n-Born) ratio and concurrently utilizes four-dimensional data sets (i.e., sources, detectors, angular projections, and emission wavelengths). We show that the forward and inversion components selected are necessary in order to achieve accurate fluorescence biodistribution reconstructions in deep tissue, over conventional FMT methods developed for the near-infrared. This approach, along with the longer light penetration in the red part of the spectrum, allowed the visualization of mCherry-labeled glioblastoma tumors in vivo achieving at least one order of magnitude increased sensitivity compared to volumetric imaging using GFP, with the potential to significantly further improve the sensitivity using novel classes of contrast agents. Herein, we present the major methodological steps, key in vivo results, and major cross-validation findings that showcase the necessity and superior performance achieved with this method, over near-infrared (NIR) optical tomography approaches and discuss the application potential of the approach for non-invasive imaging of RFPs in vivo.