ABSTRACT
Bioluminescence imaging (BLI) offers the possibility to study and image biology at molecular scale in small animals with applications in oncology or gene expression studies. Here we present a novel model-based approach to 3D animal tracking from monocular video which allows the quantification of bioluminescence signal on freely moving animals. The 3D animal pose and the illumination are dynamically estimated through minimization of an objective function with constraints on the bioluminescence signal position. Derived from an inverse problem formulation, the objective function enables explicit use of temporal continuity and shading information, while handling important self-occlusions and time-varying illumination. In this model-based framework, we include a constraint on the 3D position of bioluminescence signal to enforce tracking of the biologically produced signal. The minimization is done efficiently using a quasi-Newton method, with a rigorous derivation of the objective function gradient. Promising experimental results demonstrate the potentials of our approach for 3D accurate measurement with freely moving animal.
Subject(s)
Algorithms , Image Interpretation, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Luminescent Measurements/methods , Pattern Recognition, Automated/methods , Photography/methods , Subtraction Technique , Video Recording/methods , Animals , Image Enhancement/methods , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Bioluminescence imaging (BLI) allows detection of biological functions in genetically modified cells, bacteria, or animals expressing a luciferase (i.e., firefly, Renilla, or aequorin). Given the high sensitivity and minimal toxicity of BLI, in vivo studies on molecular events can be performed noninvasively in living rodents. To date, detection of bioluminescence in living animals has required long exposure times that are incompatible with studies on dynamic signaling pathways or nonanaesthetised freely moving animals. Here we develop an imaging system that allows: 1. bioluminescence to be recorded at a rate of 25 images/s using a third generation intensified charge-coupled device (CCD) camera running in a photon counting mode, and 2. coregistration of a video image from a second CCD camera under infrared lighting. The sensitivity of this instrument permits studies with subsecond temporal resolution in nonanaesthetized and unrestrained mice expressing firefly luciferase and imaging of calcium signaling in transgenic mice expressing green fluorescent protein (GFP) aequorin. This imaging system enables studies on signal transduction, tumor growth, gene expression, or infectious processes in nonanaesthetized and freely moving animals.
Subject(s)
Luminescent Measurements/instrumentation , Luminescent Measurements/veterinary , Luminescent Proteins/analysis , Luminescent Proteins/metabolism , Monitoring, Ambulatory/instrumentation , Monitoring, Ambulatory/veterinary , Whole Body Imaging/instrumentation , Whole Body Imaging/veterinary , Animals , Computer Systems , Equipment Design , Equipment Failure Analysis , Luminescent Measurements/methods , Mice , Mice, Nude , Monitoring, Ambulatory/methods , Movement , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Fluorescence is increasingly used for in vivo imaging and has provided remarkable results. Yet this technique presents several limitations, especially due to tissue autofluorescence under external illumination and weak tissue penetration of low wavelength excitation light. We have developed an alternative optical imaging technique by using persistent luminescent nanoparticles suitable for small animal imaging. These nanoparticles can be excited before injection, and their in vivo distribution can be followed in real-time for more than 1 h without the need for any external illumination source. Chemical modification of the nanoparticles' surface led to lung or liver targeting or to long-lasting blood circulation. Tumor mass could also be identified on a mouse model.
