We describe a Monte Carlo model of the mouse torso to optimize illumination of the mouse lung for fluorescence detection of low levels of pulmonary pathogens, specifically Mycobacterium tuberculosis. After validation of the simulation with an internally illuminated optical phantom, the entire mouse torso was simulated to compare external and internal illumination techniques. Measured optical properties of deflated mouse lungs were scaled to mimic the diffusive properties of inflated lungs in vivo. Using the full-torso model, a 2 × to 3 × improvement in average fluence rate in the lung was seen for dorsal compared with ventral positioning of the mouse with external illumination. The enhancement in average fluence rate in the lung using internal excitation was 40 × to 60 × over external illumination in the dorsal position. Parameters of the internal fiber optic source were manipulated in the model to guide optimization of the physical system and experimental protocol for internal illumination and whole-body detection of fluorescent mycobacteria in a mouse model of infection.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the most frequent causes of death worldwide. The slow growth rate of Mtb limits progress toward understanding tuberculosis including diagnosis of infections and evaluating therapeutic efficacy. Development of near-infrared (NIR) β-lactamase (BlaC)-specific fluorogenic substrate has made a significant breakthrough in the whole-animal imaging to detect Mtb infection. The reporter enzyme fluorescence (REF) system using a BlaC-specific fluorogenic substrate has improved the detection sensitivity in whole-animal optical imaging down to ~104 colony forming units (CFU) of bacteria, about 100-fold improvement over recombinant strains. However, improvement of detection sensitivity is strongly needed for clinical diagnosis of early stage infection at greater tissue depth. In order to improve detection sensitivity, we have integrated a fiber-based microendoscpe into a whole-animal imaging system to transmit the excitation light from the fiber bundle to the fluorescent target directly and measure fluorescent level using BlaC-specific REF substrate in the mouse lung. REF substrate, CNIR800, was delivered via aerosol route to the pulmonary infected mice with M. bovis BCG strain at 24 hours post-infection and groups of mice were imaged at 1–4 hours post-administration of the substrate using the integrated imaging system. In this study we evaluated the kinetics of CNIR800 substrate using REF technology using the integrated imaging system. Integration of these technologies has great promise for improved detection sensitivity allowing pre-clinical imaging for evaluation of new therapeutic agents.
The song control system of juvenile songbirds is an important model for studying the developmental acquisition and
generation of complex learned vocal motor sequences, two processes that are fundamental to human speech and
language. To understand the neural mechanisms underlying song production, it is critical to characterize the activity of
identified neurons in the song control system when the bird is singing. Neural imaging in unrestrained singing birds,
although technically challenging, will advance our understanding of neural ensemble coding mechanisms in this system.
We are exploring the use of a fiber optic microscope for functional imaging in the brain of behaving and singing birds in
order to better understand the contribution of a key brain nucleus (high vocal center nucleus; HVC) to temporal aspects
of song motor control. We have constructed a fluorescence microscope with LED illumination, a fiber bundle for
transmission of fluorescence excitation and emission light, a ~2x GRIN lens, and a CCD for image acquisition. The
system has 2 μm resolution, 375 μm field of view, 200 μm working distance, and 1 mm outer diameter. As an initial
characterization of this setup, neurons in HVC were imaged using the fiber optic microscope after injection of quantum
dots or fluorescent retrograde tracers into different song nuclei. A Lucid Vivascope confocal microscope was used to
confirm the imaging results. Long-term imaging of the activity of these neurons in juvenile birds during singing may
lead us to a better understanding of the central motor codes for song and the central mechanism by which auditory
experience modifies song motor commands to enable vocal learning and imitation.
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