FMT imaging facility
Two FMT systems are available at CMIR to image mice in vivo using a variety of NIR fluorescent imaging agents. The systems are equipped with isoflurane anesthesia and are heated to maintain subject body temperature during imaging.
There are 2 fluorescent channels available for imaging (680nm for Cy5.5-like agents and 750nm of AF750-like agents). The FMT software allows for quantification of fluorescence in a 3-dimensional region of interest. While the systems are equipped to capture planar images both in white light and fluorescence, they are also able to detect fluorescence deep within tissue and resolve probe signal in organs such as the lungs and the heart.
Typically, we combine FMT imaging, which yields a three-dimensional map of fluorochrome tracer concentration, with anatomical imaging (CT or MRI). This approach, akin to PET-CT imaging, can simultaneously deliver molecular information (FMT) and the exact anatomic structure the signal originates from (CT, as shown in figures).
Imaging is rapid, a typical FMT acquisition takes approximately 5 minutes and reconstructions of the data sets are run on the system. ROI data can be collected and exported to an Excel spreadsheet, screenshots and Z-stack movies can also be created.
FPT imaging
Fluorescence Protein Tomography (FPT) is a variation of FMT that is optimized for the in vivo 3D tomographic imaging of fluorescence proteins in mice. The method expands the everyday fluorescent protein tagging techniques to the 3D non-invasive in-vivo small animal imaging field.
The third generation of FPT system is highly versatile. Tomographic imaging can be performed (a) with and without the use of index matching fluid, (b) incorporating a single planar or multiple full-angular projections of the mouse with surface extraction, and (c) multispectral acquisitions with a sequence of filters. The system is equiped with 3 lasers (tunable Ar+, 532nm NdYAG, 593nm DPSS) to excite a wide variety of fluorescent proteins from GFP to the new red shifted fluorescent proteins, and also equiped with the standard 670 and 750 nm diode lasers to excite near infrared fluorescent probes.
Optical imaging methods are currently inadequate for non-invasive in-vivo imaging of intact developing insects, animal embryos or small animal extremities, i.e. when working at dimensions between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits in optical macroscopy (>1cm).
Mesoscopic Fluorescence Tomography (MFT) was developed to operate in the 0.5mm-1cm regime with focus on enabling in-vivo observation of common biological model organisms. The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It employs the Fermi simplification to the Fokker-Plank solution of the photon transport equation, combined with geometrical optic principles in order to allow in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
To investigate the in-vivo performance of the technique, we applied it in imaging developing Drosophila. The Drosophila represents an extensively studied species with high importance in biomedical research; yet most of the studies rely on histological sectioning, making it impractical to study the evolution of events in response to mutations and external stimuli in a high throughput fashion. Similarly, a large number of insects is required to accurately build up a time sequence of events. Using MFT we show the possibility to three dimensionally visualize GFP-expressing salivary glands and study the morphogenesis of wing-imaginal discs in-vivo and in real time over a period of six consecutive hours.
This new-found ability offers "time" in the study of developing insects and increases the ability to interrogate events in the unperturbed environment and longitudinally on the same insect.