Imaging consists of different types of microscopy (optic-, fluorescence-, confocal and electron microscopy), that allow in vitro, in vivo and ex-vivo studies of samples at high magnification, aimed at characterising subcellular structures and organisms. In vivo imaging allows to reduce the number of experimental animals by longitudinal studies, in which the same animal is used at different time points. Moreover, in vivo imaging decreases animal suffering since procedures are usually performed under deep anaesthesia.
- LEICA TCS-SP5 Inverted Confocal Microscope. This microscope has an inverted stage, equipped with different objectives (dry 5-10-20x , high NA oil immersion 40 e 63x, glycerol 63x, oil 100x; different Phase contrast objectives are also available). The microscope is equipped with 4 visible lasers emitting the following wavelengths: 405nm, 458nm, 476nm, 488nm, 496nm, 514nm, 543nm, 633nm. The signal can be detected by 5 spectral detectors (2 Hybrid and 3 PMTs). An external PMT is available for the bright field signal. A motorised stage allows mosaic acquisition and stitching (Tile Scan), as well as time lapse on different points of the sample at the same time (Mark and Find). The incubator, equipped with temperature and CO2 controller, allows to perform live cell imaging for long hours.
The software is equipped with FRET-AB, FRET-SE, FRAP, live data mode wizards.
A 8 kHz resonant resonant scanner is also available to reduce bleaching during live acquisition.
Recently, this machine has been equipped with a widefield acquisition system including a high speed fluorescence camera (Hamamatzu), Metamorph acquisition software and the Gemini system for widefield FRET. - LEICA TCS-SP5 Upright Confocal-Multiphoton Microscope. Upright configuration, long distance water immersion objectives (HCX APO L 10x/0.30 W, HCX APO L 20x/0.50 W U-V-I -/D 3.5, HCX APO L 40x/0.80 W UVI, HCX APO L 63x/0.90), fluorescence camera, 4 visible lasers emitting the following wavelengths: 405nm, 458nm, 476nm, 488nm, 496nm, 514nm, 543nm, 633nm. The signal can be detected by 4 spectral detectors (1 Hybrid and 3 PMTs) or, only for multiphoton imaging, by 2 external non-descanned detectors (2PMTs, filter cubes FITC/TRITC). The multiphoton laser is a tunable Ti-Sa pulsed Chameleon Ultra (Coherent) emitting a single laser line in the range 680 a 1080nm. In multiphoton microscopy the sample is excited point by point by a high-energy pulsed IR laser beam. Long wavelength allows: i) a deep penetration of the sample and ìì) low photobleaching. Typical applications are the observation of cells in very thick samples such as organoids, lynph nodes, thick slice tissues or directly into the animal.
For instance, it is possible to perform imaging of fluorescent neurons or microglial cells directly in the cerebral cortex of mice.
Recently, this machine has been equipped with a high NA objective set in order to be used also as a standard confocal and increase the multi-user application. - Optical Imager (OI) IVIS Spectrum (Xenogen, Alameda, USA). It is made up by a Charge Coupled Device (CCD) chilled at -90°C with quantum efficiency greater than 85% between 500 and 700 nm and greater than 30% between 400 and 800 nm. In march 2012 the instrument has been upgrade to Spectrum IVIS (Perkin Elmer, Hopkinton, USA) and equipped with excitation and emission filters and trans- illumination configuration in order to allow a completed tomographic acquisition in the fluorescence mode. The system is also provided with a license for multimodal imaging in order to combine images from different imaging modalities (TC, PET, Magnetic Resonance and OI) The Optical Imager technique is used to detect optical photons, especially in the red and infrared regions. The most relevant processes are the fluorescence emission ( excitation of fluorophore injected in organism) and bioluminescence emission (enzymatic reactions creating light). The OI technique in very sensible (at the level of single cells), repeatable, reliable, very fast and economic.
In the fluorescence mode, the instrument uses the fluorescence emission of colorant injected in living organism. Moreover, different kinds of cells (including stem cells) can be marked with fluorescent molecules allowing to study in vivo their homing. In the bioluminescence mode, OI detects light emitted in the enzymatic reactions. Recently, here in Verona, the Cerenkov Luminescence Imaging (CLI), a new technique combining Optical Imaging and Nuclear Medicine, has been discovered and developed. The CLI technique has been already used in humans. - Magnetic Resonance Imaging Tomograph (Bruker, Biospin) for small animals. MRI system is equipped with a 7 Tesla, 16 cm bore Ultra Shilded superconducting magnet (Pharmascan 70/16 US Bruker). It’is based on Bruker Advance II electronics and a Bruker B-GA9S HP gradient insertwith 380 mT/m maxim intensity. It’s equipped with five acquisition coils: birdcage coil for rats and mice, helmet surface coil for rats and mice brain and array coil- 4 channels for mouse brain. For in vivo acquisition, the system is provided with gas anesthesia, vital signs measurement and heating devices for animals. In oncological research, MR standard images are used to study tumor growth and to distinguish tumor tissue from perifocal edema. At the same time, in the Central Nervous System, MR standard images allow to evaluate lesions, blood brain permeability and atrophy. Advanced MRI application allow to measure blood flow (using the Artery Spin Labeling technique), to evaluate the functional answer to a stimulus (with the BOLD technique), the axonal connectivity (with the Diffusor Tensor Imaging technique) and the functional connectivity (resting state functional MRI). Finally, at high field, localized Magnetic Resonance Spectroscopy (MRS) allows not invasive detection and quatification of different metabolites that have crucial relevance for staging tumors and defining CNS pathologies, like N-Acetil Aspartate (NAA), choline (Cho), creatine (Cr, myo inositol (MI) and glutamate and glutamine compounds (Glu-n).
- Transmission Electron Microscope FEI TECNAI G2 (TEM). High resolution imaging system equipped with an integrated digital camera. The TEM allows the bi-dimensional observation of biological or inorganic samples with an extremely high resolution (2-3nm). TEM has a wide range of applications, from the study of pathological samples to the study of basic physiological structures. In particular, TEM allows to analyse the organisation of intracellular structures and organelles, giving a detailed image about the different organelle equipments of different cytotypes. Another application is the study of nanoparticles and aggregation in material science. The highest resolution for this machine is 650 000 x. The system is equipped with an EDAX probe for microanalysis and with different types of holders: Copper single tilt, Berillium single tilt and a tomographic holder.
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