The Laser Zentrum Hannover e.V. (LZH) has filed a patent application for a SLOT process, which was originally developed as a 3-D fluorescence process for quickly scanning larger samples. SLOT, which can be viewed as the optical equivalent to computer tomography, works with isotropic resolution, that is, with the same resolution in all three spatial planes.
Data of a murine lung. Left: Transmission image (photo-diode). Middle: Autofluorescence image (photo multiplier tube). Right: Superposition of both signals (red: PD, green: PMT). (Scale 500 µm)
It simultaneously records transmissive, scattered and fluorescent light. Samples can thus be imaged with a 3-D resolution of at least 1/1000 of the object size, in a short time. The goal of the current project is further development of this promising technology, so that an imaging speed of 20 seconds for 600 individual projections can be reached.
SLOT has several advantages over optical projection tomography: Apart from homogeneous lighting with a 300 times higher photon exploitation and a high signal-to-noise ratio of 10-90 dB, ring artefacts and speckles can be avoided, due to one-dimensional detection. Furthermore, the process can be also be used with intrinsic (absorption, scattering, auto-fluorescence) and extrinsic (fluorescence and absorption marker) contrast mechanisms.
Based on intrinsic contrast processes, high resolution, ex vivo volumetric images could be made, for example of locust heads and mouse lungs. By using absorption and auto-fluorescent imaging, the researchers at the LZH could image lung structures down to the resolution of a single alveola.
Further, SLOT can be used to determine objects on non-transparent sample surfaces. In 2012, the three-dimensional visualization of bacteria growth on the surface of dental implants was possible, including images of the in vitro development of the micro-organisms. As fluorescent staining was not necessary, these images were given a fourth dimension.
The technical basis for this process uses detection of the scattered laser light from living bio-films, or correspondingly the wavelength-dependent absorption of metabolism markers such as 2,3,5- Triphenyltetrazoliumchloride (TTC), which are implemented and enriched in the metabolically active bacteria cells in 1,3,5 Triphenylformazan (TPF).
Currently, various investigations on establishing this new imaging process are being carried out in close cooperation with the University of Veterinary Medicine Hannover and the Hannover Medical School. Scientists in the Biophotonic Imaging and Manipulation Group at the LZH are working together with industrial partners to establish SLOT as a fully-automated tomographic module, the so-called Laser-Scanning Tomographic Module (LSTM)., Built into an existing confocal and 2-photon microscope, this unit could be used to monitor the fluctuation of NAD/NADH, cAMP or calcium ions, or for the analysis of the intra- and extracellular accumulation of micro- and nanoparticles in cell aggregates. LSTM is also a promising, innovative technology for non-invasive, temporally and spatially resolved investigations on artificial tissue, which is subsequently subject to in vitro investigations.The development of LSTM is financially supported by the German Federal Ministry of Economics and Technology (BMWi) within the Framework of the Central Innovation Program SME (ZIM). Besides the LZH, project partners are the companies LaVision BioTec GmbH and Scivis wissenschaftliche Bildverarbeitung GmbH.
Michael Botts | Laser Zentrum Hannover e.V.
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