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Inclined Single Plane Illumination Microscopy (ISPIM)

2012; Elsevier BV; Volume: 102; Issue: 3 Linguagem: Inglês

10.1016/j.bpj.2011.11.1048

1542-0086

Francesco Cutrale, Enrico Gratton,

Advanced Electron Microscopy Techniques and Applications

Current need in biomedical sciences requests live imaging with higher spatial and temporal resolution in the 3D environment. Furthermore, live sample acquisition increases the technical challenges required for 3D imaging. Ideal features of a system for this purpose include high imaging speed, high contrast ratio, low photo-bleaching and photo-toxicity, good resolution in a 3D context and mosaic acquisition for large samples. Our work presents the inclined illumination implementation of Single Plane Illumination Microscopy, introduced by Stelzer et al (2004). SPIM has been gaining importance in the biomedical field over the years, with its load of technical challenges but has been limited by construction and sample preparation. Based on the same principle introduced by Stelzer, but with a configuration similar to Total Internal Reflection Fluorescence basis, Dunsby (2008) proved the possibility to build an Oblique Single Plane Illumination Microscope (OSPM), also known as Oblique Light Sheet Microscope on a completely lab-made system while avoiding any change in common sample preparation protocol. Our effort was to realize a system with the advantages of the simplification of sample preparation of the OSPM but constructed on commercial microscopes, such as IX-71 Olympus. We have increased the performance of the original OSPM system and we have realized a powerful instrument for applications to biological issues. The optimized system goes under the name of inclined Single Plane Illumination Microscope (iSPIM). Furthermore we are investigating the possibility to increase spatial resolution and obtain isotropic information by adapting the formulas for three dimensional inverse Radon transform to the iSPIM system and acquiring sample information at different angles. Acknowledgments: P41-RRO3155, NIH-R01-DK066029, P50-GM076516