Eliminating the scattering ambiguity in multifocal, multimodal, multiphoton imaging systems

• Published in: Journal of biophotonics 2012-05, Vol.5 (5-6), p.425-436
• Main Authors: Hoover, Erich E., Field, Jeffrey J., Winters, David G., Young, Michael D., Chandler, Eric V., Speirs, John C., Lapenna, Jacob T., Kim, Susy M., Ding, Shi-you, Bartels, Randy A., Wang, Jing W., Squier, Jeff A.
• Format: Article
• Language: English
• Subjects: Animals; Cellulose - metabolism; Drosophila melanogaster - cytology; Image Processing, Computer-Assisted; Luminescent Proteins - metabolism; Microscopy, Fluorescence, Multiphoton - methods; Molecular Imaging - methods; multifocal; multimodal; multiphoton; photon counting; Red Fluorescent Protein; Scattering, Radiation; Starch - chemistry; Zea mays - chemistry
• ISSN: 1864-063X; 1864-0648
• DOI: 10.1002/jbio.201100139

In this work we present how to entirely remove the scattering ambiguity present in existing multiphoton multifocal systems. This is achieved through the development and implementation of single‐element detection systems that incorporate high‐speed photon‐counting electronics. These systems can be used to image entire volumes in the time it takes to perform a single transverse scan (four depths simultaneously at a rate of 30 Hz). In addition, this capability is further exploited to accomplish single‐element detection of multiple modalities (two photon excited fluorescence and second harmonic generation) and to perform efficient image deconvolution. Finally, we demonstrate a new system that promises to significantly simplify this promising technology. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)