期刊
ELIFE
卷 11, 期 -, 页码 -出版社
eLIFE SCIENCES PUBL LTD
DOI: 10.7554/eLife.63776
关键词
tumor; bone; 3-photon microscopy; third harmonic generation; nonlinear microscopy; Human; Mouse
类别
资金
- FP7 Ideas: European Research Council [617430-DEEPINSIGHT]
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek NWO Gravitation Programme [024.001.028]
- Agence Nationale de la Recherche [ANR-11-EQPX-0029, ANR-15-CE13-0015]
- Fondation pour la Recherche Medicale [DEI201512440]
- Agence Nationale de la Recherche (ANR) [ANR-15-CE13-0015] Funding Source: Agence Nationale de la Recherche (ANR)
Three-photon excitation is an effective method for intravital microscopy in deep regions of the mouse brain. This study analyzes the benefits of high-pulse-energy infrared excitation for in-depth imaging of tumor and bone tissue, and demonstrates the possibility of simultaneous 3- and 4-photon processes.
Three-photon excitation has recently been demonstrated as an effective method to perform intravital microscopy in deep, previously inaccessible regions of the mouse brain. The applicability of 3-photon excitation for deep imaging of other, more heterogeneous tissue types has been much less explored. In this work, we analyze the benefit of high-pulse-energy 1 MHz pulse-repetition-rate infrared excitation near 1300 and 1700 nm for in-depth imaging of tumorous and bone tissue. We show that this excitation regime provides a more than 2-fold increased imaging depth in tumor and bone tissue compared to the illumination conditions commonly used in 2-photon excitation, due to improved excitation confinement and reduced scattering. We also show that simultaneous 3- and 4-photon processes can be effectively induced with a single laser line, enabling the combined detection of blue to far-red fluorescence together with second and third harmonic generation without chromatic aberration, at excitation intensities compatible with live tissue imaging. Finally, we analyze photoperturbation thresholds in this excitation regime and derive setpoints for safe cell imaging. Together, these results indicate that infrared high-pulse-energy low-repetition-rate excitation opens novel perspectives for intravital deep-tissue microscopy of multiple parameters in strongly scattering tissues and organs.
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