Abstract
Multi-photon laser scanning microscopes have many advantages over single-photon systems. However, the speed and flexibility of currently available multi-photon microscopes are limited by the use of mechanical mirrors to steer pulsed radiation for fluorophore excitation. Here, we describe the multi-photon adaptation of a confocal microscope that uses an acoustic optical deflector (AOD) for beam steering. AODs are capable of very rapid scanning and, in addition, offer the flexibility of zooming, panning, and being adjustable for slow image acquisition. Because of the highly dispersive nature of AODs, pulsed radiation must be temporally compressed by introducing negative dispersion into the beam path. More critically, pulsed radiation must also be spatially compressed by introducing lateral dispersion into the beam path. This was accomplished by using two prisms in the external beam path and by introducing a third prism element subsequent to the AOD. The end result is an AOD-based multi-photon microscope that is capable of rapid imaging of physiological events as well as slow detection of weakly fluorescent biological samples.
Full Text
The Full Text of this article is available as a PDF (484.0 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Centonze V. E., White J. G. Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J. 1998 Oct;75(4):2015–2024. doi: 10.1016/S0006-3495(98)77643-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Denk W., Strickler J. H., Webb W. W. Two-photon laser scanning fluorescence microscopy. Science. 1990 Apr 6;248(4951):73–76. doi: 10.1126/science.2321027. [DOI] [PubMed] [Google Scholar]
- John L. M., Lechleiter J. D., Camacho P. Differential modulation of SERCA2 isoforms by calreticulin. J Cell Biol. 1998 Aug 24;142(4):963–973. doi: 10.1083/jcb.142.4.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Soeller C., Cannell M. B. Construction of a two-photon microscope and optimisation of illumination pulse duration. Pflugers Arch. 1996 Jul;432(3):555–561. doi: 10.1007/s004240050169. [DOI] [PubMed] [Google Scholar]
- Xu C., Zipfel W., Shear J. B., Williams R. M., Webb W. W. Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10763–10768. doi: 10.1073/pnas.93.20.10763. [DOI] [PMC free article] [PubMed] [Google Scholar]