Abstract
Nonlinear optical scanning microscopy has become a useful tool for living tissue imaging. Biological tissues are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light travels into the sample. While performing imaging of biological scattering tissues in nonlinear excitation regime, the localization of the maximum two-photon excitation (2PE) intensity was found to shift closer to the surface and the 2PE imaging depth limit appears strongly limited by near-surface fluorescence. In this work, we computed the illumination and the photobleaching distribution to characterize the effects induced by scattering. An experimental test has been carried out by imaging, with 0.9 NA objective, thick scattering fluorescent immobile sample (polyelectrolyte gel) as a phantom for biological samples. Results confirm that under these conditions no photobleaching effects due to scattering occur close to the surface.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Born M, Wolf E (1959) Principles of optics. Cambridge University Press, Cambridge
De Grauw C, Frederix P, Gerritsen H (2002) Aberrations and penetration in in-depth confocal and two-photon-excitation microscopy. In: Diaspro A (ed) Confocal and two-photon microscopy: foundations, applications and advances. Wiley-Liss, Wilmington, pp 160–163
Denk W, Strickler J, Webb W (1990) Two photon laser scanning fluorescence microscopy. Science 248(4951):73–76
Diaspro A, Chirico G, Collini M (2006) Two-photon fluorescence exitation and related thechniques in biological microscopy. Q Rev Biophys 15:1–70
Mayer MG (1931) Uber elementarakte mit zwei quantensprungen. Ann Phys 9:273–295
Mazza D, Cella F, Vicidomini G, Krol S, Diaspro A (2007) Role of three-dimensional bleach distribution in confocal and two-photon fluorescence recovery after photobleaching experiments. Appl Opt 46:7401–7411
Mie G (1908) Beitrge zur optik trber medien, speziell kolloidaler metallsungen. Ann Phys 4:377–445
Palamidessi A, Frittoli E, Garr M, Faretta M, Mione M, Testa I, Diaspro A, Lanzetti L, Scita G, Fiore PPD (2008) Endocytic tracking of rac is required for the spatial restriction of signaling in cell migration. Cell 134:135–147
Patterson GH, Piston DW (2000) Photobleaching in two photon excitation microscopy. Biophys J 78(4):2159–2162
Sheppard WTC (1984) Theory and practice of scanning optical microscopy. Academic, New York
Theer P, Denk W (2006) On the fundamental imaging-depth limit in two-photon microscopy. J Opt Soc Am A Opt Image Sci Vis 23:3139–3149
Ying J, Liu F, Alfano RR (1999) Spatial distribution of two-photon-excited fluorescence in scattering media. Appl Opt 38:224–229
Ying J, Liu F, Alfano RR (2000) Effect of scattering on nonlinear optical scanning microscopy imaging of highly scattering media. Appl Opt 39:509–514
Zwier JM, Rooij GJV, Hofstraat JW, Brakenhoff GJ (2004) Image calibration in fluorescence microscopy. J Microsc 216:15–24
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Berlin Heidelberg
About this chapter
Cite this chapter
Lavagnino, Z., Zanacchi, F.C., Diaspro, A. (2011). Role of Scattering and Nonlinear Effects in the Illumination and the Photobleaching Distribution Profiles. In: Diaspro, A. (eds) Optical Fluorescence Microscopy. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-15175-0_4
Download citation
DOI: https://doi.org/10.1007/978-3-642-15175-0_4
Published:
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-15174-3
Online ISBN: 978-3-642-15175-0
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)