Non-Linear Microscopy - Biophotonics - page 66

2. The use of near-infrared wavelengths allows to minimize biological damage
as biological tissues in general poorly absorb infrared light. This combined
with the in-focus photobleaching allows for long-term observation of liv-
ing samples. Because of the wide two-photon cross section of most of the
fluorescent molecules, it is possible to excite different dyes at once with
only one laser line, reducing the average power delivered on the sample.
Furthermore, the use of IR light permits deeper penetration into the sam-
ple (up to 0.5 mm) as both absorption and scattering are reduced with
this wavelength. Finally, because of the fact that the separation between
excitation and emission (excitation in the IR, emission in the UV-visible)
is usually wider than in conventional excitation, it is considerably eas-
ier do discriminate between the actual emission and scattered/reflected
excitation light.
The advances of TPE is strictly connected to the developments in an-
other non-linear microscopy technique, second harmonic generation imaging.
It offers a practically non-invasive tool for deep tissue imaging as it does not
require fluorescent labelling of the samples and it retain the 3D investigation
and optical sectioning properties of multi-photon excitation. Also in this case
all the advantages using IR light for the illumination are conserved.
Since relevant generation of second harmonic signal is associated with the
presence of tissue constituents as collagen, SHG may play an important role
in skin disease diagnosis and in the medical research field in general, being a
promising endoscopy tool.
The combination of MPE microscopy and SHG imaging provides both
a deep insight into biological matter and offers possibilities in the nano-
manipulation of living cells and tissues and in the 3D micro- and nano-surgery
field. The range of application of these techniques is rapidly increasing in the
biomedical, biotechnological and biophysics sciences and it is now facing the
clinical applications.
References
1. W. Kaiser, C.G.B. Garrett, Phys. Rev. Lett. 7 , 229 (1961)
2. S. Singh, L.T. Bradley, Phys. Rev. Lett. 12 , 162 (1964)
3. D.M. Friedrich, J. Chem. Educ. 59 , 472 (1982)
4. D.M. Friedrich, W.M. McClain, Annu. Rev. Phys. Chem. 31 , 559 (1980)
5. P. R. Callis, Annu. Rev. Phys. Chem. 48 , 271 (1997)
6. W. Denk, J.H. Strickler, W.W. Webb, Science 248 (4951), 73 (1990)
7. A. Diaspro, IEEE Eng. Med. Biol. Mag. 15 , 29 (1996)
8. A.J. Koster, J. Klumperman, Nat. Rev. Mol. Cell. Biol. (Suppl), SS6 (2003)
9. U. Durig, J.K. Gimzewski, D.W. Pohl, Phys. Rev. Lett. 57 (19), 2403 (1986)
10. G. Binnig, C.F. Quate, Ch. Gerber, Phys. Rev. Lett. 56 (9), 930l (1986)
11. E. Ruska, M. Knoll, Z. Tech. Phys. 12 , 389 (1931)
12. J.B. Pawley (ed.), Handbook of Biological Confocal Microscopy . (Plenum, New
York, 2005)