Non-Linear Microscopy - Biophotonics - page 50

Fig. 4.1. Perrin-Jablonski diagram representing the possible pathways for one mole-
cule to relax back to the ground state once excited
must match the molecular energy gap, multi-photon processes allow using
longer excitation wavelength. Multi-photon microscopy generally use infrared
(IR) light for the excitation, resulting in the increase of penetration depth in
turbid media and in the decrease of the photo-toxicity in biological samples.
Because of the fact that the molecule is memoryless of the way in which
excitation is accomplished, the two-photon induced fluorescence retains the
same characteristics of conventional emission process. Therefore, in multi-
photon excitation, the fluorescent emission is generally at shorter wavelengths
than the one absorbed by the molecule.
4.3.2 Confocal Principles and Laser Scanning Microscopy
In conventional wide-field microscopy a large portion of the sample is entirely
illuminated with a light source and viewed directly by eye or through any
image collection system (charged coupled device (CCD), for instance). With
this method the sample undergoes continuous excitation and all the points
of the sample, both in-focus and out-of-focus, will contribute to the image.
As a consequence, the out-of-focus contribution will appear blurred in the
image, resulting in the decrease of the axial resolution and in the hazing of the
collected image. In this context, as reported by Minsky in 1961, the maximum
performance of a microscopy imaging system should be met if it would be
possible to investigate point by point the observed sample in order to collect
at each position the light scattered or emitted by that point alone, rejecting
all the contributions from the other parts of the sample, especially from those
belonging to different focal planes [48]. Even if it is not possible to eliminate
every undesired ray because of multiple scattering, it is straightforward to
remove all the rays that do not focus on the point of interest, by using a