Biomedical Engineering Reference
In-Depth Information
laser beam [41]. For many years the application of these non-linear processes
was limited to the spectroscopic studies of inorganic samples. The first ob-
servation of TPE of organic dyes is dated to 1970 [42], while in 1976 Berns
reported a two-photon effect as a result of focusing an intense laser beam onto
chromosomes of living cells [43].
The application of these principles to the microscopy field required 20
more years. Even if the original idea of generating 3D microscopy images
by means of non-linear effects was first suggested and attempted in the
1970s by Sheppard, Kompfner, Gannaway and Choudhury of the Oxford
group [44,45], the practical realization of a 'two-photon' microscope is related
to the pioneering work of W. Denk in W.W. Webb Laboratories (Cornell
University, Ithaca, NY), which was responsible for spreading the technique
that revolutionized fluorescence microscopy imaging [6].
4.3 Principles of Confocal and Two-Photon Fluorescence
Microscopy
4.3.1 Fluorescence
The term fluorescence is related to the capability of certain molecules to emit
light (in a time scale of 10
−
9
s) when they are illuminated with a proper
wavelength. More precisely, the energy required to prime fluorescence is the
energy that is necessary to produce a molecular transition to an electronic
excited state [46,47]. In other words, if
λ
is the wavelength of the light delivered
on the sample, the energy provided by photons
E
=
hc/λ
(where
h
=6
.
6
×
10
−
34
J s is the Plank's constant and
c
=3
10
8
ms
−
1
is the speed of light)
should be equal to the molecular energy gap ∆
E
g
between the ground state
and one vibrational or rotational level of the electronic excited state:
×
∆
E
g
=
E
=
hc/λ.
(4.1)
Once the molecule has adsorbed the photon, it has several pathways for
relaxing back to the ground state, including non-radiative phenomena, phos-
phorescence (associated to the forbidden transition to the triplet state) and
fluorescence (Fig. 4.1). In fluorescence the internal conversion from the lower
vibrational level of the excited state is not associated with light emission while
the relaxation to the ground state is achieved by emitting one photon. For this
reason, the fluorescence emission is generally shifted towards a longer wave-
length than the one used for exciting the molecule. This phenomenon is known
as Stokes' shift and it ranges from 50 to 200 nm depending on the fluorescent
molecule in consideration. Conventional imaging techniques use ultraviolet or
visible light for the excitation. In multi-photon excitation the jump between
the ground state and the excited one is due to the simultaneous absorption of
two or more photons [5]. As the sum of the energy of the absorbed photons
