Nanoparticles for in-vitro and in-vivo biosensing and imaging

48 Principles
ω xy = 0.320λ √
2NA
ifNA ≤ 0.7
ω xy = 0.325λ √
2NA 0.91
ifNA ≻ 0.7
ω z = 0.532λ [
]
1
√
2 n − √ n 2 − NA 2
(2.24)
(2.25)
On the other hand, by integrating the Gaussian Lorentzian beam we obtain V G−L
T P E
= πω 4 0 /λ ∼ = 0.113µm 3 for a 1.2 NA lens at λ = 900nm [32].
2.4.2 OPE versus TPE
For optical sectioning, i.e. for microscopy applications, the most important consequence
of two-photon excitation, TPE, or non-linear excitation in general, is the fact that the
molecular excitation is limited to a sub-femtoliter region around the focal plane. While
in one-photon excitation, OPE, the excitation volume is not confined in the focal plane
(see Figure 2.10).
Figure 2.10: Excited volumes by two photon laser source of λ= 760nm (upper) and by one photon laser
source λ= 380nm (lower) in fluorescein solution.
Under OPE the optical sectioning is obtained by spatial filtering the emission in front
of the detector in the so called confocal detection scheme. The rejection of out of focus
plane is achieved by placing a screen with a pinhole in front of the detector. The focal
point of the objective lens forms an image onto the pinhole screen: the specimen plane
and the pinhole screen are conjugated planes (and hence the name confocal). The ability
of a confocal microscope to create sharp optical sections makes it possible to build 3D