Biomedical Engineering Reference
In-Depth Information
(a)
(b)
1.0
1.0
I
(
r, z
)
2
I
(
r, z
)
0.8
0.8
0.6
0.6
0.4
0.4
ω
r
ω
z
0.2
0.2
z
0.0
0.0
r
-1000 -500
0
500
1000
-4000 -2000
0
2000
4000
r
(nm)
z
(nm)
(c)
0.320
λ
0.532
λ
1
NA
≤
0.7
ω
z
=
2
NA
2
n -
n
2
- NA
2
ω
r
0.325
λ
NA
>
0.7
2
NA
0.91
FIgurE 2.8
(a) Axial and later views of intensity point spread function (
I
(
r
,
z
) in black) in the excitation volume and
squared point spread function (
I
(
r
,
z
)
2
in gray). Squaring the
I
(
r
,
z
) results in minimal wings relative to center. (b) Radial
(on the left) and axial (on the right) profiles of
I
(
r
,
z
) (black line) and
I
(
r
,
z
)
2
(gray line). Both axial and lateral PSFs are
intended for two-photon fluorescence and give the best approximation for the PSFs associated with SHG imaging. The
simulated profiles have been obtained using the following parameters:
NA
= 1, λ = 800 nm,
n
= 1.33 (water).
Spatial resolution is not the only feature to be considered when choosing the excitation objective.
Several specifications have to be taken into account when choosing an objective lens:
•
Numerical aperture:
As a general rule, the NA of the excitation objective should not be <0.5, in
order to provide a photon density in the focus sufficient for two-photon excitation of the speci-
men. Higher NA objectives are preferable, since they increase both microscope spatial resolution
and detected signal intensity (i.e., signal to noise). As a drawback, high-NA objectives correspond
in general to higher magnification and, hence, to smaller fields of view. Furthermore, high-NA
objectives require oil or water immersion. They could also introduce additional spurious effects
in the polarization when using a polarization-scanning equipment, because of the phenomenon
of scrambling occurring with very high NAs.
•
Objective transmission:
The spectral range commonly used in an SHG microscope is in the near
infrared. The excitation objective should have a good transmittance in the excitation wavelength
range used (typically 700-1000 nm).
•
Working distance:
Very often high NA objectives have working distances limited to 100-200 μm,
imposing a limit on deep imaging inside a biological tissue. The working distance of the objective
should be chosen according to the morphology of the sample to be investigated. For example, for
in vivo
deep tissue imaging, long working distances are required. A water immersion objective
with NA ranging from 0.8 to 1 and a working distance ranging from 2 to 3 mm is a good solution
for this purpose.
•
Back aperture:
The size of the objective back aperture has to be chosen carefully and considering
the dimension of the exciting laser beam. In fact, it is very important that the excitation beam
slightly overfills the objective back aperture. In this way, all the NA of the objective can be used,
maximizing the resolution achievable with the objective (Figure 2.9a). The focal spot size criti-
cally depends on the ratio of the beam size and the objective back aperture, as shown in the insets
of Figure 2.9. On the other hand, while overfilling guarantees the NA of the objective, it also
reduces transmitted power.
