Biomedical Engineering Reference
In-Depth Information
effective PSF can be reduced by a factor of 3 instead of 2 used for SHG (Squier and Muller 2001). The
lateral resolution can be given by
1
3
0.51
λ
0.3
λ
,
r
=
⋅
≈
(14.10)
NA
NA
and the axial resolution can be given by
1
3
n
NA
λ
0.57
n
NA
λ
.
d
z
=
⋅
≈
(14.11)
2
2
Again, the restriction of THG excitation can both help to increase the spatial resolution and reduce
the out-of-focus photodamage. The resolution of THG microscopy is higher than with either confocal or
SHG microscopy due to its higher-order nonlinearity. The virtual optical sectioning of bulky tissues can
be easily achieved by making use of this high spatial resolution. A comparison of the 3D spatial resolu-
tion and excitation properties among conventional, confocal, SHG, and THG microscopy is shown in
Table 14.1.
High 3D resolution is required to provide cellular or subcellular information for performing virtual
optical sectioning of bio-tissues. Using a Cr:F laser for excitation and a high-NA objective for tight
focusing, submicron lateral and axial resolution can be theoretically achieved with both SHG and THG
microscopy (Table 14.1). In practice, the spatial resolution depends not only on the wavelength and NA,
but also the spatial mode of the laser beam and the system alignment. With the optimized system align-
ment, a submicron 3D resolution can be achieved by our HGM system, for performing depth-resolved
optical sectioning, and 3D images can be reconstructed from the obtained stack of HGM images versus
TABLE 14.1
Comparisons of Some Important Properties of Conventional Microscopy, Confocal Microscopy,
SHG Microscopy, and THG Microscopy
Conventional
Microscopy
Confocal Microscopy
SHG Microscopy
THG Microscopy
Order of optical process
Linear
Linear
Second-order
nonlinearity
Third-order
nonlinearity
Illumination
Wide-field
Point (scanning)
Point (scanning)
Point (scanning)
Out-of-focus excitation
Yes
Yes
No
No
Spatial filter
No
Confocal pinhole
Localized
excitation
Localized
excitation
Lateral resolution
0 6.
λ
NA
0 3.
λ
NA
0 3.
λ
NA
0 .
λ
NA
Axial resolution
0 64
2
.
λ
0 7
.
nλ
NA
0 57
.
nλ
NA
nλ
NA
2
2
2
2
n
−
n
−
NA
Optical sectioning of
bulky tissues
No
Yes
Yes
Yes
Out-of-focus
photodamages
Yes
Yes
No
No
Energy deposition
Exists for single-
photon fluorescence
Exists for single-photon
fluorescence
No
No
Level transition
Real-level transition
for single-photon
fluorescence
Real-level transition for
single-photon
fluorescence
Virtual-level
transition
Virtual-level
transition
