Biomedical Engineering Reference
In-Depth Information
laser (1230 nm) was used for excitation, similar survival rates were found in nonimaged embryos versus
embryos under 10 min continuous observation with >120 mW average power and >21.6 J total exposure
per embryo. The total number of tested embryos was 146, which is greater than the minimum of 30, and is
enough to provide statistical significance for this study. Compared with the Ti:S and 1047 nm excitation,
the maximum tolerance of 140 mW excitation power is much higher than 1 mW for Ti:S and 13 mW for
1047 nm, and this indicates much reduced photodamage under the Cr:F excitation due to much reduced
multiphoton absorption with a lower excitation photon energy (1230 nm; 1.01 eV) (Chen et al. 2002).
In addition to the average power of the laser radiation, the pulse energy and peak intensity of a laser
pulse are also determined by the repetition rate and the pulse duration (Equations 14.1 and 14.2)
P
peak
=
energy per pulse
pulse duration
(14.1)
P
= ×
ion rate of laser pulses
energy per pulse
number of pulses per second
ave
(14.2)
(repetit
=
1/number of pulses per second).
To maintain cell vitality, the repetition rate and the pulse duration should be carefully controlled to
reduce the pulse energy and peak intensity to a safe level. Increasing the repetition rate can decrease the
pulse energy, while simultaneously decreasing pulse width can help retrieve enough peak intensity for suf-
ficient excitation of nonlinear signals. In addition, if the peak intensity is consistent with the safety require-
ment, it may limit the signal intensity, penetrability, and the frame rate of the imaging. This problem can
also be solved by increasing the repetition rate, since under the same scanning speed, a single point on a
sample can be excited more times and more signals can be collected and integrated to increase the signal
intensity. This is different from the fluorescence-based technique, where a relaxation time is required for
the excited upper-state elections to return to the ground state. There is no relaxation time restricting the
selection of the repetition rate for virtual-transition harmonic generation processes. We chose a Cr:F laser
with a repetition rate of 110 MHz and pulse duration of 140 fs for maintaining both the excitation effi-
ciency and cell vitality of combined SHG/THG microscopy. A compact fiber pumped femtosecond Cr:F
laser with more than 1 GHz repetition rate and 500 mW average laser power has been demonstrated (Liu
et al. 2005) to a suitable laser source for nonlinear optical endoscopy (Chan et al. 2005).
The Cr:F laser is applied with the 110 MHz repetition rate and 140 fs pulse duration for the excitation
of combined SHG/THG microscopy. The noninvasiveness of this imaging system is also tested in
in vivo
studies of various animal models in addition to the viability test of mammalian embryos. Previously,
in Cr:F-based SHG/THG imaging of zebrafish embryos (Sun et al. 2004; Chen et al. 2006), the zebra-
fish embryonic brain development has been continuously observed in the same embryo. After 20 h of
nonstop continuous imaging (100-140 mW, >7000 J exposure per embryo), all observed embryos were
shown to have developed normally to their larval stages. In a previous
in vivo
SHG/THG virtual biopsy
study of Syrian hamster oral mucosa (Tai et al. 2006), after 3 h of continuous observation of the same
area (150 mW, 1620 J), the observed hamster buccal tissues were then excised immediately for patho-
logical examination. There was no evidence of coagulation necrosis in the buccal squamous epithelium
and no subepithelial stroma appeared under examination of all the studied animals. All the results of
previous
in vivo
studies strongly indicate that the harmonic generation microscopy (HGM) system is
noninvasiveness in nature and can satisfy the safety requirements for clinical trials.
14.1.3 improvement of Penetrability
The reduction of the attenuation of both excitation irradiation and generated harmonic generation sig-
nals should improve the penetrability of the Cr:F-based SHG/THG microscopy in bio-tissues. In a work
of comparing the penetration depth between Ti:S-based and Cr:F-based SHG microscopy (Yasui et al.
