Biomedical Engineering Reference
In-Depth Information
(Guo et al. 1996, 1999; Campagnola et al. 1999; Moreaux et al. 2000b; Brown et al. 2003). At the same
time, the broad tuning range of 700-1000 nm covers the range of efficient two-photon excitation of most
endogenous fluorophores in bio-tissues. Thus, Ti:S lasers have been adopted for various 2PF microscopic
applications (Moreaux et al. 2000a; Campagnola et al. 2002; Zoumi et al. 2002). However, since THG
radiation has a higher frequency (3ω) and a shorter wavelength (
λ
/3) than SHG radiation (2ω;
λ
/2) due
to its higher nonlinearity, pulse lasers with longer wavelengths than that used in SHG microscopy are
more appropriate and often used for excitation. For instance, Yelin et al. used a Ti:S laser with a wave-
length of 800 nm for THG microscopy (Yelin et al. 2002). The generated THG at 267 nm fell within
the deep UV region and suffered strong absorption and scattering in the bio-tissues. In 1996, Alfano
(Guo et al. 1996) applied a Nd:YAG laser with an emission wavelength of 1064 nm for THG excitation
in chicken tissues. THG signals at 354 nm were found in the chicken skin. To shift the wavelength of
THG to the visible region so as to avoid serious absorption, lasers with longer wavelengths, including an
optical parametric amplifier (OPA) (wavelength: 1200 nm; repetition rate: 250 KHz) pumped by a Ti:S
amplifier (Muller et al. 1998; Squier et al. 1998; Debarre et al. 2006), a Cr:F laser at 1230 nm with a rep-
etition rate of 110 MHz (Chu et al. 2001, 2003; Sun et al. 2003; Yu et al. 2007; Lee et al. 2009), an optical
parametric oscillator (OPO) (wavelength: 1500 nm; repetition rate: 80 MHz) synchronously pumped by
a Ti:S laser (Yelin and Silberberg 1999; Canioni et al. 2001), and a fiber laser at 1560 nm with a repetition
rate of 50 MHz (Millard et al. 1999). Under the excitation of these lasers, the generated THG signals all
fall within the visible range of 400-520 nm and showed reduced absorption and scattering in the bio-
tissues. However, in addition to the attenuation of the THG signals, the attenuation of the excitation
laser radiation in bio-tissues should also be taken into consideration since it can lead to the degradation
of the excitation intensity, decreasing the penetrability and absorption-induced photodamage.
In an earlier study, Anderson and Parish measured the curves of both the scattering and absorp-
tion constant of human skin (Anderson and Parish 1981). The attenuation (combination of scattering
and absorption) was found to reach a minimum value around 1200-1300 mm, which is the so-called
penetration window, while the attenuation demonstrated a serious increase with increasing wavelength
(>1300 nm) due to strong water absorption. This indicates that lasers with wavelengths above 1300 nm
may suffer higher attenuation and cannot effectively help to improve the penetrability in human skin.
Therefore, a femtosecond Cr:F laser with a wavelength of 1230 nm, well within the penetration window,
could be an optimal laser source for reducing the attenuation of both THG signals and laser radiation
to increase the imaging penetrability. Using the Cr:F laser for the excitation of combined SHG/THG
microscopy, both the wavelengths of SHG (615 nm) and THG (410 nm) fall within the visible range (Sun
et al. 2004), which also makes signal detection much easier.
14.1.2 Reduction of Photodamage
Using a high-intensity femtosecond laser source for excitation, the issue of photodamage induced by
multiphoton absorption has to be seriously taken into consideration. It is important to reduce the photo-
damage in bio-tissues, especially for clinical trials. However, if the maximum applicable light intensity is
limited to reduce photodamage, the signal intensity and penetrability can also be compromised. For the
in vivo
Ti:S-based (730-960 nm) 2PF techniques (Konig 2008), an average power of 30 mW was needed
for imaging up to a depth of 100 μm. The irradiation of living cells with 730-800 nm beams of >1 mW
average power (total exposure per cell = 0.2 J) was found to inhibit cloning efficiency (Konig et al. 1997).
Excitation energy levels much higher than 1 mW can be assumed to be invasive. Besides, even when
using a light intensity as high as 30 mW, the imaging depth of the Ti:S-based 2PF techniques is limited
to 200 μm, meaning that Ti:S lasers do not meet the requirements for clinical trials. In a previous study
of mammalian embryos, Squirrell et al. moved the excitation wavelength for 2PF microscopy to 1047 nm
(Squirrell et al. 1999). The imaged embryos were found to maintain their viability through time-lapsed
observations (five optical scanning sections collected every 15 min) with a 13 mW average power and 2 J
total exposure. In previous studies of mouse embryos (Hsieh et al. 2008; Chen et al. 2010), as the Cr:F
