Multiphoton Imaging - Biomedical Optical Imaging Technologies: Design and Applications - page 242

pulsed lasers are being used for multiphoton microscopy due to their high peak
intensities.
A femtosecond laser emits optical pulses with a duration of a few femtoseconds
(1 fs D 10 15 s) that are also termed as ultrashort pulses. The generation of such
ultrashort pulses is achieved with a technique called mode locking, and the lasers
are then termed as mode-locked lasers. The laser resonator contains either an active
element (an optical modulator) or a nonlinear passive element (a saturable absorber),
which causes the formation of an ultrashort pulse circulating in the laser resonator.
Under the steady state operation, the various effects influencing the pulse are in a
balance so that the pulse parameters are unchanged after each completed round-
trip. Each time the pulse hits the output coupler mirror of the laser, a pulse is
emitted. Assuming a single circulating pulse, the pulse repetition period corresponds
to the resonator round-trip time (typically several nanoseconds), whereas the pulse
duration is much lower, from a few femtoseconds to picoseconds range. Due to very
short pulse width, the peak power of a mode-locked laser can be orders of magnitude
higher than the average power.
Most widely used and current industrial standard for multiphoton microscopy is
the femtosecond pulsed Ti:sapphire laser. The wavelength range is only limited by
the bandwidth of the Ti:sapphire gain medium to between approximately 690 and
1,070 nm. This wavelength range is also termed as a therapeutic window, since it
can penetrate deep into biological tissue without causing significant photodamage
and is not absorbed by water which may result in heating. For THG microscopy, a
Ti:sapphire laser seems to be ideal, but strong absorption of the signal generated
in the UV region limits its application to thin biological specimens. To avoid
UV absorption of the generated THG signal in thick biological samples, THG
microscopy is done with wavelengths longer than 1,200 nm. Cr:forsterite laser at
excitation wavelength of 1,230 nm and repetition rate of 110 MHz is widely used
for THG microscopy [ 45 , 61 ]. Other lasers used for THG microscopy include optical
parametric oscillator (OPO) working at a wavelength of 1,500 nm and repetition rate
of 80 MHz, synchronously pumped by a femtosecond Ti:sapphire laser [ 15 , 62 ]; an
optical parametric amplifier (OPA) at 1,200-nm and 250-kHz repetition rate pumped
by a Ti:sapphire laser [ 50 , 51 ]; and a fiber laser at 1,560 nm with a repetition rate of
50 MHz [ 63 ].
Some new laser sources being used for multiphoton microscopy are Yb:glass,
Nd:glass, Cr:LiSAF, and fiber lasers. Femtosecond fiber lasers are the next genera-
tion of compact laser systems that are being used for multiphoton imaging purposes
due to their small footprint as compared with their counterpart femtosecond lasers
that are usually very bulky and require external cooling systems. The only drawback
of a fiber laser is its lack of wavelength tuning ability that is the hallmark of a
femtosecond laser like Ti:sapphire laser. Fiber lasers have a number of qualities
which make them attractive for ultrashort pulse generation via active or passive
mode locking. The gain bandwidth of rare-earth-doped fibers is quite large, typically
tens of nanometers that allows the generation of femtosecond pulses. The high
gain efficiency of active fibers makes it possible to operate such lasers with fairly
low pump powers and to allow intracavity optical elements with relatively high