Multiphoton Imaging - Biomedical Optical Imaging Technologies: Design and Applications - page 243

optical losses. For example, certain optical filters or arrangements for dispersion
compensation could not be used in bulk lasers. Fiber lasers can be fabricated
with low cost and can be very compact and rugged. Mode-locked fiber lasers
are commonly based on telecommunication components, which have been very
carefully developed for reliable long-term operation and have a moderate cost.
The performance of picosecond and femtosecond mode-locked fiber lasers and
amplifiers in terms of pulse energy, peak power, and pulse quality can be severely
limited by the strong nonlinearities of fibers and sometimes also by the chromatic
dispersion. Other problems can result from uncontrolled birefringence of fibers, if
they are not polarization-maintaining.

7.4 A Multiphoton Imaging System Design

A multiphoton microscope has many common features with a confocal laser
scanning microscope. Multiphoton excitation system can be developed by coupling
a femtosecond laser into a confocal scanning microscope and using a nondescanned
port for efficient detection of the nonlinear signal [ 64 ]. The functionality of
multiphoton microscopes can be enhanced by implementing efficient detection
schemes to allow different signal detection in the transmission and/or excitation
direction. In this section, system design of a multiphoton laser scanning microscope
is described, and various components of the system are discussed.
A customized multiphoton microscope system is described that can be used
to image biological tissue in reflection mode. Such a microscope is suitable for
thick tissue and in vivo imaging in biomedical applications. For simultaneous
detection of second harmonic and fluorescence signal, two channels of detection are
used. The emission signal is divided into different spectral ranges by appropriate
dichroic mirrors and optical filters. Similarly, spectral separation can be applied
for detecting second and third harmonic signals along with fluorescence. Backscat-
tered coherently generated harmonics are usually much weaker than the forward
generated signals; therefore, forward-detection of harmonics can be accomplished
with lower excitation intensities. The detection in transmission requires a more
extensive modification of the scanning microscope setup. The easiest way of
building a transmission mode harmonic generation microscope is by using a high
NA condenser that is available on some commercial microscope models [ 64 , 65 ].
The design and instrumentation of nonlinear microscopes have been extensively
reviewed [ 66 - 68 ].

7.4.1 System Design

Optical design of a multiphoton microscope capable of TPEF, SHG, and THG
detection in reflection mode is shown in Fig. 7.3 . A high repetition rate femtosecond