Multiphoton Imaging - Biomedical Optical Imaging Technologies: Design and Applications - page 235

Fig. 7.2 Optical setup of a typical multiphoton imaging microscope. A femtosecond pulsed laser
beam is scanned onto the sample, and signal is detected in the forward and/or backward direction
in various modes of the microscope. The terms used for the optical components are M mirror,
NDF neutral density filter, DBS dichroic beam splitter, F filters, L lens, and PMT photomultiplier
tube

The light source is usually a femtosecond pulsed laser in the near infrared
spectrum to excite most of the ultraviolet to visible light-absorbing dyes and
fluorophores. The resonant processes of SHG, THG, and CARS are also excited
by such a laser. The commonly used multiphoton wavelengths are in the range of
700-1,300 nm that are applicable in the biomedical field. This wavelength range
is used for its long range penetration in the tissue, less scattering, and absorption
by the water molecules, therefore, resulting in negligible heating of the tissue.
In the following subsections, TPEF, SHG, THG, and CARS microscopy methods
are described.

7.3.1 Two-Photon Excited Fluorescence (TPEF)

TPEF is by far the most commonly used multiphoton excitation process, in which
a molecule simultaneously absorbs two photons in a single quantum event [ 6 ]. The
combined energy of the two photons allows the use of lasers in the near infrared
spectrum to excite dyes and fluorophores. The fluorescence emission induced by this