Multiphoton Imaging - Biomedical Optical Imaging Technologies: Design and Applications - page 244

Fig. 7.3 Optical setup of the multiphoton microscope. M mirror, DBS dichroic beam splitter, NDF
neutral density filter, F filter, and PMT photomultiplier tube
pulsed laser such as a Ti:sapphire laser is used for excitation of nonlinear signal.
A neutral density filter (NDF) is used to control the intensity of the laser beam. The
laser beam is then expanded using a spatial filter and beam expander optics in order
to fill the entrance aperture of the microscope objective. Some part of the laser beam
is directed toward an autocorrelator for beam diagnostics such as measurement of
pulse width of the femtosecond laser. The beam is directed toward the scanning
mirrors that are mounted in a box with their electronic drive units. A set of two
lenses is used to deliver the laser beam from the scanning mirrors to the microscope
objective. These two lenses form what is known as a telecentric optical system. The
idea of telecentricity is to keep the scanning beam stationary at the entrance pupil of
the microscope objective. One of these lenses is called the scan lens that is placed
close to the scan mirrors, and the other lens, known as the tube lens, is placed before
the objective. The tube lens is designed to correct for aberrations of the objective;
thus, it is important to match the lens and objective from the same manufacturer. A
dichroic beam splitter (DBS) is used for the collection of backscattered light from
the sample. The laser beam transmits through the DBS and through the microscope
objective and illuminates the sample. The backscattered light is collected with the
same objective and is now reflected by the DBS that is optimized for transmission of
infrared light and reflection of shorter wavelengths (that depends on the excitation
wavelength). The light reflected from the DBS is passed through an infrared filter
to block any scattered laser light. A set of two lenses is used to collect the signal
coming from the sample and to image the pupil plane of the objective onto the