Multiphoton Imaging - Biomedical Optical Imaging Technologies: Design and Applications - page 245

detectors. This is done so that the scanning beam remains stationary at the detector.
Another dichroic beam splitter is used to separate the fluorescence and SHG signal
simultaneously. The transmitted signal is passed through an arrow band pass filter
to select the fluorescence, and reflected light is passed through a narrow band pass
filter to select the second harmonic signal. The two signals are detected by a set of
photomultiplier tubes (PMT) for their fast response and high sensitivity. The PMT
signal is converted into a voltage signal using transimpedance amplifiers and fed into
the data acquisition board in a computer. The pixel and line clock signals are used
to synchronize the data acquisition with the scanner system. A fast data acquisition
card is used to receive the pulses simultaneously from the two detectors and is
synchronized with the reading of x and y positions from the scanning mirrors. This
renders the two simultaneously acquired images with different nonlinear contrast
mechanisms. For obtaining optical sections at different depths, the microscope
objective is translated along the optical axis with a piezo scanner.
The setup of multiphoton nonlinear microscope can be divided into four main
functional units: the femtosecond laser source, beam scanning unit, the optical
microscope, and the detection system synchronized with the laser beam scanning.
In the following sections, these units are described in detail.

7.4.2 Optical Parts

A fiber laser was used to excite the SHG signal in the samples. The IMRA fiber
laser model Femtolite FX-100 is based on a passively mode-locked Er-doped fiber
oscillator followed by an Er-doped fiber amplifier. The laser produces 110 fs pulses
at a rate of 75 MHz and average power of 100 mW at a fixed wavelength of 800 nm.
This laser is suitable in biomedical imaging systems for its compact design and
turnkey solution. The laser beam was monitored with an autocorrelator (model
AA-110D, Del Mar Photonics) to measure its pulse width. A neutral density filter
(CSND-1-50.0M, CVI Melles Griot) was used to control the laser beam power. A
Faraday isolator (ISO-04-780-MP-W, Newport Corp.) was used to block any back
reflections into the laser. The mirrors used for laser beam delivery are BBDS-PM-
1037-C (CVI Melles Griot).
The beam was expanded with a five axis spatial filter set (model 910A, along
with a pinhole: 910PH-20 and objective lens: M10x, from Newport Corp.).The laser
beam goes into a scanner consisting of a resonant scan mirror and a galvo-mirror
set. The fast scanner is a resonant scanning mirror (CRS 8 KHz, General Scanning)
oscillating at 8 kHz, and the slow scanning mirror is at 30 Hz (VM500PLUS,
General Scanning). Combination of these two mirror sets allows to achieve a scan
rate of 30 frames per second at 512 512 pixel image and 15 frames per second at
1; 024 1; 024 pixel image.
A scan lens, in this case, a 10 wide-field microscope eyepiece (NT36-130,
Edmund Optics) and a tube lens (PLCX-50.0-77.3-UV-800, CVI Melles Griot),
are used in a telecentric arrangement between the scan mirrors and the microscope