Coherent Raman scattering (CRS) microscopy is gaining acceptance as a valuable addition to the imaging toolset of biological researchers. Optimal use of this label-free imaging technique benefits from a basic understanding of the physical principles and technical merits of the CRS microscope. This tutorial offers qualitative explanations of the principles behind CRS microscopy and provides information about the applicability of this nonlinear optical imaging approach for biological research.
The nonlinear optical interference of two successively generated coherent anti-Stokes Raman scattering (CARS) signals from two different samples placed in series is demonstrated for the imaging performance, in which a collinear phase matching geometry is used. The relative phase of two CARS signals is controlled by a phase-shifting unit made of dispersive glass materials of which the thickness can be precisely varied. The clear interference fringes are observed as the thickness of the phase-shifting unit changes. The interference effect is then utilized to achieve a better quality CARS image of a biological tissue taken from a mouse skin. Placing the tissue in the second sample position and performing raster scans of the laser beams on it, we can acquire a CARS image of higher contrast compared to the normal image obtained without interferometric implementation.
Nonlinear interference of two successively generated coherent anti-Stokes Raman scattering (CARS) signals from thin glass slabs is demonstrated, in which a collinear phase matching geometry is tried. We used 76 MHz mode-locked Nd:YAG pulsed laser at 1064 nm and its frequency-doubled optical parametric down-converted signal at 817.2 nm as Stokes and pump beams, respectively. The pulse duration time is around 7 ps for both incident laser beams. The relative phase of two CARS signals is controlled by a phase shifting element made of dispersive glass material of which thickness can be varied. The clear interference fringes are observed as the thickness of the phase shifting element changes. The interference effect is utilized to achieve better CARS image quality. We first try imaging the polystyrene beads immersed in water to estimate how the nonlinear interference could improve the contrast of the beads image. Performing the raster scan of the laser beams on the sample, we can get the CARS image and investigate the image quality as a function of the relative phase and amplitude.
We have demonstrated a new design of a four-pass dye laser amplifier that can suppress parasitic oscillation in a multi-pass amplifier. Amplified spontaneous emission (ASE) in the output beam of the amplifier is reduced with a diffraction grating. A high-peak power pulsed dye laser beam is obtained by amplifying a continuous-wave narrow-bandwidth dye laser with power gain greater than 2 X 106. The eventual bandwidth of the pulses is measured to be approximately 130 MHz. The obtained ASE ratio of the four- pass amplifier system is < 1.5%. Also, we have obtained a high-resolution coherent anti-Stoke Raman spectrum of oxygen in air using the laser system.
Using 4-pass dye amplifier pumped by a frequency doubled Nd:YAG laser, we obtained high peak power pulsed dye laser beam with good efficiency of 42% at total pumping power 77 mJ. The bandwidth of 120 MHz was measured.
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