Multimodal Nonlinear Optical Microscopy

Coherent anti-Stokes Raman Scattering (CARS), multi-photon excited (MPE) fluorescence, and second/third harmonic generation (SHG/THG) microscopy.

Layered structure depth profile images of liquid crystal samples between two cover slips obtained by the three photon excited fluorescence technique.

Experimental setup for the multimodal nonlinear optical microscopy of multiphoton excitation fluorescence (MPEF) and second harmonic generation (SHG) imaging. A femtosecond pulse from a tunable Ti:Sapphire oscillator (680~1080nm, 140fs, 80MHz, Chameleon Ultra II, Coherent Inc.) is introduced into a confocal laser scanning unit (Fluoview FV-300, Olympus Inc.) and focused onto a sample with a high NA objective lens of an inverted microscope (IX-81, Olympus Inc.). The half wave plate and the Glan-laser polarizer allow control of power and polarization. Two or three photon excitation fluorescence, second harmonic signals and transmission light can be collected by three different detection channels with photon multiplier tubes (H5784-20, Hamamatsu), i.e. forward, backward, or transmission detection systems. The polarization at sample position can be controlled by using a ferroelectric liquid crystal polarization rotator, which changes polarization direction by applying a different voltage. [CL: collecting lens, DM: dichroic mirror, FLCPR: ferroelectric liquid crystal polarization rotator, FM: flip mirror, GLP: Glan laser polarizer, HWP: broadband half wave plate, LPF: long pass filter, M: mirror, OL: objective lens, RP: rotating polarizer, SPF: short pass filter]

Experimental setup for coherent anti-Stokes Raman scattering polarizing microscopy (CARS-PM). A femtosecond pulse from a Ti:Sapphire oscillator (Chameleon Ultra-II, Coherent Inc.) is divided into two pulses after a prism-pair pulse compressor, one of which generates supercontinuum for a Stokes pulse using a photonic crystal fiber module (Femtowhite-800, NKT photonics) and the other generates pump/probe pulses. A Faraday isolator protects the Ti:Sapphire laser from the back-reflection from the PCF and the half wave plate and the Glan-laser polarizer allows control of laser power and polarization. After spatially and temporally recombining the pump and Stokes pulses by a long pass filter and two delay lines, they are introduced into the confocal laser scanning unit (Fluoview FV-300, Olympus Inc.). Both the pulses are focused onto the sample with a high NA objective lens of an inverted microscope (IX-81, Olympus Inc.). CARS signals and transmitted light can be collected by three different detection channels with photon multiplier tubes (H5784-20, Hamamatsu), i.e., forward CARS (F-CARS), backward CARS (Epi-CARS), or transmission detectors. The polarization at the sample position can be controlled by using a ferroelectric liquid crystal polarization rotator, which changes polarization direction by applying a different voltage. [AL: achromatic lenses, BPF: band pass filter, BS: beam splitter, CL: collecting lens, DM: dichroic mirror, FI: Faraday Isolator, FLCPR: ferroelectric liquid crystal polarization rotator, GLP: Glan laser polarizer, HWP: broadband half wave plate, LPF: long pass filter, OL: objective lens, PCF: photonic crystal fiber, RP: rotating polarizer, SPF: short pass filter]

Taewoo Lee and Rahul Trivedi aligning optical beam paths from a femtosecond Ti:Sapphire laser for CARS imaging.
 

For more information about Multimodal Nonlinear Optical Microscopy, contact Ivan Smalyukh.

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