Enabling the detection of UV signal in multimodal nonlinear microscopy with catalogue lens components.

Using an optical system made from fused silica catalogue optical components, third-order nonlinear microscopy has been enabled on conventional Ti:sapphire laser-based multiphoton microscopy setups. The optical system is designed using two lens groups with straightforward adaptation to other microscope stands when one of the lens groups is exchanged. Within the theoretical design, the optical system collects and transmits light with wavelengths between the near ultraviolet and the near infrared from an object field of at least 1 mm in diameter within a resulting numerical aperture of up to 0.56. The numerical aperture can be controlled with a variable aperture stop between the two lens groups of the condenser. We demonstrate this new detection capability in third harmonic generation imaging experiments at the harmonic wavelength of ∼300 nm and in multimodal nonlinear optical imaging experiments using third-order sum frequency generation and coherent anti-Stokes Raman scattering microscopy so that the wavelengths of the detected signals range from ∼300 nm to ∼660 nm.

Dispersion of the resonant nonlinear optical susceptibility obtained with femtosecond time-domain coherent anti-Stokes Raman scattering.

We propose and experimentally demonstrate a method that is capable of resolving both real and imaginary parts of third-order nonlinearity (χ(3)) in the vicinity of Raman resonances. Dispersion of χ(3) can be obtained from a medium probed within microscopic volumes with a spectral resolution of better than 0.10 cm(-1).

Three-dimensional nonlinear microspectroscopy and imaging of soft condensed matter.

We report on the realization of a sensitive microspectroscopy and imaging approach based on a three-color femtosecond coherent anti-Stokes Raman scattering (CARS) technique with high spectral, time, and spatial resolution. Independently tunable, high-repetition rate optical parametric oscillators were used to attain a dynamic range of 5 orders of magnitude for time-domain CARS signal. The attained sensitivity permitted tracing the decay of weak and structurally complex Raman active modes in soft condensed matter. Application of this approach to imaging of the biological specimen shows a great potential in quantitative characterization of live biological media with an ability to access inter- and intra-molecular interactions.

Imaging skeletal muscle using second harmonic generation and coherent anti-Stokes Raman scattering microscopy.

We describe experimental results on label free imaging of striated skeletal muscle using second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy. The complementarity of the SHG and CARS data makes it possible to clearly identify the main sarcomere sub-structures such as actin, myosin, acto-myosin, and the intact T-tubular system as it emanates from the sarcolemma. Owing to sub-micron spatial resolution and the high sensitivity of the CARS microscopy technique we were able to resolve individual myofibrils. In addition, key organelles such as mitochondria, cell nuclei and their structural constituents were observed revealing the entire structure of the muscle functional units. There is a noticeable difference in the CARS response of the muscle structure within actin, myosin and t-tubule areas with respect to laser polarization. We attribute this to a preferential alignment of the probed molecular bonds along certain directions. The combined CARS and SHG microscopy approach yields more extensive and complementary information and has a potential to become an indispensable method for live skeletal muscle characterization.

Femtosecond optical parametric oscillator based on periodically poled stoichiometric LiTaO3 crystal.

Efficient optical parametric oscillation is demonstrated in periodically poled stoichiometric lithium tantalate crystal pumped by a mode-locked Ti:sapphire laser. The optical parametric oscillator (OPO) delivers a maximum average power of more than 345 mW in signal and 180 mW in idler beams. The OPO is continuously tunable across the 940-1350 nm wavelength range in its signal branch, delivering nearly transform-limited 160-180 fs pulses. Despite the onset of high absorption loss in the crystal in the mid-IR, more than 40 mW of power is obtained for the idler beam tunable within the 4.3-4.6 microm wavelength range. The high parametric gain in the crystal allows tunable OPO operation at a repetition rate as high as 760 MHz with 50 mW of output power.

Multimodal nonlinear optical imaging of collagen arrays.

We report multimodal nonlinear optical imaging of fascia, a rich collagen type I sheath around internal organs and muscle. We show that second harmonic generation (SHG), third harmonic generation (THG) and Coherent anti-Stokes Raman scattering (CARS) microscopy techniques provide complementary information about the sub-micron architecture of collagen arrays. Forward direction SHG microscopy reveals the fibrillar arrangement of collagen type I structures as the main matrix component of fascia. SHG images detected in the backward direction as well as images of forward direction CARS microscopy show that the longitudinal collagen fiber bundles are further arranged in sheet-like bands. Forward-THG microscopy reveals the optically homogeneous content of the collagen sheet on a spatial scale of the optical wavelength. This is supported by the fact that the third harmonic signal is observed only at the boundaries between the sheets as well as by the CARS data obtained in both directions. The observations made with THG and CARS microscopy are explained using atomic force microscopy images.

High-sensitivity vibrational imaging with frequency modulation coherent anti-Stokes Raman scattering (FM CARS) microscopy.

We demonstrate a new approach to coherent anti-Stokes Raman scattering (CARS) microscopy that significantly increases the detection sensitivity. CARS signals are generated by collinearly overlapped, tightly focused, and raster scanned pump and Stokes laser beams, whose difference frequency is rapidly modulated. The resulting amplitude modulation of the CARS signal is detected through a lock-in amplifier. This scheme efficiently suppresses the nonresonant background and allows for the detection of far fewer vibrational oscillators than possible through existing CARS microscopy methods.

Broadly tunable dual-wavelength light source for coherent anti-Stokes Raman scattering microscopy.

The signal and idler beams from a picosecond, synchronously pumped optical parametric oscillator (OPO) provide the two colors necessary for coherent anti-Stokes Raman scattering (CARS) microscopy. The OPO provides a continuously tunable frequency difference between the two beams over a broad range of Raman shifts (100-3700 cm(-1)) by varying the temperature of a single nonlinear crystal. The near-infrared output (900-1300 nm) allows for deep penetration into thick samples and reduced nonlinear photodamage. Applications of this light source to in vivo cell and ex vivo tissue imaging are demonstrated.

Towards CARS Endoscopy.

We provide a proof-of-principle demonstration of CARS endoscopy. The design utilizes a single mode optical fiber with a focusing unit attached to the distal end. Picosecond pump and Stokes pulse trains in the near infrared are delivered through the fiber with nearly unaltered spectral and temporal characteristics at intensities needed for endoscopy. CARS endoscopic images are recorded by collecting the epi-CARS signal generated at the sample and raster scanning the sample with respect to the fiber. This CARS endoscope prototype represents an important step towards in situ chemically selective imaging for biomedical applications.