Three-Photon Adaptive Optics for Mouse Brain Imaging.

Three-photon microscopy (3PM) was shown to allow deeper imaging than two-photon microscopy (2PM) in scattering biological tissues, such as the mouse brain, since the longer excitation wavelength reduces tissue scattering and the higher-order non-linear excitation suppresses out-of-focus background fluorescence. Imaging depth and resolution can further be improved by aberration correction using adaptive optics (AO) techniques where a spatial light modulator (SLM) is used to correct wavefront aberrations. Here, we present and analyze a 3PM AO system for in vivo mouse brain imaging. We use a femtosecond source at 1300 nm to generate three-photon (3P) fluorescence in yellow fluorescent protein (YFP) labeled mouse brain and a microelectromechanical (MEMS) SLM to apply different Zernike phase patterns. The 3P fluorescence signal is used as feedback to calculate the amount of phase correction without direct phase measurement. We show signal improvement in the cortex and the hippocampus at greater than 1 mm depth and demonstrate close to diffraction-limited imaging in the cortical layers of the brain, including imaging of dendritic spines. In addition, we characterize the effective volume for AO correction within brain tissues, and discuss the limitations of AO correction in 3PM of mouse brain.

Pressure-assisted sintering and characterization of Nd:YAG ceramic lasers.

Spark plasma sintering (SPS) is an advanced one-stage, rapid, near-net shape densification technique combining uniaxial pressure with resistive heating. Various transparent ceramics have been successfully fabricated by SPS, despite the existence of inherent carbon contamination and residual pores. Due to the disk-shape of SPS-processed samples, the technique may be suited for producing thin-disk ceramic laser materials. Nevertheless, an in-depth study of these materials has never been reported. With that goal in mind, the major focus of this study was to characterize the laser performance of Nd:YAG ceramics fabricated by one-stage SPS under conventional (60 MPa) and high (300 MPa) applied pressures. In addition to measuring the lasing slope efficiency and threshold, the passive losses associated with each sample were also evaluated. Surprisingly, it was found that in-line transmittance spectra do not provide accurate predictions of laser performance due to the nature of residual porosity. Moreover, homogeneity and beam quality were assessed, and comparisons were drawn between conventional and high-pressure SPS ceramics. This study lays the groundwork for the future of laser materials fabricated by SPS or similar pressure-assisted techniques.

Deep three-photon imaging of the brain in intact adult zebrafish.

Behaviors emerge from activity throughout the brain, but noninvasive optical access in adult vertebrate brains is limited. We show that three-photon (3P) imaging through the head of intact adult zebrafish allows structural and functional imaging at cellular resolution throughout the telencephalon and deep into the cerebellum and optic tectum. With 3P imaging, considerable portions of the brain become noninvasively accessible from embryo to sexually mature adult in a vertebrate model.

Comparing the effective attenuation lengths for long wavelength in vivo imaging of the mouse brain.

Light attenuation in thick biological tissues, caused by a combination of absorption and scattering, limits the penetration depth in multiphoton microscopy (MPM). Both tissue scattering and absorption are dependent on wavelengths, which makes it essential to choose the excitation wavelength with minimum attenuation for deep imaging. Although theoretical models have been established to predict the wavelength dependence of light attenuation in brain tissues, the accuracy of these models in experimental settings needs to be verified. Furthermore, the water absorption contribution to the tissue attenuation, especially at 1450 nm where strong water absorption is predicted to be the dominant contributor in light attenuation, has not been confirmed. Here we performed a systematic study of in vivo three-photon imaging at different excitation wavelengths, 1300 nm, 1450 nm, 1500 nm, 1550 nm, and 1700 nm, and quantified the tissue attenuation by calculating the effective attenuation length at each wavelength. The experimental data show that the effective attenuation length at 1450 nm is significantly shorter than that at 1300 nm or 1700 nm. Our results provide unequivocal validation of the theoretical estimations based on water absorption and tissue scattering in predicting the effective attenuation lengths for long wavelength in vivo imaging.

In vivo label-free confocal imaging of the deep mouse brain with long-wavelength illumination.

Optical microscopy is a valuable tool for in vivo monitoring of biological structures and functions because of its non-invasiveness. However, imaging deep into biological tissues is challenging due to the scattering and absorption of light. Previous research has shown that 1300 nm and 1700 nm are the two best wavelength windows for deep brain imaging. Here, we combined long-wavelength illumination of ~1700 nm with reflectance confocal microscopy and achieved an imaging depth of ~1.3 mm with ~1-micrometer spatial resolution in adult mouse brains, which is 3-4 times deeper than that of conventional confocal microscopy using visible wavelength. We showed that the method can be added to any laser-scanning microscopy with simple and low-cost sources and detectors, such as continuous-wave diode lasers and InGaAs photodiodes. The long-wavelength, reflectance confocal imaging we demonstrated is label-free, and requires low illumination power. Furthermore, the imaging system is simple and low-cost, potentially creating new opportunities for biomedical research and clinical applications.

Two-photon Shack-Hartmann wavefront sensor.

We introduce a simple wavefront sensing scheme for aberration measurement of pulsed laser beams in near-infrared wavelengths (<2200 nm), where detectors are not always available or are very expensive. The method is based on two-photon absorption in a silicon detector array for longer wavelengths detection. We demonstrate the simplicity of such implementations with a commercially available Shack-Hartmann wavefront sensor and discuss the detection sensitivity of this method.

Nonresonant background suppression for coherent anti-Stokes Raman scattering microscopy using a multi-wavelength time-lens source.

We demonstrate a robust, all-fiber, two-wavelength time-lens source for background-free coherent anti-Stokes Raman scattering imaging. The time-lens source generates two picosecond pulse trains simultaneously: one at 1064 nm and the other tunable between 1040 nm and 1075 nm (~400 mW for each wavelength). When synchronized to a mode-locked Ti:Sapphire laser, the two wavelengths are used to obtain on- and off-resonance coherent anti-Stokes Raman scattering images. Real-time subtraction of the nonresonant background in the coherent anti-Stokes Raman scattering image is achieved by the synchronization of the pixel clock and the time-lens source. Background-free coherent anti-Stokes Raman scattering imaging of sebaceous glands in ex vivo mouse tissue is demonstrated.

Adaptive optics in multiphoton microscopy: comparison of two, three and four photon fluorescence.

We demonstrate adaptive optics system based on nonlinear feedback from 3- and 4-photon fluorescence. The system is based on femtosecond pulses created by soliton self-frequency shift of a 1550-nm fiber-based femtosecond laser together with micro-electro-mechanical system (MEMS) phase spatial light modulator (SLM). We perturb the 1020-segment SLM using an orthogonal Walsh sequence basis set with a modified version of three-point phase shifting interferometry. We show the improvement after aberrations correction in 3-photon signal from fluorescent beads. In addition, we compare the improvement obtained in the same adaptive optical system for 2-, 3- and 4-photon fluorescence using dye pool. We show that signal improvement resulting from aberration correction grows exponentially as a function of the order of nonlinearity.

Tunable WDM sampling pulse streams using a spatial phase modulator in a biased pulse shaper.

We generate transform-limited WDM optical sampling pulse bursts by filtering ultrashort pulses from a mode-locked laser. A phase spatial light modulator (SLM) is used in a biased pulse shaper to circumvent the need to modulate with 2π phase wraps, which are known to limit the phase response. The arrangement compresses and retimes user-selectable bandwidths from the optical short pulse source with precise control of pulse bandwidth, pulse stream rates, and duty cycle.

Variable optical attenuator and dynamic mode group equalizer for few mode fibers.

Variable optical attenuation (VOA) for three-mode fiber is experimentally presented, utilizing an amplitude spatial light modulator (SLM), achieving up to -28dB uniform attenuation for all modes. Using the ability to spatially vary the attenuation distribution with the SLM, we also achieve up to 10dB differential attenuation between the fiber's two supported mode group (LP01 and LP11). The spatially selective attenuation serves as the basis of a dynamic mode-group equalizer (DME), potentially gain-balancing mode dependent optical amplification. We extend the experimental three mode DME functionality with a performance analysis of a fiber supporting 6 spatial modes in four mode groups. The spatial modes' distribution and overlap limit the available dynamic range and performance of the DME in the higher mode count case.

Nyquist-WDM filter shaping with a high-resolution colorless photonic spectral processor.

We employ a spatial-light-modulator-based colorless photonic spectral processor with a spectral addressability of 100 MHz along 100 GHz bandwidth, for multichannel, high-resolution reshaping of Gaussian channel response to square-like shape, compatible with Nyquist WDM requirements.

Generation of WDM adaptive-rate pulse bursts by cascading narrow/wideband tunable optical dispersion compensators.

We demonstrate passive generation of optical pulse trains with each pulse having distinct center carrier and spectra using tunable group delay (GD) staircase transfer functions. The GD steps result from opposite and equal magnitude GD slopes from narrowband and wideband tunable optical dispersion compensators. We use this technique to split the spectrum of a femtosecond pulse to a pulse burst with precise control of pulse time separation.

Tunable fiber ring laser with an intracavity high resolution filter employing two-dimensional dispersion and LCoS modulator.

We demonstrate a tunable fiber ring laser employing a two-dimensional dispersion arrangement filter, with the lasing determined by a liquid crystal on silicon (LCoS) spatial light modulator. Lasing wavelengths can be tuned discontinuously across the communication C-band at an addressable resolution of less than 200 MHz. We introduce full characterization of the laser output including phase and amplitude stability and short and long-term bandwidth measurements.

A photonic spectral processor employing two-dimensional WDM channel separation and a phase LCoS modulator.

We present a Photonic Spectral Processor (PSP) that provides both fine spectral resolution and broad bandwidth support by dispersing light over two-dimensional space using the crossed-grating approach. The PSP uses a hybrid guided wave/free-space optics arrangement, where a waveguide grating router implemented in silica waveguides disperses the light in one dimension with a 100 GHz FSR and a bulk 1200 gr/mm diffraction grating disperses the light along the second (crossed) dimension. The diffracted light is focused by a lens onto a liquid-crystal on silicon, two-dimensional, phase-only, spatial light modulator, which we use to prescribe phase and amplitude to the signal's spectral components. With the 2-D PSP arrangement we are able to address frequency components at 0.2 GHz/column with an optical resolution of 3.3 GHz covering 40 C-band channels.

All-channel tunable optical dispersion compensator based on linear translation of a waveguide grating router.

We propose and demonstrate a compact tunable optical dispersion compensation (TODC) device with a 100 GHz free spectral range capable of mitigating chromatic dispersion impairments. The TODC is based on longitudinal movement of a waveguide grating router, resulting in chromatic dispersion compensation of ±1000 ps/nm. We employed our TODC device for compensating 42.8 Gbit/sec differential phase-shifting keying signal, transmitted over 50 km fiber with a -2 dB power penalty at 10⁻9.