Fringe-free, background-free, collinear third-harmonic generation frequency-resolved optical gating measurements for multiphoton microscopy

A background-free, fringe-free form of frequency-resolved optical gating using the third-harmonic signal generated from a glass coverslip is used to characterize 100 fs pulses at the focus of a 0.65 NA objective.     

Milliwatt-peak-power pulse characterization at 1.55 microm by wavelength-conversion frequency-resolved optical gating

A novel wavelength-conversion configuration based on four-wave mixing in an optical fiber has been used to generate a frequency-resolved optical gating (FROG) trace identical to that obtained from second-harmonic generation (SHG). The use of an optical fiber waveguide permits enhanced measurement sensitivity compared with that of conventional SHG-FROG and has been used for complete characterization of 1-mW peak-power picosecond pulses at 1.55 microm from an unamplified semiconductor laser diode gain switched at 10 GHz.     

In situ broadband pulse compression for multiphoton microscopy using a shaper-assisted collinear SPIDER

The characterization and control of the phase of broadband femtosecond pulses in nonlinear microscopy are successfully demonstrated with a collinear configuration of spectral shear interferometry for direct electric field reconstruction (SPIDER). A femtosecond-pulse shaper is used as a dispersionless interferometer for the measurement of the spectral phase and to actively compress a broadband supercontinuum from a photonic crystal fiber. This allows in situ online phase management and enables the application of quantum control spectroscopy in microenvironments.     

Characterization of a broadband pulse for phase controlled multiphoton microscopy by single beam SPIDER

We present what we believe to be a new version of spectral phase interferometry for direct electric field reconstruction (SPIDER) using only a single-phase and polarization controlled laser beam. Two narrow pulses and one broadband pulse are selected out of an ultrafast laser pulse by a polarization and phase control technique to generate second harmonic generation (SHG) signals, which are equivalent to a spectral shear interferogram in the conventional SPIDER method. The spectral phase of the broadband laser pulse is extracted analytically with double quadrature spectral interferometry (DQSI). An arbitrary spectral phase can be retrieved with great precision and compensated in situ at the sample position of a microscope. This new method requires no separate reference beam and is suitable for nonlinear optical microscopy with a phase controlled laser pulse.     

Multifocal multiphoton microscopy

We present a real-time, direct-view multiphoton excitation fluorescence microscope that provides three-dimensional imaging at high resolution. Using a rotating microlens disk, we split the near-infrared light of a mode-locked titanium-sapphire laser into an array of beams that are transformed into an array of high-aperture foci at the object. We typically scan at 225 frames per second and image the fluorescence with a camera that reads out the images at video rate. For 1.4 aperture oil and 1.2 water immersion lenses at 780-nm excitation we obtained axial resolutions of 0.84 and 1.4mum , respectively, which are similar to that of a single-beam two-photon microscope. Compared with the latter setup, our system represents a 40-100-fold increase in efficiency, or imaging speed. Moreover, it permits the observation with the eye of high-resolution two-photon images of (live) samples.     

Single-beam homodyne SPIDER for multiphoton microscopy

We report a new version of spectral phase interferometry for direct electric field reconstruction (SPIDER) requiring only a single phase-shaped laser beam. A narrowband probe pulse is selected out of a broadband ultrafast laser pulse by a phase pulse-shaping technique and mixed with the original broadband pulse to generate a second-harmonic generation (SHG) signal. Using another SHG signal solely generated by the broadband pulse as a local oscillator, the spectral phase of the broadband laser pulse can be analytically retrieved by a combination of double-quadrature spectral interferometry and homodyne optical technique for SPIDER (HOT SPIDER). An arbitrary spectral phase at the sample position of a microscope can be compensated with a precision of 0.05 rad over the FWHM of the laser spectrum. It is readily applicable to a nonlinear microscopy technique with a phase-controlled broadband laser pulse.     

Time-frequency analysis for pulse driven ultrasonic microscopy for biological tissue characterization

The authors have proposed a new type of ultrasonic microscopy for biological tissue characterization. The system is driven by a nanosecond pulse voltage, the generated acoustic wave being reflected at the front and rear side of the sliced tissue. In this report, a time-frequency analysis was applied to determine the sound speed thorough the tissue. Frequency dependence of sound speed was obtained with a myocardium of a rat sliced into 10 microm. As the reflected waveform had a significant amount of oscillating component, the waveform was once subjected to the deconvolution process. As the result, two reflections were clearly separated in time domain. Then these two reflections were separately analyzed by time-frequency analysis. Each reflection was extracted by using a proper window function. Phase angles of these reflections at the same frequency were compared. A sound speed micrograph at an arbitrary frequency in between 50 and 150 MHz was successfully obtained. A tendency was found that the sound speed slightly increases with frequency.     

Highly sensitive differential tomographic technique for real-time ultrashort pulse characterization

We demonstrate a highly sensitive real-time optical pulse characterization technique based on differential chronocyclic tomography. The spectral intensity and phase of the pulse under test are reconstructed analytically from two experimental traces measured simultaneously in the spectral domain. The high sensitivity and accuracy are made possible by lock-in detection of the differential spectra in the simplified chronocyclic tomography. An accuracy of approximately 0.04 rad of spectral phase recovery is achieved with a 10-Hz refresh rate and 10-microW sensitivity. We also show that the measurement technique is applicable to pulses as short as approximately 100 fs.     

Starch-based second-harmonic-generated collinear frequency-resolved optical gating pulse characterization at the focal plane of a high-numerical-aperture lens

We report the use of starch as an ideal nonlinear medium with which to perform collinear frequency-resolved optical gating measurements of ultrashort pulses at the focal plane of a high-numerical-aperture (NA) lens. We achieved these measurements by simply sandwiching starch granules (suspended in water) between two coverslips and placing them within the focal plane of a high-NA lens. The natural nonlinear characteristics of starch allow the correct phase matching of pulses at the focal plane of a high-NA lens at different wavelengths. This elegant arrangement overcomes all the complexity and problems that were previously associated with pulse characterization within a multiphoton microscope.     

新型多光子成像技术研究进展

相比于传统的光学成像技术,近年来获得快速发展的新型多光子成像技术具有穿透深度大,组织光损伤小,信噪比高,且可方便进行光学层析成像的特点,故而被广泛应用于包括脑、肿瘤、胚胎在内的多种活体组织成像中。本综述回顾了新型多光子成像技术的诞生与发展历程,包括微型化双光子成像技术、双光子内窥技术和三光子成像技术,概括分析了其基本原理与成像特点,讨论了这一领域具有代表性的最新研究成果,重点总结了其在生物学基础研究领域和临床医学诊断中的主要应用,并展望了其未来的应用与发展前景。可以预见,随着激光器和光探测技术的不断进步,多光子成像技术将会得到更大的发展与更加广泛的应用。     

Time-decorrelated multifocal array for multiphoton microscopy and micromachining

We demonstrate a temporally decorrelated, multifocal multiphoton microscope. Using an etalon, we split the 800-nm light from either an ultrashort-pulsed Ti:Al (2)O (3) oscillator or a Ti:Al (2)O (3) regenerative amplifier into an array of beamlets that are delayed with respect to one another in time. The collimated beams overlap at slightly different input angles at the entrance pupil of a 1.25-numerical aperture oil-immersion objective to produce an array of foci that are temporally decorrelated at the focal plane of the objective. The temporal decorrelation eliminates any interference among the foci and permits multifocal multiphoton imaging with the resolution of single-point illumination.     

High-speed spectrally resolved multifocal multiphoton microscopy

We present a spectrally resolved multifocal multiphoton microscopy that is capable of performing fast 2-dimensional (2-D) spectral measurements of fluorescent samples with optical sectioning. One galvanometer mirror is used to scan the array of excitation foci across the sample along one direction (x) for two-photon excitation. Fluorescence emission from the excited lines on the sample is spectrally fanned out with a prism along the y direction, and a CCD array is used to acquire the spectrally resolved image. Another galvanometer mirror scans the excitation foci lines along the y direction step by step to obtain 3-dimensional (3-D) spectral data cube of the sample. A proof-of-principle experiment is performed with fluorescent microspheres of different colors. Spectrally resolved images of 512×512 pixels can be obtained by acquiring only 128 raw images when a 4×4 excitation foci array is used.     

Agitation-free multiphoton microscopy of oblique planes

The scanning two-photon fluorescence microscope produces optically sectioned images from the focal plane. It is sometimes desirable to acquire images from other planes of the specimen that are inclined with respect to the focal plane. In this Letter, we discuss the issues concerned with acquiring such images together with the effects of the inclination angle on image resolution and sectioning strength. To obtain images from oblique planes at high speed, a two-photon system was built wherein a novel optical system is used to provide aberration-free scanning.     

Temporal characterization of mid-IR free-electron-laser pulses by frequency-resolved optical gating

We performed what we believe are the first practical full-temporal-characterization measurements of ultrashort pulses from a free-electron laser (FEL). Second-harmonic-generation frequency-resolved optical gating (FROG) was used to measure a train of mid-IR pulses distorted by a saturated water-vapor absorption line and showing free-induction decay. The measured direction of time was unambiguous because of prior knowledge regarding free-induction decay. These measurements require only 10% of the power of the laser beam and demonstrate that FROG can be implemented as a pulse diagnostic simultaneously with other experiments on a FEL.     

Depth-resolved multiphoton polarization microscopy by third-harmonic generation

We achieve depth-resolved polarization microscopy by measuring third-harmonic generation induced by a tightly focused circularly polarized beam. In crystals exhibiting strong birefringence this signal is dominated by positively phase-matched third-harmonic generation. This process occurs in only optically anisotropic media, in which the birefringence compensates for the phase mismatch between the fundamental and the third harmonic induced by dispersion. Both the intensity and the polarization of the emitted signal provide information on the local optical anisotropy. We demonstrate the technique by imaging biogenic crystals in sea urchin larval spicules.     

Spectral-domain optical coherence phase and multiphoton microscopy

We describe simultaneous quantitative phase contrast and multiphoton fluorescence imaging by combined spectral-domain optical coherence phase and multiphoton microscopy. The instrument employs two light sources for efficient optical coherence microscopic and multiphoton imaging and can generate structural and functional images of transparent specimens in the epidirection. Phase contrast imaging exhibits spatial and temporal phase stability in the subnanometer range. We also demonstrate the visualization of actin filaments in a fixed cell specimen, which is confirmed by simultaneous multiphoton fluorescence imaging.     

Hybrid reflecting objectives for functional multiphoton microscopy in turbid media

Most multiphoton imaging of biological specimens is performed using microscope objectives optimized for high image quality under wide-field illumination. We present a class of objectives designed de novo without regard for these traditional constraints, driven exclusively by the needs of fast multiphoton imaging in turbid media: the delivery of femtosecond pulses without dispersion and the efficient collection of fluorescence. We model the performance of one such design optimized for a typical brain-imaging setup and show that it can greatly outperform objectives commonly used for this task.     

Fully differential rates for femtosecond multiphoton double ionization of neon

We have investigated the full three-dimensional momentum correlation between the electrons emitted from strong field double ionization of neon when the recollision energy of the first electron is on the order of the ionization potential. The momentum correlation in the direction perpendicular to the laser field depends on the time difference of the two electrons leaving the ion. Our results are consistent with double ionization proceeding through transient double excited states that field ionize.     

Aberration correction for multiphoton microscopy using covariance matrix adaptation evolution strategy

Multiphoton microscopy is the enabling tool for biomedical research, but the aberrations of biological tissues have limited its imaging performance. Adaptive optics(AO) has been developed to partially overcome aberration to restore imaging performance. For indirect AO, algorithm is the key to its successful implementation. Here, based on the fact that indirect AO has an analogy to the black-box optimization problem, we successfully apply the covariance matrix adaptation evolution strategy(CMA-ES...     

Miniaturized probe based on a microelectromechanical system mirror for multiphoton microscopy

A factor that limits the use of multiphoton microscopy (MPM) in clinical and preclinical studies is the lack of a compact and flexible probe. We report on a miniaturized MPM probe employing a microelectromechanical system (MEMS) scanning mirror and a double-clad photonic crystal fiber (DCPCF). The use of a MEMS mirror and a DCPCF provides many advantages, such as size reduction, rapid and precise scanning, efficient delivery of short pulses, and high collection efficiency of fluorescent signals. The completed probe was 1 cm in outer diameter and 14 cm in length. The developed probe was integrated into an MPM system and used to image fluorescent beads, paper, and biological specimens.