Compensation-free, all-fiber-optic, two-photon endomicroscopy at 1.55 μm

We present an all-fiber-optic scanning multiphoton endomicroscope with 1.55 μm excitation without the need for prechirping femtosecond pulses before the endomicroscope. The system consists of a 1.55 μm femtosecond fiber laser, a customized double-clad fiber for light delivery and fluorescence collection, and a piezoelectric scan head. We demonstrate two-photon imaging of cultured cells and mouse tissue, both labeled with indocyanine green. Free-space multiphoton imaging with near-IR emission has previously shown benefits in reduced background fluorescence and lower attenuation for the fluorescence emission. For fiber-optic multiphoton imaging there is the additional advantage of using the soliton effect at the telecommunication wavelengths (1.3-1.6 μm) in fibers, permitting dispersion-compensation-free, small-footprint systems. We expect these advantages will help transition multiphoton endomicroscopy to the clinic.     

In vivo video rate multiphoton microscopy imaging of human skin

We present a multiphoton microscopy instrument specially designed for in vivo dermatological use that is capable of imaging human skin at 27 frames per second with 256 pixels × 256 pixels resolution without the use of exogenous contrast agents. Imaging at fast frame rates is critical to reducing image blurring due to patient motion and to providing practically short clinical measurement times. Second harmonic generation and two-photon fluorescence images and videos acquired at optimized wavelengths are presented showing cellular and tissue structures from the skin surface down to the reticular dermis.     

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.     

Improving signal levels in intravital multiphoton microscopy using an objective correction collar

Multiphoton microscopy has enabled biologists to collect high-resolution images hundreds of microns into biological tissues, including tissues of living animals. While the depth of imaging exceeds that possible from any other form of light microscopy, multiphoton microscopy is nonetheless generally limited to depths of less than a millimeter. Many of the advantages of multiphoton microscopy for deep tissue imaging accrue from the unique nature of multiphoton fluorescence excitation. However, the quadratic relationship between illumination level and fluorescence excitation makes multiphoton microscopy especially susceptible to factors that degrade the illumination focus. Here we examine the effect of spherical aberration on multiphoton microscopy in fixed kidney tissues and in the kidneys of living animals. We find that spherical aberration, as evaluated from axial asymmetry in the point-spread function, can be corrected by adjustment of the correction collar of a water immersion objective lens. Introducing a compensatory positive spherical aberration into the imaging system decreases the depth-dependence of signal levels in images collected from living animals, increasing signal by up to 50%.     

Adaptive control of femtosecond pulse propagation in optical fibers

Nonlinear effects present fundamental obstacles to the propagation of femtosecond pulses of detectable energy in single-mode optical fibers, inducing severe distortion even after a very short (a few meters) propagation distance. We show here that adaptive pulse shaping can overcome these limitations by synthesizing pulses that are self-correcting for higher-order nonlinear effects when they are launched in the fiber. This approach would not only affect optical communications but also yield benefits in various disciplines requiring optimized fiber-based femtosecond pulse delivery, for example, nonlinear imaging techniques such as multiphoton microscopy, material processing, and medical diagnostics.     

Optimization of second-harmonic generation microscopy

We describe the principles and characteristics of second-harmonic generation imaging (SHGI) and explore various methods for optimization of the technique. Second-harmonic imaging is optimized for ultrashort laser pulses, high numerical aperture microscope objectives, a highly sensitive non-descanned large area detector, pseudo-phase-matching, and specimens with large second-order non linearity or which exhibit surface plasmon enhanced phenomena. We also compare and contrast the techniques of SHGI and two-photon excited fluorescence imaging.     

Multiphoton endoscopy

Despite widespread use of multiphoton fluorescence microscopy, development of endoscopes for nonlinear optical imaging has been stymied by the degradation of ultrashort excitation pulses that occurs within optical fiber as a result of the combined effects of group-velocity dispersion and self-phase modulation. We introduce microendoscopes (350-1000 microm in diameter) based on gradient-index microlenses that effectively eliminate self-phase modulation within the endoscope. Laser-scanning multiphoton fluorescence endoscopy exhibits micrometer-scale resolution. We used multiphoton endoscopes to image fluorescently labeled neurons and dendrites.     

Simultaneous Spatial and Temporal Focusing in Nonlinear Microscopy

Simultaneous spatial and temporal focusing (SSTF), when combined with nonlinear microscopy, can improve the axial excitation confinement of wide-field and line-scanning imaging. Because two-photon excited fluorescence depends inversely on the pulse width of the excitation beam, SSTF decreases the background excitation of the sample outside of the focal volume by broadening the pulse width everywhere but at the geometric focus of the objective lens. This review theoretically describes the beam propagation within the sample using Fresnel diffraction in the frequency domain, deriving an analytical expression for the pulse evolution. SSTF can scan the temporal focal plane axially by adjusting the GVD in the excitation beam path. We theoretically define the axial confinement for line-scanning SSTF imaging using a time-domain understanding and conclude that line-scanning SSTF is similar to the temporally-decorrelated multifocal multiphoton imaging technique. Recent experiments on the temporal focusing effect and its axial confinement, as well as the axial scanning of the temporal focus by tuning the GVD, are presented. We further discuss this technique for axial-scanning multiphoton fluorescence fiber probes without any moving parts at the distal end. The temporal focusing effect in SSTF essentially replaces the focusing of one spatial dimension in conventional wide-field and line-scanning imaging. Although the best axial confinement achieved by SSTF cannot surpass that of a regular point-scanning system, this trade-off between spatial and temporal focusing can provide significant advantages in applications such as high-speed imaging and remote axial scanning in an endoscopic fiber probe.     

Simultaneous visualization of spatial and chromatic aberrations by two-dimensional Fourier transform spectral interferometry

We demonstrate the use of a simple tool to simultaneously visualize and characterize chromatic and spherical aberrations that are present in multiphoton microscopy. Using two-dimensional Fourier transform spectral interferometry, we measured these aberrations, deducing in a single shot spatiotemporal effects in high-numerical-aperture objectives.     

Dorsal Skin Fold Chamber for High Resolution Multiphoton Imaging

The advantages offered by multiphoton microscopy enabled this technique to be applied for in vivo understanding of physiological processes. However, multiphoton intravital microscopy also requires associated technologies to be developed. In this work, we detailed the design of a dorsal skin fold chamber made of titanium alloy that allow high resolution multiphoton fluorescence and second harmonic generation (SHG) microscopy to be achieved. We demonstrate that our apparatus is capable of obtaining high resolution, images of blood vessels and collagen matrix in nude mice. Such an imaging chamber will allow physiological processes to be investigated at high resolution     

Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging

We performed multiphoton fluorescence (MF) and second-harmonic generation (SHG) imaging on human basal cell carcinoma samples. In the dermis, basal cell carcinomas can be identified by masses of autofluorescent cells with relatively large nuclei and marked peripheral palisading. In the normal dermis, SHG from dermal collagen contributes largely to the multiphoton signal. However, within the cancer stroma, SHG signals diminish and are replaced by autofluorescent signals, indicating that normal collagen structures responsible for SHG have been altered. To better delineate the cancer cells and cancer stroma from the normal dermis, a quantitative MF to SHG index is developed. We demonstrate that this index can be used to differentiate cancer cells and adjacent cancer stroma from the normal dermis. Our work shows that MF and SHG imaging can be an alternative for Mohs' surgery in the real-time guidance of the secure removal of basal cell carcinoma.     

多光子皮肤成像技术及其应用

多光子成像技术是一种层析能力好、信噪比高的新型光学成像技术。在皮肤光学三维检测中,多光子技术已经应用于无创在体成像,且已得到产业化开发。本文将首先介绍多光子皮肤检测系统的若干核心技术,即双光子自发荧光技术、二次谐波成像技术、荧光寿命成像技术、相干反斯托克斯-拉曼成像技术等,然后简要介绍多光子成像系统在皮肤疾病成像检测上的应用,最后分析该系统的优势和未来可能的发展趋势。     

Photolytic-interference-free, femtosecond two-photon fluorescence imaging of atomic hydrogen

We discuss photolytic-interference-free, high-repetition-rate imaging of reaction intermediates in flames and plasmas using femtosecond (fs) multiphoton excitation. The high peak power of fs pulses enables efficient nonlinear excitation, while the low energy nearly eliminates interfering single-photon photodissociation processes. We demonstrate proof-of-principle, interference-free, two-photon laser-induced fluorescence line imaging of atomic hydrogen in hydrocarbon flames and discuss the method's implications for certain other atomic and molecular species.     

Repetition rate multipliers for efficient implementation of multiphoton and pump-probe fluorescence microscopy

With the advances in pulsed laser systems, microscopic imaging techniques such as multiphoton and pump-probe fluorescence microscopy have developed into effective tools for investigating intensity and time-resolved phenomena inside biological systems. However, pulsed lasers used in these techniques usually are commercial systems with repetition frequencies of around 80 MHz. While these systems have proven to be adequate for multiphoton and pump-probe microscopic imaging applications, the temporal separation of the laser pulse train (around 12.5 ns) is long compared to the fluorescence lifetimes of many common fluorescence species. In this work, we present the designs of repetition rate multipliers based on passive optical components that can be used to increase the efficiency in multiphoton and pump-probe fluorescence microscopy. Depending on the lifetime of fluorescence molecules under investigation, the passive repetition rate multiplier can increase the duty cycle of multiphoton or pump-probe microscopy up to fourfold.     

Peak Multiphoton Excitation of mCherry Using an Optical Parametric Oscillator (OPO)

mCherry is a red fluorescent protein which is bright, photostable, and has a low molecular weight. It is an attractive choice for multiphoton fluorescence imaging; however, the multiphoton excitation spectrum of mCherry is not known. In this paper we report the two photon excitation spectrum of mCherry measured up to 1190 nm in the near infrared (NIR) region. Skin tissues of transgenic mice that express mCherry were used in the experiments. mCherry in the tissues was excited with a Titanium:Sapphire laser and an optical parametric oscillator pumped by the Titanium:Sapphire laser. We found that the peak excitation of mCherry occurs at 1160 nm.     

Quantum spatial superresolution by optical centroid measurements

Quantum lithography (QL) has been suggested as a means of achieving enhanced spatial resolution for optical imaging, but its realization has been held back by the low multiphoton detection rates of recording materials. Recently, an optical centroid measurement (OCM) procedure was proposed as a way to obtain spatial resolution enhancement identical to that of QL but with higher detection efficiency (M. Tsang, Phys. Rev. Lett. 102, 253601 (2009)). Here we describe a variation of the OCM method with still higher detection efficiency based on the use of photon-number-resolving detection. We also report laboratory results for two-photon interference. We compare these results with those of the standard QL method based on multiphoton detection and show that the new method leads to superresolution but with higher detection efficiency.     

Analytical Results of Eigenstates and Eigenenergies for Three Kinds of Models Describing N-mode Multiphoton Process

We obtain the exact analytical results of all the eigenvalues and eigenstates for three kinds of models describing N-mode multiphoton process without using the assumption of the Bethe ansatz. The exact analytical results of all the eigenstates and eigenvalues are in terms of a parameter λ for three kinds of models describing N-mode multiphoton process. The parameter is shown to be determined by the roots of a polynomial and is solvable analytically or numerically. Moreover, these three kinds of models can be processed with the same procedure.     

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.     

Quantum imaging with incoherent photons

We propose a technique to obtain subwavelength resolution in quantum imaging with potentially 100% contrast using incoherent light. Our method requires neither path-entangled number states nor multiphoton absorption. The scheme makes use of N photons spontaneously emitted by N atoms and registered by N detectors. It is shown that for coincident detection at particular detector positions a resolution of lambda/N can be achieved.     

Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence

A multiphoton microscope employing second-harmonic generation (SHG) and two-photon excited fluorescence (TPF) is used for high-resolution ex vivo imaging of rabbit cornea in a backscattering geometry. Endogenous TPF and SHG signals from corneal cells and extracellular matrix, respectively, are clearly visible without exogenous dyes. Spectral characterization of these upconverted signals provides confirmation of the structural origin of both TPF and SHG, and spectral imaging facilitates the separation of keratocyte and epithelial cells from the collagen-rich corneal stroma. The polarization dependence of collagen SHG is used to highlight fiber orientation, and three-dimensional SHG tomography reveals that approximately 88% of the stromal volume is occupied by collagen lamellae.