Improved depth resolution in video-rate line-scanning multiphoton microscopy using temporal focusing

By introducing spatiotemporal pulse shaping techniques to multiphoton microscopy it is possible to obtain video-rate images with depth resolution similar to point-by-point scanning multiphoton microscopy while mechanically scanning in only one dimension. This is achieved by temporal focusing of the illumination pulse: The pulsed excitation field is compressed as it propagates through the sample, reaching its shortest duration (and highest peak intensity) at the focal plane before stretching again beyond it. This method is applied to produce, in a simple and scalable setup, video-rate two-photon excitation fluorescence images of Drosophila egg chambers with nearly 100,000 effective pixels and 1.5 microm depth resolution.     

Self-compression of femtosecond pulses in argon with a power close to the self-focusing threshold

Self-compression of femtosecond pulses in noble gases with an input power close to the self-focusing threshold has been investigated experimentally and theoretically. It is demonstrated that either multiphoton ionization （MPI） or space-time focusing and self-steepening effects can induce pulse shortening, but they predominate at different beam intensities during the propagation. The latter effects play a key role in the final pulse self-compression. By choosing an appropriate focusing parameter, action distance of the space-time focusing and self-steepening effects can be lengthened, which can promote a shock pulse structure with a duration as short as two optical cycles. It is also found that, for our calculation cases in which an input pulse power is close to the self-focusing threshold, either group velocity dispersion （GVD） or multiphoton absorption （MPA） has a negligible influence on pulse characteristics in the propagation process.     

High average power Yb:CaF2 femtosecond amplifier with integrated simultaneous spatial and temporal focusing for laser material processing

A watt level, 10-kz repetition rate chirped pulse amplification system that has an integrated simultaneous spatial and temporal focusing (SSTF) processing system is demonstrated for the first time. SSTF significantly reduces nonlinear effects normally detrimental to beam control enabling the use of a low numerical aperture focus to quickly treat optically transparent materials over a large area. The integrated SSTF system has improved efficiency compared to previously reported SSTF designs, which combined with the high-repetition rate of the laser, further optimizes its capability to provide rapid, large volume processing.     

Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging

We demonstrate stimulated emission depletion (STED) microscopy implemented in a laser scanning confocal microscope using excitation light derived from supercontinuum generation in a microstructured optical fiber. Images with resolution improvement beyond the far-field diffraction limit in both the lateral and axial directions were acquired by scanning overlapped excitation and depletion beams in two dimensions using the flying spot scanner of a commercially available laser scanning confocal microscope. The spatial properties of the depletion beam were controlled holographically using a programmable spatial light modulator, which can rapidly change between different STED imaging modes and also compensate for aberrations in the optical path. STED fluorescence lifetime imaging microscopy is demonstrated through the use of time-correlated single photon counting.     

Addressable discrete-line-scanning multiphoton microscopy based on a spatial light modulator

We developed a novel addressable discrete-line-scanning multiphoton microscope with high lateral and axial resolutions based on a spatial light modulator. Our discrete-line-focus design eliminates the cross talk that occurs in conventional one-dimensional line-scanning multiphoton microscopies. Additionally, a phase-only spatial light modulator is able to scan only a sample's target area by generating a specific discrete line focus according to the shape and location of the target area. Compared with other multiphoton microscopies, this technique shortens scanning time and minimizes photodamage by concentrating scanning energy and dwell time on the area of interest.     

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.     

Remotely scanned multiphoton temporal focusing by axial grism scanning

A simple technique for remote scanning of the focal plane in temporal focusing multiphoton microscopy is demonstrated both theoretically and experimentally. A new on-axis light propagation optical setup design enables this scanning, which was considered not feasible in previous studies. The focal plane is axially displaced by the movement of a remote optical device, consisting of a double prism grating, and optionally a cylindrical lens. The displacement is linear, and its slope is inversely proportional to the square of the optical system's magnification.     

Independence of Maximum Single Molecule Fluorescence Count Rate on the Temporal and Spectral Laser Pulse Width in Two-Photon FCS

We investigate the fluorescence emission characteristics of standard dye tetramethylrhodamine (TMR) in two-photon fluorescence correlation spectroscopy for different temporal and spectral properties of the femtosecond excitation pulses. After determining the second-order dispersion of our setup, including the microscope objective, a pulse stretcher was employed to control the temporal width at the location of the specimen. As expected, the fluorescence per molecule and therefore the signal-to-noise ratio of an FCS-measurement can be improved at constant average excitation power by altering either the temporal or spectral width of the excitation pulses. We found however, that the maximum achievable molecular brightness is largely independent of the temporal and spectral width in the regime analyzed. This observation confirms the current working hypothesis for two-photon fluorescence correlation spectroscopy that bleaching and saturation, and thus, the inherent properties of the dye system, are the dominant effects limiting the quality of measurements. As a practical consequence, elaborate optimization of temporal and spectral laser pulse width, e.g. by introducing pulse stretchers in the beam path, is less critical than previously expected.     

A UV–Visible–NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution

This article describes the design and characterization of a wide-field, time-domain fluorescence lifetime imaging microscopy (FLIM) system developed for picosecond time-resolved biological imaging. The system consists of a nitrogen-pumped dye laser for UV–visible–NIR excitation (337.1–960 nm), an epi-illuminated microscope with UV compatible optics, and a time-gated intensified CCD camera with an adjustable gate width (200 ps-10^-3 s) for temporally resolved, single-photon detection of fluorescence decays with 9.6-bit intensity resolution and 1.4-μm spatial resolution. Intensity measurements used for fluorescence decay calculations are reproducible to within 2%, achieved by synchronizing the ICCD gate delay to the excitation laser pulse via a constant fraction optical discriminator and picosecond delay card. A self-consistent FLIM system response model is presented, allowing for fluorescence lifetimes (0.6 ns) significantly smaller than the FLIM system response (1.14 ns) to be determined to 3% of independently determined values. The FLIM system was able to discriminate fluorescence lifetime differences of at least 50 ps. The spectral tunability and large temporal dynamic range of the system are demonstrated by imaging in living human cells: UV-excited endogenous fluorescence from metabolic cofactors (lifetime ∼1.4 ns); and 460-nm excited fluorescence from an exogenous oxygen-quenched ruthenium dye (lifetime ∼400 ns). Received: 23 February 2003 / Published online: 22 May 2003 RID="*" ID="*"Corresponding author. Fax: +1-734/9361-905, E-mail: mycek@umich.edu     

Self-aligned fiber-based dual-beam source for STED nanolithography

A fiber-based source that can be exploited in a stimulated emission depletion(STED) inspired nanolithography setup is presented. Such a source maintains the excitation beam pulse, generates a ring-shaped depletion beam, and automatically realizes dual-beam coaxial alignment that is critical for two beam nanolithography. The mode conversion of the depletion beam is realized by using a customized vortex fiber, which converts the Gaussian beam into a donut-shaped azimuthally polarized beam. The pulse width and repetition frequency of the excitation beam remain unchanged, and its polarization states can be controlled. According to the simulated point spread function of each beam in the focal region, the full width at half-maximum of the effective spot size in STED nanofabrication could decrease to less than 28.6 nm.     

Adaptive phase compensation for ultracompact laser scanning endomicroscopy

We present an approach to laser scanning endomicroscopy that requires no moving parts and can be implemented with no distal scanners or optics, permitting extremely compact endoscopic probes to be developed. Our approach utilizes a spatial light modulator to correct for phase variations across a fiber imaging bundle and to encode for arbitrary wavefronts at the distal end of the fiber bundle. Thus, it is possible to realize both focusing and beam scanning at the output of the fiber bundle with no distal components. We present proof of principle results to illustrate three-dimensional scanning of the focal spot and exemplar images of a United States Air Force resolution test chart.     

基于数字微镜的快速轴向扫描系统

为解决双光子荧光显微成像系统轴向扫描问题,提出一种基于数字微镜(DMD)的快速轴向扫描系统。该系统采用DMD选择光路,不同光路放置不同焦距的透镜组对光束发散角产生不同的改变,经物镜聚焦后得到不同深度的轴上扫描点。对该系统的轴向扫描距离、扫描点位置及衍射效率进行了理论计算仿真,结果表明:扫描系统采用4个模块以及5个模块时其轴向扫描距离均可达到1 mm,4模块系统中透镜的焦距为297.3 mm,5模块系统中透镜焦距为361.47 mm。轴向扫描点除边缘点外线性分布, 轴向扫描频率达到几十kHz,满足脑神经成像的要求。     

Subdiffraction resolution in far-field fluorescence microscopy

We overcame the resolution limit of scanning far-field fluorescence microscopy by disabling the fluorescence from the outer part of the focal spot. Whereas a near-UV pulse generates a diffraction-limited distribution of excited molecules, a spatially offset pulse quenches the excited molecules from the outer part of the focus through stimulated emission. This results in a subdiffraction-sized effective point-spread function. For a 1.4 aperture and a 388-nm excitation wavelength spatial resolution is increased from 150 +/- 8 nm to 106 +/- 8 nm with a single offset beam. Superior lateral resolution is demonstrated by separation of adjacent Pyridine 2 nanocrystals that are otherwise indiscernible.     

双光子技术在三维成像和三维存储技术中的应用

对双光子过程的空间分辨能力进行了理论分析。阐明了双光子技术在三维成像和三维存储中的独特优势。着重介绍了双光子技术与扫描共焦显微术、近场显微术相结合进行三维成像 ,以及一种多焦点多光子显微术和用连续光源激发的双光子三维成像技术的研究和进展情况。对建立在共焦显微镜基础之上的双光子三维光存储和微细加工方面的研究也作了回顾与展望     

Flow imaging by use of femtosecond-laser-induced two-photon fluorescence

A novel technique is demonstrated for the imaging of turbulent flows in which a single window to the flow is the only optical access required. A femtosecond laser is used to excite two-photon fluorescence in a disodium-fluorescein-seeded water jet. The fluorescence signal is generated at only the focal point of the laser because of the highly nonlinear nature of the two-photon absorption, and it is collected in a direction counterpropagating to the excitation beam. Tight focusing of the laser is used to limit the probe volume, and the two-dimensional mean and rms concentration images are collected by raster scanning the laser.     

Fiber-optic scanning two-photon fluorescence endoscope

We report on the development of a miniature, flexible, fiber-optic scanning endoscope for two-photon fluorescence imaging. The endoscope uses a tubular piezoelectric actuator for achieving two-dimensional beam scanning and a double-clad fiber for delivery of the excitation light and collection of two-photon fluorescence. Real-time imaging of fluorescent beads and cancer cells has been performed.     

Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy

We investigate a novel concept to efficiently generate multiphoton induced fluorescence from organic molecules. The concept is based on frustrating the energy transfer between a fluorescent donor and one or more acceptors in conjugated molecules. The nonlinearity is not based on higher order molecular susceptibilities but entirely on their linear properties. Therefore, in contrast to nonresonant multiphoton absorption, this method does not require high local intensities. Likewise, the production of visible fluorescence does not require an infrared excitation wavelength. Hence, when applied to scanning microscopy this property is predicted to increase spatial resolution. Instead of the ∼10 GW/cm² required in non-resonant multiphoton excitation, focal intensities ∼10 MW/cm² are expected to produce an equally strong nonlinear signal. The predicted resolution is up to 30% greater than that of an ideal confocal microscope operating at the same fluorescence wavelength. The resolution improvement over non-resonant two-photon absorption microscopes is about two-fold in all directions.     

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.     

平场复用多焦点结构光照明超分辨显微成像

多焦点结构光照明显微技术(multifocal structured illumination microscopy, MSIM)能在50μm的成像深度内和1Hz的成像速度下实现两倍于衍射极限分辨率的提升,相比传统的宽场结构光照明显微技术,具有较大的成像深度和层析能力,更适合应用于厚样品的长时程三维超分辨成像.然而, MSIM存在成像速度慢、图像处理过程复杂等问题.本文提出了一种基于平场复用多焦点结构光照明的快速超分辨显微成像方法和系统(flat-field multiplexed MSIM, FM-MSIM),通过在照明光路中插入光束整形器件,将高斯光束转变为均为分布的平顶光束,提高激发点阵的强度均匀性和扩大视场;通过将每个衍射受限的激发点沿y方向延长,形成新的多路复用多焦点阵照明图案,提高能量利用率,减少扫描步数,进而提高成像速度和信噪比;结合基于多重测量矢量模型的稀疏贝叶斯学习图像重构算法,简化图像重构步骤,在保证空间分辨率的同时实现至少4倍于传统MSIM的成像速度.在此基础上,利用搭建的FM-MSIM系统进行了BSC细胞微管样片和小鼠肾切片标准样片的超分辨成像实验,实验结果证明...     

随机扫描多光子荧光显微成像系统

多光子荧光显微成像是生物学研究的有力手段,但目前的成像速度难以满足神经成像中快事件检测的需要。针对这一问题,提出了一套随机扫描快速多光子荧光显微成像系统。系统采用二维声光偏转器快速扫描飞秒激发光束,能够以每点10μs的速度对特定的感兴趣区域进行跳跃式扫描,即随机扫描,使得有效的扫描速度大为提高。引入单棱镜补偿方法解决应用声光偏转器带来的色散问题。以170 nm荧光小球为样品,测得系统的横向分辨力为0.3μm,纵向分辨力为1.3μm。给出了随机扫描系统和商品化多光子荧光显微镜对同一个荧光细胞的成像结果,证明了新系统的成像能力。