Simultaneous imaging of multiple focal planes using a two-photon scanning microscope

Despite all the advances in nonlinear microscopy, all existing instruments are constrained to obtain images of one focal plane at a time. In this Letter we demonstrate a two-photon absorption fluorescence scanning microscope capable of imaging two focal planes simultaneously. This is accomplished by temporally demultiplexing the signal coming from two focal volumes at different sample depths. The scheme can be extended to three or more focal planes.     

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.     

Focal plane tuning in wide-field-of-view microscope with Talbot pattern illumination

We have developed a focal plane tuning technique for use in focus-grid-based wide-field-of-view microscopy (WFM). In WFM, the incidence of a collimated beam on a mask with a two-dimensional grid of aperture produced the Talbot images of the aperture grid. The Talbot pattern functioned as a focus grid and was used to illuminate the sample. By scanning the sample across the focus grid and collecting the transmission, we can generate a microscopy image of the sample. By tuning the wavelength of the laser, we can tune the focal plane of the WFM and acquire images of different depth into the sample. Images of a green algae microscope slide were acquired at different focal planes for demonstration.     

长时程双光子成像技术

双光子成像(Two-Photon Imaging)技术以其优越特性被广泛用于活细胞动态三维成像,但光功率极高的短脉冲光对焦平面荧光分子严重的光漂白极大地影响了双光子长时间成像的图像质量,针对双光子荧光漂白问题,本文提出一种优化光照的双光子(Optimized Lighting-Two Photon,OL-TP)成像技术。通过预扫描获取双光子图像分析高低阈值,以预设的高低阈值为标准优化一幅图像中不同区域的光照时长,利用扫描过程中记录的荧光信息和光照时间信息可以重建OL-TP图像,既保证信噪比又降低荧光漂白。重建的OL-TP图像与传统双光子图像基本一致,信噪比略有降低,但图像并未失真。对110 nm的荧光小球样本分别连续取30幅普通双光子和优化光照的双光子图像,到第30幅图时,重建后的优化光照双光子图像比普通双光子图像荧光漂白降低了28.86%。OL-TP通过优化光照时间大幅降低双光子成像的荧光漂白,使双光子荧光显微镜能够更好地对生物样本进行长时间观测。     

基于声透镜成像系统的光声层析成像

光声成像是采用“光激发-超声波成像”的新型成像技术，它是检测强散射介质内部光吸收分布的一种有效医学影像技术.用短脉冲激光照射强散射介质(如生物组织)，强散射介质由于光声效应产生超声信号，使用具有成像能力的声透镜把声压分布成像于像面上，然后利用具有空间分辨能力的阵列光声传感器，把同一像面上的光声信号强度记录下来，最后根据光声信号强度的空间分布进行图像重组.根据成像系统物像共轭原理，同一物平面的光声信号到达像面的时延相等，而不同物面的光声信号到达同一个探测器平面的时延各不相同，因此，利用BOXCAR的门控积分 关键词： 光声层析成像 声透镜 光声信号     

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.     

Dipole‐like backscatter stimulated Raman scattering for in vivo imaging

Stimulated Raman scattering (SRS) scanning microscopy has the potential to enable label‐free in vivo imaging for research and clinical medicine. Volume SRS from focus occurs in the forward scattered direction. Therefore, multiple scattering events are required to direct the light out of the tissue, reducing imaging depth and resolution. Here, a method called Stokes interference SRS (SISRS) is introduced that operates by the addition to the standard pump and stimulated emission probe beams a third beam called the donut beam. The donut is close in wavelength to the probe beam and, after passage through a π phase plate, forms an annular beam in the focal plane with bright nodes above and below focus. The donut beats with the probe beam, and when they destructively interfere with each other, the microscope's 3‐D stimulated emission focal spot is reduced to subwavelength dimensions. A subwavelength focal volume emits a dipole pattern of SRS with forward and backscatter lobes, enabling high‐resolution single‐backscatter imaging from deep within tissues. The reduction of the focal volume also increases the resolution of the scanning image creating imaging beyond the diffraction limit. SISRS imaging may provide in vivo label‐free Raman images comparable with that achieved in stained in vitro tissues in all planes of section. Copyright © 2014 John Wiley & Sons, Ltd.     

Multifocal multiphoton endoscope

We report a miniaturized resonant/non-resonant multi-fiber raster scanner that is paired with a gradient-index lens assembly to achieve a compact and flexible multifocal multiphoton endoscope capable of longitudinal parallel image acquisition. Multiphoton images are obtained simultaneously at three axial depths, separated by ≥4.8 μm, by incorporating three axially offset double clad optical fibers into the miniaturized scanner. The fabricated endoscope has an outer diameter of 3 mm, a rigid length of 4 cm, and acquires images at 4 frames/s per focal plane, with lateral and axial resolutions for two-photon imaging of 0.8 and 10 μm, respectively.     

High-speed 3D imaging based on structured illumination and electrically tunable lens

In this Letter, we present a high-speed volumetric imaging system based on structured illumination and an electrically tunable lens(ETL), where the ETL performs fast axial scanning at hundreds of Hz. In the system,a digital micro-mirror device(DMD) is utilized to rapidly generate structured images at the focal plane in synchronization with the axial scanning unit. The scanning characteristics of the ETL are investigated theoretically and experimentally. Imaging experiments on pollen samples are performed to verify the optical cross-sectioning and fast axial scanning capabilities. The results show that our system can perform fast axial scanning and threedimensional(3D) imaging when paired with a high-speed camera, presenting an economic solution for advanced biological imaging applications.     

小鼠卵母细胞染色体三维双光子荧光图像的轴向衰减

双光子荧光显微镜作为一种高分辨光学仪器,已经被广泛应用于生物样品的非侵入式三维光学成像中。相比共聚焦显微镜,双光子荧光显微镜拥有更深的探测深度。然而,即便如此,在对较厚的生物样品进行非侵入式光学三维成像时,样品的成像质量也往往会随着探测深度的增加而下降。在临床和生物学领域对研究母性遗传起重要作用的小鼠卵母细胞拥有较大的直径(80～100 μm),吸收和散射效应较为明显。本文研究小鼠卵母细胞染色体的三维双光子荧光图像随探测深度增加图像质量的衰减程度。通过对所得图像进行轴向衰减矫正,利用体积作为参数,将矫正前后小鼠卵母细胞内染色体三维双光子荧光图像进行对比。结果表明,由于吸收和散射效应,卵母细胞存在较严重的光学轴向衰减问题,因此,对用双光子荧光三维成像手段获得的小鼠卵母细胞图像进行衰减矫正是有必要的。这为进一步精确定量的研究卵母细胞内染色体的三维构像打下良好的基础。     

Experimental characterization of a two-photon memory

We demonstrate the recording of 100 planes of digital images in a page-oriented two-photon memory and characterize the images in terms of signal-to-noise ratio and bit error rate. Possible error sources in the recording are discussed, and methods for compensating for some of these effects are presented. Looking at the distributions of the normalized bit intensities, we are able to estimate the minimum achievable bit error rate for this system.     

固体浸没透镜出射光场偏振特性研究

在固体浸没透镜近场光存储中, 光的偏振对近场光场分布具有很大的影响. 采用三维时域有限差分法, 对聚焦线偏振高斯光束通过固体浸没透镜后焦平面上各分量场分布及距离焦平面不同位置光斑图样进行了模拟. 结果表明在焦平面上及距离焦平面较近的近场区域, 出射光场在与入射光偏振垂直方向出现显著的场增强现象, 从而使光斑图样呈近似椭圆分布; 而在距焦平面较远的远场区域光斑图样趋向圆形对称分布.     

声透镜对多层样品的光声层析成像

由于光声效应产生光声压分布图像,所以当强散射介质中的模拟吸光组织在受到短脉冲激光照射时,该声压分布会经声透镜成像在像平面上。在像平面上利用线性超声探测器阵列获取光声信号并传递给高速数据采集卡进行数据采集,可由程序重构出光声图像。设计的光声层析成像系统可以采集记录一定深度的数据,成像时只要在所采集到的数据中选取不同列数即可同时获得强散射介质多层样品不同层面的光声图像。实验成功地获得了强散射介质内多层样品不同层面的光声层析图像。该成像方法无需进行复杂的算法重建,且可以同时实现多层样品不同切面的光声成像。     

Optical method of determination of stress at a point

A method of determining stress at a point is suggested here. The effect of bending of a wave front that is due to variations of the refractive index is used to measure different aspects of stresses. A Fourier lens with a cross slit at its front focal plane is used to form interference fringes at planes near its back focal plane. The sample, illuminated by a plane-parallel coherent beam of light, is placed close to a cross slit, and the change in fringe pattern due to axial shift of the spectrum planes of the slits is measured to relate it to the state of stress.     

Magnetic properties of epitaxial-grown exchange-coupled FePt/FeRh bilayer films

The propagation of a laser beam through a micro-lens array (MLA) was simulated using Finite Time Difference Domain (FDTD) method. The intensity distribution at different output planes away from the micro-lens array surface was investigated. As compared to the focal plane, the intensity distribution observed at those out-of-focus planes varies, which is attributed to the interference and diffraction of output laser beams. The simulated results were counter checked by placing a physical MLA under an illumination of a 488 nm continuous wave Argon Ion laser and images were captured for different output planes. Both simulation and experimental results show a great similarity in terms of the distribution patterns. By changing the lens sag height with respect to the lens diameter, the full width at half maximum (FWHM) of the focused laser spot and its corresponding maximum energy flux were analyzed. A FWHM of 160 nm can be achieved by proper selection of lens sag height. It is also found that the energy flux is proportional to numerical aperture (NA).     

Measurement of focal length for a spherical lens with a joint Fourier transform spectrum interferometer

A method of testing the monochromatic focal length of a spherical lens by using a joint Fourier transform spectrum interference technique is proposed and experimentally demonstrated. The focal length is determined by the spacing of the interference fringes resulting from two identical input images and is measured at the back focal plane of the tested lens. The system is easy to adjust and realizes automatic measurement with high accuracy.     

Impurity states and interlayer tunneling in high temperature superconductors

We argue that the scanning tunneling microscope (STM) images of resonant states generated by doping Zn or Ni impurities into Cu-O planes of BSCCO are the result of quantum interference of the impurity signal coming from several distinct paths. The impurity image seen on the surface is greatly affected by interlayer tunneling matrix elements. We find that the optimal tunneling path between the STM tip and the metal (Cu, Zn, or Ni) d(x(2)-y(2)) orbitals in the Cu-O plane involves intermediate excited states. This tunneling path leads to the fourfold nonlocal filter of the impurity state in Cu-O plane that explains the experimental impurity spectra. Applications of the tunneling filter to the Cu vacancy defects and "direct" tunneling into Cu-O planes are also discussed.     

3D behaviour of Frieden filters in confocal imaging

The three-dimensional (3D) focal behaviour of the super-resolving Frieden filters is investigated numerically. It is shown that, as the central bright spot is sharpened, super-giant secondary maximums are formed on the optic axis. These lobes are much higher that the well-known side-lobes inherent to spatial filtering that surround the restricted, utilisable field, whose characteristics in the meridional plane are depicted for various values of the space-bandwidth parameter and for various numbers of terms that compose the window function. The two-terms filter is found to present, for the first time to my knowledge, some axial apodizing properties. To be compatible with practical realisation, the use of this class of filters in a single- and two-photon confocally scanned system is discussed in terms of 3D super-resolution with an intentionally limited light-power loss. It is shown that these filters match particularly well with recently designed axial apodizers for the transmission-mode confocal scanning microscope and provide a 3D intensity point-spread volume reduction of variable amount as high as 37 percent. The filtering process is shown to vary significantly with the mode of operation.     

Visualisation of mitochondria in living neurons with single- and two-photon fluorescence laser microscopy

In this work we investigated the relative merits of conventional single-photon confocal laser scanning fluorescence microscopy (CLSM) and two-photon laser scanning fluorescence microscopy (2p-LSM) for the study of mitochondria in living neurons. Dorsal root ganglion neurons were loaded with the mitochondrion-specific fluorescent dye JC-1, the ratio between red (J-aggregates) and green (monomer) fluorescence of which reflects mitochondrial membrane potential. Cells were illuminated at 488 nm for single-photon excitation or at 870 nm for two-photon excitation. In both modalities we found that mitochondria showed: (i) similar appearance; (ii) similar fluorescence ratio values over both whole cell bodies and individual mitochondria; and (iii) similar responses to mitochondrial uncoupler, which dropped the ratio values by 50%. However, 2p-LSM exhibited several advantages over CLSM: (i) better signal/noise ratio in the green emission channel; (ii) less phototoxicity upon repetitive scanning in the focal plane; and (iii) no significant loss of image quality upon repetitive scans in the z direction. We conclude that, while both techniques enable visualisation of individual mitochondria in living cells, 2p-LSM has significant advantages for physiological work requiring time-lapse experiments or four-dimensional reconstructions of mitochondria.     

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.