Fast handheld two-photon fluorescence microendoscope with a 475 microm x 475 microm field of view for in vivo imaging

A fast handheld two-photon fiber-optic fluorescence endoscope for three-dimensional (3D) in vivo cellular imaging is developed. The compact handheld probe of the two-photon endoscope can simply be placed into contact with the target tissue to reveal clear 3D surface and subsurface histological images without biopsy. The new system has, to the best of our knowledge, the largest field of view (FOV) of 475 microm x 475 microm and a 3D imaging volume larger than 475 microm x 475 microm x 250 microm. A real-time two-photon fluorescence image is displayed at 0.4 mm(2)/s. The lateral and axial resolutions of the two-photon fluorescence endoscope are better than 1 and 14.5 microm, respectively.     

In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope

We introduce a compact two-photon fluorescence microendoscope based on a compound gradient refractive index endoscope probe, a DC micromotor for remote adjustment of the image plane, and a flexible photonic bandgap fiber for near distortion-free delivery of ultrashort excitation pulses. The imaging head has a mass of only 3.9 g and provides micrometer-scale resolution. We used portable two-photon microendoscopy to visualize hippocampal blood vessels in the brains of live mice.     

Two-photon fluorescence endoscopy with a micro-optic scanning head

A major obstacle in the race to develop two-photon fluorescence endoscopy is the use of complicated bulk optics to transmit an ultrashort-pulsed laser beam and return the emitted fluorescence signal. We describe an all-fiber two-photon fluorescence microendoscope based on a single-mode optical fiber coupler, a microprism, and a gradient-index rod lens. It is found that the new endoscope exhibits an axial resolution of 3.2 microm and is capable of imaging transverse cross sections of internal cylindrical structures as small as approximately 3.0 mm in diameter. This device demonstrates the potential for developing a real-time diagnostic tool for biomedical research without the need for surgical biopsy and may find applications in photodynamic therapy, microsurgery, and early cancer detection.     

Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation

We report on scanning far- and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited fluorescence images are obtained for 50-200 mW of average power at the sample. A nearly quadratic dependence of fluorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning far-field microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.     

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。     

Integrated multimodal endomicroscopy platform for simultaneous en face optical coherence and two-photon fluorescence imaging

We report an all-fiber-optic scanning, multimodal endomicroscope capable of simultaneous optical coherence tomography (OCT) and two-photon fluorescence (TPF) imaging. Both imaging modalities share the same miniature fiber-optic scanning endomicroscope, which consists of a double-clad fiber with a core operating in single mode at both the OCT (1310 nm) and two-photon excitation (1550 nm) wavelengths, a piezoelectric two-dimensional fiber-optic beam scanner, and a miniature aspherical compound lens suitable for simultaneous acquisition of en face OCT and TPF images. A fiber-optic wavelength division multiplexer was employed in the integrated platform to combine the low coherence OCT light source and the femtosecond two-photon excitation laser into the same optical path. Preliminary imaging results of cell cultures and mouse tissue ex vivo demonstrate the feasibility of simultaneous real-time OCT and TPF imaging in a scanning endomicroscopy setting for the first time.     

Pupil-segmentation-based adaptive optical correction of a high-numerical-aperture gradient refractive index lens for two-photon fluorescence endoscopy

The intrinsic aberrations of high-NA gradient refractive index (GRIN) lenses limit their image quality as well as field of view. Here we used a pupil-segmentation-based adaptive optical approach to correct the inherent aberrations in a two-photon fluorescence endoscope utilizing a 0.8 NA GRIN lens. By correcting the field-dependent aberrations, we recovered diffraction-limited performance across a large imaging field. The consequent improvements in imaging signal and resolution allowed us to detect fine structures that were otherwise invisible inside mouse brain slices.     

衍射透镜补偿声光偏转器扫描飞秒激光的色散

在飞秒激光随机扫描双光子显微成像系统中使用宽带二维声光偏转器扫描飞秒激光,可以增大扫描角度至74 mrad,增大双光子显微成像范围。但宽带二维声光偏转器在大角度扫描时引入的色散较大,造成成像范围边缘的光斑严重畸变,边缘光斑直径达2.3 μm,影响边缘视场的成像质量。为了提高成像质量,设计了一种新的色散补偿方法,基于衍射透镜组成的开普勒望远系统,可以同时补偿不同扫描角度的不同色散。经过色散补偿后成像边缘的光斑直径小于1 μm,使系统获得大范围扫描成像的同时,所有扫描角度的色散都能够得到很好的补偿,在整个视场范围内光斑直径小于1 μm,实现更均匀的荧光激发,均匀成像。     

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。     

Fast-scanning two-photon fluorescence imaging based on a microelectromechanical systems two- dimensional scanning mirror

Towards overcoming the size limitations of conventional two-photon fluorescence microscopy, we introduce two-photon imaging based on microelectromechanical systems (MEMS) scanners. Single crystalline silicon scanning mirrors that are 0.75 mm x 0.75 mm in size and driven in two dimensions by microfabricated vertical comb electrostatic actuators can provide optical deflection angles through a range of approximately16 degrees . Using such scanners we demonstrated two-photon microscopy and microendoscopy with fast-axis acquisition rates up to 3.52 kHz.     

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.     

DHL细胞中5-ALA代谢PpIX的双光子荧光光谱

双光子激发生物组织荧光,激发光仅作用于焦点区域,对生物样品的光漂白性和光毒性都很小,因而双光子荧光显微技术已成为细胞生物学研究的一种新技术。文章采用波长为820 nm飞秒激光激发孵育有5-ALA的DHL细胞,在激光扫描显微镜的Lambda模式中获得单个DHL细胞的双光子荧光光谱,并测量DHL细胞内积聚的卟啉九(PpIX)特征荧光值。获得了浓度分别为2, 4和10 mmol·L-1的5-ALA溶液中,细胞代谢的PpIX含量随孵育时间的变化情况。DHL细胞内积聚的PpIX处于动态变化过程,并呈现出两阶段性的特点：细胞内积聚的PpIX含量随着孵育时间增长而增加,在3 h附近达到最大值,随后随着孵育时间增长反而下降。结果表明,基于激光扫描显微的双光子荧光光谱可成为DHL细胞等白血病细胞摄取5-ALA并生成PpIX的动力学研究的有效方法。     

Acetylene-Substituted Two-Photon Absorbing Molecules With Rigid Elongated Pi-Conjugation: Synthesis,Spectroscopic Properties and Two-Photon Fluorescence Cell Imaging Applications

Two asymmetrical molecules with substituted acetylene as central rigid elongated conjugation are reported as potential chromophores for two-photon microscopic imaging. These molecules consist of a typical D–π–A structure, have different donors (D), the same π-conjugated center (π) and the same acceptor (A). Structural characterization and spectroscopic properties, including single-photon (linear) absorption, quantum yields, single-photon fluorescence, and two-photon absorption spectra, were studied in solvents with different polarity. These acetylene-substituted molecules were found to have high two-photon absorption cross-sections (for example, 690 GM for molecule 1 in toluene), which were determined by a two-photon induced fluorescence method using a femtosecond Ti: sapphire laser as excitation source. Single- and two-photon cellular imaging experiments demonstrate that the substituted acetylene derivatives could be one kind of promising two-photon fluorescence probes for cellular imaging.     

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.     

Rapid-scanning forward-imaging miniature endoscope for real-time optical coherence tomography

We developed a miniature endoscope that is capable of rapid lateral scanning and is suitable for real-time forward-imaging optical coherence tomography (OCT). The endoscope has an outer diameter of 2.4 mm, consisting of a miniature tubular lead zirconate titanate (PZT) actuator, a single-mode fiber-optic cantilever, and a graded-index lens. Rapid lateral scanning at 2.8 kHz is achieved when the fiber-optic cantilever is resonated with the PZT actuator. This allows OCT imaging to be performed by fast lateral beam scanning followed by slow depth scanning, which is different from the conventional OCT imaging sequence. Real-time OCT imaging with the endoscope operated in the new image acquisition sequence at 6 frames/s is demonstrated.     

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.     

Fabrication of a micro-optical lens using femtosecond laser 3D micromachining for two-photon imaging of bio-tissues

We report, for the first time to our knowledge, on the application of a micro-optical lens fabricated by three-dimensional (3D) femtosecond laser direct writing for two-photon fluorescence imaging of biological tissues. We show that the two-photon fluorescence images of a plant leaf tissue acquired with the micro-optical lens are comparable to that of a 5× objective lens. Our result represents an important step towards the application of micro-optical components fabricated by femtosecond laser micromachining in miniaturized nonlinear fluorescence microscopy applications, such as two-photon endoscopy.     

Plasmonic-enhanced two-photon fluorescence with single gold nanoshell

Single gold nanoshell with mutilpolar plasmon resonances is proposed to enhance two-photon fluorescence efficiently.The single emitter single nanoshell configuration is studied systematically by employing the finite-difference time-domain method.The emitter located inside or outside the nanoshell at various positions leads to a significantly different enhancement effect.The fluorescent emitter placed outside the nanoshell can achieve large fluorescence intensity given that both the position and orientation of the emission dipole are optimally controlled.In contrast,for the case of the emitter placed inside the nanoshell,it can experience substantial two-photon fluorescence enhancement without strict requirements upon the position and dipole orientations.Metallic nanoshell encapsulating many fluorescent emitters should be a promising nanocomposite configuration for bright two-photon fluorescence label.The results provide a comprehensive understanding about the plasmonic-enhanced two-photon fluorescence behaviors,and the nanocomposite configuration has great potential for optical detecting,imaging and sensing in biological applications.     

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.     

Simultaneous multichannel nonlinear imaging: combined two-photon excited fluorescence and second-harmonic generation microscopy

Simultaneous two-photon excited fluorescence (TPF) and second-harmonic generation (SHG) imaging is demonstrated using a single femtosecond laser and a scanning microscope. This composite nonlinear microscopic technique was applied to imaging DNA and chromosomes, and it was shown that the two different interaction mechanisms provide complementary information on the structure and nonlinear properties of these biological materials, beyond that achievable using either TPF or SHG imaging alone. The use of separate modes of detection, in reflection and transmission respectively, and the simultaneous nature of the acquisition of the two images allows pure TPF and SHG images in precise registration to be obtained.