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%.     

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     

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.     

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。     

Fourier-transform coherent anti-Stokes Raman scattering microscopy

We report a novel Fourier-transform-based implementation of coherent anti-Stokes Raman scattering (CARS) microscopy. The method employs a single femtosecond laser source and a Michelson interferometer to create two pulse replicas that are fed into a scanning multiphoton microscope. By varying the time delay between the pulses, we time-resolve the CARS signal, permitting easy removal of the nonresonant background while providing high resolution, spectrally resolved images of CARS modes over the laser bandwidth (approximately 1500 cm(-1)). We demonstrate the method by imaging polystyrene beads in solvent.     

Optical sectioning by multiexcitonic ladder climbing in colloidal quantum dots

Optical sectioning is performed by collecting the fluorescent emission of two-exciton states in colloidal quantum dots. The two-exciton state is created by two consecutive resonant absorption events, thus requiring unprecedented low excitation energy and peak powers as low as 10(5) W/cm(2). The depth resolution is shown to be equivalent to that of standard multiphoton microscopy, and it was found to deteriorate only slowly as saturation of the two-exciton state is approached, owing to signal contribution from higher excitonic states.     

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.     

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     

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.     

4Pi microscopy with linear fluorescence excitation

Practical 4Pi microscopy has so far exclusively relied on multiphoton excitation of fluorescence, because the nonlinear suppression of contributions from higher-order sidelobes was mandatory for unambiguous axial superresolution. We show that novel lenses of 74 degrees semiaperture angle enable biological 4Pi microscopy with regular one-photon fluorescence excitation, thus increasing the signal and reducing system complexity and cost. An axial resolution of 95 nm, corresponding to a more than fourfold improvement over confocal microscopy, is verified in the imaging of microtubules in mammalian cells.     

Near-infrared laser scanning confocal microscopy and its application in bioimaging

Near-infrared (NIR) fluorescence imaging is an important imaging technology in deep-tissue biomedical imaging and related researches, due to the low absorption and scattering of NIR excitation and/or emission in biological tissues. Laser scanning confocal microscopy (LSCM) plays a significant role in the family of fluorescence microscopy. Due to the introduction of pinhole, it can provide images with optical sectioning, high signal-to-noise ratio and better spatial resolution. In this study, in order to combine the advantages of these two techniques, we set up a fluorescence microscopic imaging system, which can be named as NIR-LSCM. The system was based on a commercially available confocal microscope, utilizing a NIR laser for excitation and a NIR sensitive detector for signal collection. In addition, NIR fluorescent nanoparticles (NPs) were prepared, and utilized for fluorescence imaging of the ear and brain of living mice based on the NIR-LSCM system. The structure of blood vessels at certain depth could be visualized clearly, because of the high-resolution and large-depth imaging capability of NIR-LSCM.     

滑动焦点光声显微成像方法

在高分辨光声显微成像系统中,只有在有限焦深范围内的吸收体可以获得高分辨率高信噪比的成像,而实际的生物组织往往具有不规则的轮廓,导致成像结果分辨率和信噪比不均匀。提出了一种基于稀疏扫描轮廓的滑动焦点光声显微成像方法,首先在固定的焦点上稀疏扫描轮廓,人工选择轮廓点获取轮廓的稀疏矩阵,然后通过双调和(Biharmonic)插值获取完整的轮廓矩阵,并将轮廓矩阵转化为焦点位置移动矩阵,最后进行二次扫描,根据焦点位置移动矩阵在每次扫描之前调整焦点位置,使得在整个扫描过程中,样品始终处于焦深的范围,从而获得分辨率和信噪比均匀的光声显微图像。通过模型实验和在体皮下血管成像、颅下血管成像,证明了该方法可以有效地提高光声成像的质量。     

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.     

Optical biopsy of liver fibrosis by use of multiphoton microscopy

We demonstrate the application of multiphoton microscopy in diagnosing toxin- (CCl4-) induced liver fibrosis in mice. Although hepatocyte autofluorescence does not vary significantly, different degrees of necrosis and stellate cell proliferation at necrotic sites in livers with fibrosis (ex vivo) can be detected easily from multiphoton-induced autofluorescence images by use of 780-nm excitation. Our result suggests that multiphoton microscopy can be developed into an effective technique for the detection and diagnosis of liver fibrosis in vivo.     

多光子成象术中深层图象损耗恢复的研究

本文在分析混浊介质对共焦三维扫描图象衰减的基础上,建立了多光子激发荧光扫描的深层图象恢复的数学模型.依照此模型对深层图象进行依赖组织光学参量的恢复,并给出了猪表皮三维扫描成象的恢复图象.     

Multimodal nonlinear optical polarizing microscopy of long-range molecular order in liquid crystals

We demonstrate orientation-sensitive multimodal nonlinear optical polarizing microscopy capable of probing orientational, polar, and biaxial features of mesomorphic ordering in soft matter. This technique achieves simultaneous imaging in broadband coherent anti-Stokes Raman scattering, multiphoton excitation fluorescence, and multiharmonic generation polarizing microscopy modes and is based on the use of a single femtosecond laser and a photonic crystal fiber as sources of the probing light. We show the viability of this technique for mapping of three-dimensional patterns of molecular orientations and show that images obtained in different microscopy modes are consistent with each other.     

Pseudopulse near-field subsurface tomography

Decisive success has been achieved in developing the subsurface near-field scanning tomography that overcomes the Rayleigh diffraction limit of a resolution. It is related to the transformation of the multifrequency inverse scattering problem to that for a complex-valued synthesized pulse (pseudopulse). It leads to the integral equation that has maxima in the depth dependence of its kernel and, hence, to the much better depth resolution of tomography. Moreover, the noise related to surface scattering is mainly suppressed in such an approach. This idea is realized here in the microwave subsurface tomography of 3D inhomogeneous dielectric structures. For homogeneous dielectric targets, this approach is applied to obtain holography images of their shape.     

Resolution improvement by nonconfocal theta microscopy

We present a novel scanning fluorescence microscopy technique, nonconfocal theta microscopy (NCTM), that provides almost isotropic resolution. In NCTM, multiphoton absorption from two orthogonal illumination directions is used to induce fluorescence emission. Therefore the point-spread function of the microscope is described by the product of illumination point-spread functions with reduced spatial overlap, which provides the resolution improvement and the more isotropic observation volume. We discuss the technical details of this new method.     

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...     

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.