Second-harmonic-generation microscope with a microlens array scanner

We propose a multifocus scanning technique in second-harmonic-generation (SHG) microscopy in which the SHG detection efficiency and the image acquisition rate are improved by several tens of times compared with typical single-focus scanning techniques. Because of a microlens array scanner, the laser beam of a mode-locked Ti:sapphire laser is split into ~100 beamlets, and the same number of foci are formed in the specimen. Parallel scanning of the foci enables us to observe real-time images of the specimen. The imaging properties and the usefulness of the microscope are demonstrated by SHG images of living HeLa cells and rat cardiac myocytes.     

Improved sectioning in a slit scanning confocal microscope

We describe a simple implementation of a slit scanning confocal microscope to obtain an axial resolution better than that of a point-scanning confocal microscope. Under slit illumination, images of a fluorescent object are captured using an array detector instead of a line detector so that out-of-focus light is recorded and then subtracted from the adjacent images. Axial resolution after background subtraction is 2.2 times better than the slit confocal resolution, and out-of-focus image suppression is calculated to attenuate with defocus faster by 1 order of magnitude than in the point confocal case.     

Hybrid Rayleigh,Raman and two‐photon excited fluorescence spectral confocal microscopy of living cells

A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low‐wavenumber‐resolution Raman imaging, Rayleigh scatter imaging and two‐photon fluorescence (TPE) spectral imaging, fast ‘amplitude‐only’ TPE‐fluorescence imaging and high‐spectral‐resolution Raman imaging. This multi‐dimensional fluorescence–Raman microscopy platform enables rapid imaging along the fluorescence emission and/or Rayleigh scatter dimensions. It is shown that optical contrast in these images can be used to select an area of interest prior to subsequent investigation with high spatially and spectrally resolved Raman imaging. This new microscopy platform combines the strengths of Raman ‘chemical’ imaging with light scattering microscopy and fluorescence microscopy and provides new modes of correlative light microscopy. Simultaneous acquisition of TPE hyperspectral fluorescence imaging and Raman imaging illustrates spatial relationships of fluorophores, water, lipid and protein in cells. The fluorescence–Raman microscope is demonstrated in an application to living human bone marrow stromal stem cells. Copyright © 2009 John Wiley & Sons, Ltd.     

基于成像机理的小波包变换多聚焦图像融合

由于可见光成像系统的聚焦范围有限,因而在成像过程中,除聚焦良好的物体能生成清晰的图像外,该物体前后一定距离外的所有物体都将呈现不同程度的模糊.为了获得场景内所有物体均清晰的图像,在分析了多聚焦图像成像机理的基础上,提出了一种基于小波包变换的融合方法.它是将成像系统先聚焦在一部分对象上,得到其清晰的图像;然后再将其聚焦在另一部分对象上,得到另一清晰的图像;最后把这两幅实验图像加以融合,从而获得场景内所有物体均清晰的图像.实验结果表明,基于小波包变换的融合方法能够将信号的频带进行多层次划分,对高频成分也能进一步地分解,可有效综合多聚焦图像.     

Addressable, large-field second harmonic generation microscopy based on 2D acousto-optical deflector and spatial light modulator

We present an addressable, large-field second harmonic generation microscope by combining a 2D acousto-optical deflector with a spatial light modulator. The SLM shapes an incoming mode-locked, near-infrared Ti:Sapphire laser beam into a multifocus array, which can be rapidly scanned by changing the incident angle of the laser beam using a 2D acousto-optical deflector. Compared to the single-beam-scan technique, the multifocus array scan can increase the scanning rate and the field-of-view size with the multi-region imaging ability.     

Combined Raman and continuous-wave-excited two-photon fluorescence cell imaging

We demonstrate a confocal optical microscope that combines cw two-photon-excited fluorescence microscopy with confocal Raman microscopy. With this microscope fast image acquisition with fluorescence imaging can be used to select areas of interest for subsequent chemical analysis with spontaneous Raman imaging. The distribution of the UV-absorbing fluorophore Hoechst 33342 in the apoptotic HeLa cells is measured in the combined cw two-photon-excited fluorescence and Raman microscopy modes. The 647-nm line of a Kr-ion laser is used to excite both the Raman scattering and the two-photon-excited fluorescence emission. The lateral and axial resolutions in the two imaging modes are compared by use of the Gaussian beam approximation and backprojection of the focal volume through the confocal pinhole.     

Surface‐sensitive Raman microscopy with total internal reflection illumination

A Raman microscope using a total internal reflection (TIR) annular illumination geometry through a ZnSe solid immersion lens (SIL) is described. Spectra of a thin‐film sample of the transparent organic conductor poly(3,4‐ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS) on a polyethylene terephthalate (PET) substrate are presented and compared with those from a conventional confocal Raman configuration. These spectra demonstrate a significant increase in surface selectivity upon the use of TIR illumination, as the decay length of the evanescent excitation field limits the depth of sample probed in this configuration. Spectral interference from the underlying PET substrate layer is thus greatly reduced. An increase in surface selectivity is also demonstrated for spectra acquired through the SIL with uniform illumination. Raman images of a micropatterned PEDOT:PSS film acquired with TIR illumination are also reported. Enhanced lateral resolution is realized in this configuration because of the immersion effect of the SIL, and the sampling depth is limited to 150 nm by the choice of illumination geometry. This results in analysis volumes on the order of tens of femtoliters, nearly two orders of magnitude smaller than typically achieved in conventional confocal Raman microscopes. This approach yields Raman spectra and images with surface selectivity significantly greater than can be achieved in confocal Raman, and provides a valuable tool for the microanalysis of thin surface films. Published in 2010 by John Wiley & Sons, Ltd.     

Multifocal multiphoton microscopy based on a spatial light modulator

We present a new multifocal multiphoton microscope that employs a programmable spatial light modulator to generate dynamic multifocus arrays which can be rapidly scanned by changing the incident angle of the laser beam using a pair of galvo scanners. Using this microscope, we can rapidly select the number and the spatial density of focal points in a multifocus array, as well as the locations and shapes of arrays according to the features of the areas of interest in the field of view without any change to the hardware.     

A detection of atmospheric relative humidity profile by UV Raman lidar

A new spectroscopic filter constructed with a high-spectral-resolution grating and two narrow-band mirrors is designed to separate the elastic scattering and the vibrational Raman scattering spectra in an ultraviolet (UV) Raman lidar system. The density of humidity and water vapor mixing ratio are calculated from the vibrational Raman scattering signals of N₂ and H₂O. Water vapor mixing ratio is retrieved from this development. With this measured water vapor mixing ratio, the relative humidity is calculated with atmospheric temperature profile obtained by another Raman temperature lidar. Preliminary experiments and comparison results between lidar and radiosonde showed that the UV Raman lidar system has the capability for profiling the water vapor mixing ratio up to a height of 2 km with less than 10% of the uncertainty under the conditions of laser energy of 300 mJ and signal-averaging time of 10 min.     

红细胞显微激光共焦拉曼散射光谱扫描技术研究

采用显微激光共焦拉曼散射光谱扫描系统对活态红细胞进行拉曼光谱(点测定、线扫描、二维扫描)测定及成像的技术与方法进行了研究,并对514 nm激光对红细胞在拉曼扫描中的影响进行了评价。通过扫描前后细胞的拉曼光谱变化和亮场图像变化确定了在不同扫描模式下既可获得较好的拉曼散射信号,又不会影响细胞生命活动与功能的合适扫描参数的设置。对于点扫描模式,样品激光功率是重点调节的参数,一般应小于1.5 mW。对于线扫描模式,照射激光功率和扫描间隔(步进)是要重点关注的参数。小扫描间隔意味着激光能量相对聚集,易对细胞造成损伤;大扫描间隔可以较好地降低激光对细胞的损伤,但是空间分辨率会因此而下降。对于线扫描,建议扫描间隔大于0.5 μm、照射激光功率小于0.7 mW。对于二维扫描,除照射激光功率、扫描间隔需要调节外,其他扫描参数也要作相应调节以降低激光对红细胞的影响,可适当降低样品温度和增加共焦孔径尺寸降低二维扫描过程中激光对红细胞的影响。1.0 μm扫描间隔、0.7 mW的照射激光功率和500 μm共焦孔径,以及样品温度适当调低可得到较好的二维红细胞拉曼图像。对于所有扫描模式,如果得到的红细胞的拉曼信号足够强,也可适当降低曝光积分时间以降低激光对红细胞的影响。实验前进行实验过程的优化对活态细胞的拉曼测试也非常重要。     

拉曼光谱编码的荧光液相生物芯片检测系统研究

随着医疗诊断需求的增加,生物分子检测技术越来越受到人们的重视,液相生物芯片技术作为一种高通量,多通道的分子检测手段在近几年得到了飞速发展。通过层层自组装方法制备以微片为载体的拉曼光谱编码液相生物芯片,并利用自行搭建的一套高灵敏度、高分辨率的光学系统,实现对液相生物芯片的定性与定量分析。光学系统由拉曼光谱检测系统与荧光显微成像系统耦合而成。在拉曼光谱检测系统中激光器发射出785 nm波长的激光,通过二向色镜,带反反射镜与物镜汇聚到样品上,样品产生的拉曼散射光,经物镜,带反反射镜,二向色镜与拉曼滤波片,最后通过凹透镜聚焦到光谱仪的狭缝上,光谱仪色散实现在线阵CCD上拉曼光谱的获取。荧光显微成像系统应用光学成像原理,通过调节凹透镜与405 nm的激发光之间的距离,使激发光通过物镜均匀的照射到样品之上,样品激发出的荧光,通过物镜,带反反射镜,二向色镜,滤波片与相应的凹透镜,最后成像到面阵CCD上。改进传统便携式拉曼光谱检测系统光路并选用相应波段的带反反射镜与焦距20倍的物镜完成拉曼光谱检测系统与荧光显微成像系统的耦合。为了减少两路系统之间的相互影响选用合适的二向色镜以及滤波片,在提高耦合系统获取数据的准确性中有着重要的作用。该系统通过对反应之后的液相生物芯片进行拉曼光谱检测,以完成对每个编码玻片的定性识别,即解码;同时激发反应后液相生物芯片的荧光并采集荧光强度图,根据每个解码玻片上的荧光强度值完成对目标检测物的定量分析。区别于传统荧光编码液相生物芯片, 拉曼光谱编码具有稳定性更强,光谱分辨率更高等优点。该光学系统集拉曼光谱检测系统与荧光显微成像系统于一体,解决了目前未有基于拉曼编码的液相生物芯片的检测系统的问题,并且可同时对多种目标物进行识别和定量分析,提升了实验结果的准确性。     

Multifocus image fusion and denoising scheme based on homogeneity similarity

A novel image fusion algorithm based on homogeneity similarity is proposed in this paper, aiming at solving the fusion problem of clean and noisy multifocus images. Firstly, the initial fused image is acquired with one multiresolution image fusion method. The pixels of the source images, which are similar to the corresponding initial fused image pixels, are considered to be located in the sharply focused regions. By this method, the initial focused regions are determined. In order to improve the fusion performance, morphological opening and closing are employed for post-processing. Secondly, the homogeneity similarity is introduced and used to fuse the clean and noisy multifocus images. Finally, the fused image is obtained by weighting the neighborhood pixels of the point of source images which are located at the focused region. Experimental results demonstrate that, for the clean multifocus image fusion, the proposed method performs better than some popular image fusion methods in both subjective and objective qualities. Furthermore, it can simultaneously resolve the image restoration and fusion problem when the source multifocus images are corrupted by the Gaussian white noise, and can also provide better performance than the conventional methods.     

High-contrast microscopy of semiconductor and metal sites in integrated circuits by detection of optical feedback

High-contrast microscopy of semiconductor and metal sites in integrated circuits is demonstrated with laser-scanning confocal reflectance microscopy, one-photon (1P) optical-beam-induced current (OBIC) imaging, and detection of optical feedback by means of a commercially available semiconductor laser that also acts as an excitation source. The confocal microscope has a compact in-line arrangement with no external photodetector. Confocal and 1P OBIC images are obtained simultaneously from the same focused beam scanned across the sample plane. Image pairs are processed to generate exclusive high-contrast distributions of semiconductor, metal, and dielectric sites in a GaAs photodiode array sample.     

Subdiffraction‐limited chemical imaging of patterned phthalocyanine films using tip‐enhanced near‐field optical microscopy

A tip‐enhanced near‐field optical microscope, based on a shear‐force atomic force microscope with plasmonic tip coupled to an inverted, confocal optical microscope, has been constructed for nanoscale chemical (Raman) imaging of surfaces. The design and validation of the instrument, along with its application to near‐field Raman mapping of patterned organic thin films (coumarin‐6 and Cu(II) phthalocyanine), are described. Lateral resolution of the instrument is estimated at 50 nm (better than λ/10), which is roughly dictated by the size of the plasmonic tip apex. Additional observations, such as the distance scaling of Raman enhancement and the inelastic scattering background generated by the plasmonic tip, are presented. Copyright © 2016 John Wiley & Sons, Ltd.     

Ultrafast, large-field multiphoton microscopy based on an acousto-optic deflector and a spatial light modulator

We present an ultrafast, large-field multiphoton excitation fluorescence microscope with high lateral and axial resolutions based on a two-dimensional (2-D) acousto-optical deflector (AOD) scanner and spatial light modulator (SLM). When a phase-only SLM is used to shape the near-infrared light from a mode-locked titanium:sapphire laser into a multifocus array including the 0-order beam, a 136 μm × 136 μm field of view is achieved with a 60× objective using a 2-D AOD scanner without any mechanical scan element. The two-photon fluorescence image of a neuronal network that was obtained using this system demonstrates that our microscopy permits observation of dynamic biological events in a large field with high-temporal and -spatial resolution.     

茶氨酸拉曼光谱分析

采用显微共焦拉曼光谱技术测试研究了L-茶氨酸,得到了L-茶氨酸的拉曼谱图,发现在250～1 700和2 800～3 000cm-1能够观测到明显的拉曼信号;通过对谱峰进行归属和分析,得到了不同波数范围内的特征振动模式;其中,在321,900,938,1 153,1 312,1 358,1 454和1 647cm-1找到8个较强的拉曼信号,可作为L-茶氨酸的特征峰。结果表明:拉曼光谱可能成为一种直接、准确和快速的检测茶氨酸的方法。     

Confocal scanning laser ophthalmoscopy improvement by use of Mueller-matrix polarimetry

A new technique for improving the signal-to-noise ratio and the contrast in images recorded with a confocal scanning laser system is presented. The method is based on the incorporation of a polarimeter into the setup. After the spatially resolved Mueller matrix of a sample was calculated, images for incident light with different states of polarization were reconstructed, and both the best and the worst images were computed. In both the microscope and the opthalmoscope modes, the best images are better than the originals. In contrast, the worst images are poorer. This technique may be useful in different fields such as confocal microscopy and retinal imaging.     

共焦扫描光学显微镜的高分辨率

讨论了共焦扫描光学显微镜的高分辨率性质,指出共焦扫描显微镜由于采用点探测器,因而视场大大减小,信噪比大大提高,同时每幅图像逐点扫描形成,在光学系统信息能力不变的前提下,系统的空间域通带宽度增加和时域通带宽度减小。因而可成高分辨率的像,特别是其独特的深度分辨率特性使得可以实现光学断层扫描成像。给出了所研制的共焦扫描荧光显微镜所获得光学断层扫描图像     

Mapping the intracellular distribution of carbon nanotubes after targeted delivery to carcinoma cells using confocal Raman imaging as a label-free technique

The uptake of carbon nanotubes (CNTs) by mammalian cells and their distribution within cells is being widely studied in recent years due to their increasing use for biomedical purposes. The two main imaging techniques used are confocal fluorescence microscopy and transmission electron microscopy (TEM). The former, however, requires labeling of the CNTs with fluorescent dyes, while the latter is a work-intensive technique that is unsuitable for in situ bio-imaging. Raman spectroscopy, on the other hand, presents a direct, straightforward and label-free alternative. Confocal Raman microscopy can be used to image the CNTs inside cells, exploiting the strong Raman signal connected to different vibrational modes of the nanotubes. In addition, cellular components, such as the endoplasmic reticulum and the nucleus, can be mapped. We first validate our method by showing that only when using the CNTs' G band for intracellular mapping accurate results can be obtained, as mapping of the radial breathing mode (RBM) only shows a small fraction of CNTs. We then take a closer look at the exact localization of the nanotubes inside cells after folate receptor-mediated endocytosis and show that, after 8-10 h incubation, the majority of CNTs are localized around the nucleus. In summary, Raman imaging has enormous potential for imaging CNTs inside cells, which is yet to be fully realized.     

About the SDS inclusion in PDMS/TEOS ORMOSIL: a vibrational spectroscopy and confocal Raman scattering study

Organically modified silicates (ORMOSILs) are attractive materials due to their vast applicability and easy synthesis. The doping of these materials with sodium dodecyl sulfate (SDS) is interesting in the search for good protonic conductors. The inclusion of different concentrations of SDS in ORMOSIL membranes is investigated in the present work using Raman and infrared spectroscopy, confocal Raman microscopy and confocal imaging microscopy. The spectroscopic measurements allow us to assign the vibrational modes to the chemical groups of the structures of SDS and ORMOSIL. Furthermore, these measurements show that these materials are composites, as no interactions are observed between the SDS and the ORMOSIL matrix. The confocal Raman and confocal imaging techniques are useful to study qualitatively the SDS insertion on the surface of ORMOSIL. It was observed that the SDS sizes are very nonuniform. Copyright © 2011 John Wiley & Sons, Ltd.