用于激光共聚焦显微镜的光谱扫描机构

随着生物医学技术的发展,组织样本经常被多种荧光标记物标记,需要通过光谱成像的方法区分出样本中不同的成分。本文在共聚焦显微镜基础上,介绍了一种由精密丝杠和步进电机控制的狭缝机构实现光谱成像的方法,讨论了狭缝缝片的具体设计和狭缝运动精度对光谱带宽和波长准确度的影响。     

多光谱阴道镜的微型化多通道滤光片设计

设计了一种可与阴道镜中的图像传感器集成的微型化多通道滤光片,以使阴道镜具有光谱成像能力.采用微光刻技术及真空多层镀膜技术制成微滤光片,内部微滤光单元的通光波长与病灶标志物反射光谱或荧光光谱的特征峰相对应,与图像传感器集成后,能够获取几幅与光谱特征峰对应的特征波长图像,这些图像凸显了病变组织与正常组织的差异,提供了宫颈组织上皮表面形态变化及上皮组织内病灶标志物含量变化的信息.已加工出通光波长为λ1＝630 nm、λ2＝460 nm、λ3=515 nm、λ4＝577 nm的微滤光片,微滤光单元面积为10 μm×10 μm,各通道带宽约为40 nm,透射率为30％～40％.实验显示微滤光片的光学性能已达到光谱成像的基本要求,与阴道镜集成后,能有效提高其诊断的灵敏度与特异性,减少活检频率.     

显微荧光光谱成像仪的研究与设计

将光谱分析技术快速、灵敏、准确等独特的优点,与光学成像技术的定位记录特点融合、构成光谱成像技术,可以提供分析试样中各种化学或生化成份的分布图像,因而可以获得定性、定量和定位综合、更丰富的分析信息。报导在商售落射荧光显微镜基础上,实现显微镜荧光光谱成像技术的理论基础、系统组成、设计技术关键等一系列研究工作,设计研制成了可在250~680nm波长范围内自动扫描的光纤激发显微荧光光谱成像仪样机,并获得了满意的实验结果。     

中/长波红外双衍射级次共路Offner成像光谱仪

为提高成像光谱仪的工作波长范围,提出了基于双波段焦平面探测器(FPAs)的双衍射级次全共路Offner成像光谱仪结构。该结构中凸面光栅的一级衍射光和二级衍射光完全重叠共路传输,并可由焦平面处的双波段红外焦平面探测器IR FPAs实现级次的自然分离和同时探测。分析了该结构的工作原理和设计方法,基于几何光线追迹法仿真了谱线弯曲和色畸变特性,基于Huygens点扩散函数(PSF)仿真了光谱响应函数(SRF)并导出了光谱带宽。实验显示:双衍射级次共路Offner成像光谱仪的工作波段为3~6μm(二级衍射)和6~12μm(一级衍射),谱线弯曲和色畸变均小于0.5个像元宽度,光谱带宽分别为13.2~14.3nm(二级衍射)和28.3~33.3nm(一级衍射),两个工作波段内的衍射效率均大于或等于20%。整个系统结构简单紧凑、光谱范围宽,满足对地物或深空目标的中等分辨率的中远红外光谱探测需求。     

一种采用微硅片狭缝的新型微小型光纤光谱仪

研制了一种采用微硅片狭缝代替传统机械狭缝的微小型光纤光谱仪。采用MEMS(微机电系统)工艺制造出了体积小、厚度薄的一体式微硅片狭缝,并分析了微硅片狭缝的狭缝不平直度对微小型光纤光谱仪分辨率的影响,通过测试系统分辨率的实验,验证了采用微硅片狭缝的可行性。同时,对微小型光纤光谱仪的光谱带宽和像元分辨力进行了讨论,为波长的标定提供了一种理论依据。通过对采集的汞灯谱线的分析和MATLAB的精确计算,验证了所提出的通用波长-像元迭代公式的正确性,从而研制出了一种采用微硅片狭缝的半峰全宽为0.85 nm,波长标定精度小于0.2 nm,体积为50 mm×46 mm×14 mm的微小型光纤光谱仪。     

超光谱成像仪的精细光谱定标

为了精细标定棱镜色散超光谱成像仪1024×80光谱像元的中心波长和响应带宽,建立了一套光谱定标装置,提出了实现1nm光谱定标精度的定标方法。首先,介绍了产生谱线弯曲与谱线倾斜的原因,确定了精细光谱定标的方法和数据处理算法;然后,利用光谱定标装置测定了全部光谱响应像元的离散单色光响应值,利用高斯方程拟合了相对光谱响应曲线;最后,建立了中心波长矩阵表和带宽矩阵表,采用多项式拟合算法确定了空间视场像元的色散方程和光谱通道谱线弯曲方程,实验测定了温度变化谱线漂移结果。另外,还对光谱定标精度对辐射定标精度的影响进行了分析。光谱定标结果表明:超光谱成像仪的光谱定标精度达到了±1nm,各谱段带宽平均为8.75nm;色散方程及谱线弯曲与设计结果相符,谱线弯曲值为14～19nm,平均值为17nm;1nm的定标精度对辐射定标精度的影响分别小于1%(3000K黑体)和0.25%(6000K黑体),满足超光谱成像仪1nm光谱定标精度的要求。     

用于沿海水色探测的机载紫外-可见-近红外高光谱成像仪

本文主要设计一种新型的可用于机载的紫外-可见-近红外高光谱成像系统,从而为沿海水色环境与污染观测提供一种有效的观测仪器。首先,根据探测目标特点确定了仪器系统的性能设计参数,选择了Dyson成像光谱系统来满足系统在宽谱段上的高信噪比和高光学性能;但Dyson成像光谱系统的结构过于紧凑,因此对Dyson成像光谱系统进行了研究,调整了狭缝、像面和光学元件的位置,使它们在轴向和垂直轴向上均具备足够的间隔,并在这种大空气间隔下分析了系统的完善消像差条件。通过光程分析和弯月透镜的加入,使改进型Dyson系统在0.278的数值孔径和320～1 000 nm的宽波段上具备良好的成像结果,全视场全波段MTF值在探测器奈奎斯特频率下(38.5 lp/mm)高于0.5,研制原理样机的光谱分辨率为3.375 nm,满足设计要求。该系统可为沿海水色环境的高光谱观测提供良好的工程应用基础。     

近红外光谱用于过程分析

1 近红外光谱分析技术的崛起 近红外光是介于可见光和中红外光（常称作红外光）之间的一个波段（700～2550nm），当一束复合光穿射样品后，如果被照射样品的分子选择性地吸收某些波段的光，则产生吸收光谱。吸收光的频率与分子自身的振动能态有关。已知在700～2550nm范围内的近红外光谱带对应于分子基频振动的倍频和组合频，其吸收信号弱，谱带宽，重叠严重，但与有关化学基团是密切相关的。     

基于LCTF多光谱成像的人体指甲特征光谱提取

利用由液晶可调谐滤光片(LCTF)和CCD相机组成的多光谱成像系统在450～1 000 nm光谱范围内每隔10 nm采集人体指甲样本,得到包含56个波段的人体指甲多光谱图像。通过参考白板比较测量法进行反射率反演,得到指甲的准确反射率信息,分别利用主成分分析法(PCA)和波段指数法实现样本图像的降维,得到两个特征空间,并利用光谱角度填图法(SAM)在两个特征空间内对人体指甲进行分类,分类准确度分别为92.5%及82.9%。因此,由主成分分析法得到的特征空间可以作为人体指甲的特征光谱,为指甲多光谱图谱分析和人体健康评估提供了可靠的依据。     

基于PCA的时间分辨油荧光光谱分析及优化

激光诱导荧光技术可广泛应用于油污染的监测中,然而普通的油荧光光谱技术只能实现油污染监测的粗分类,无法区分原油与燃料油的荧光特征。本文基于主成分分析方法(PCA)的时间分辨油荧光分类方法,实验测量了20种油样本的时间分辨荧光光谱特征,给出了对应的荧光寿命和时间分辨油荧光光谱的时序特征。在此基础上,利用前三个主成分构成的三维特征矢量空间,通过分析不同采集时刻下油样本矢量间相关距离的变化,对油样本的时间分辨荧光光谱进行聚类分析。为了体现油荧光变化的时序性,引入矢量距离的离散度参量,提出基于PCA进行时间分辨油荧光光谱分析的优化方法。实验结果表明,基于时间分辨油荧光光谱识别可实现原油与燃料油的光谱时序特征区分,具备良好的油荧光分类效果。     

Analysis of fluorescence from algae fossils of the Neoproterozoic Doushantuo formation of China by confocal laser scanning microscope

Chinese algae fossils can provide unique information about the evolution of the early life. Thin sections of Neoproterozoic algae fossils, from Guizhou, China, were studied by confocal laser scanning microscopy, and algae fossils were fluorescenced at different wavelengths when excited by laser light of 488 nm, 476 nm, and 568 nm wavelength. When illuminated by 488 nm laser light, images of the algae fossils were sharper and better defined than when illuminated by 476 nm and 568 nm laser light. The algae fossils fluoresce at a wide range of emission wavelengths. The three-dimensional images of the fluorescent algae fossils were compared with the transmission images taken by light microscope. We found that the fluorescence image of the confocal laser scanning microscope in a single optical section could pass for the transmission image taken by a light microscope. We collected images at different sample depths and made a three-dimensional reconstruction of the algae fossils. And on the basis of the reconstruction of the three-dimensional fluorescent images, we conclude that the two algae fossils in our present study are red algae.     

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.     

Autofluorescence spectroscopy and imaging of Platymonas subcordiformis irradiated by diode laser based on LSCM

Autofluorescence spectra and optical imaging of Platymonas subcordiformis after irradiation of diode laser were observed via laser scanning confocal microscopy (LSCM). With 488 nm Ar(+) laser excitation, the horizontal and vertical dimensions of a cup-shaped chloroplast of the irradiation group increased about 10% compared with the control group. The fluorescence spectra were similar between irradiation group and control group with a maximum fluorescence band around 682 nm, whereas the former has a higher intensity. Image of a small circular substance with stronger two-photon autofluorescence (TPA) was obtained when using two-photon excitation wavelength of 800 nm in single-channel mode. Further analysis by the 800 nm excitation based on two independent-channels mode showed an emission band of the small circular substance around 376-505 nm, which corresponded to the eyespot of P. subcordiformis. In lambda scanning mode, with two-photon wavelength of 800 nm excitation, six fluorescence peaks that are located at 465, 520, 560, 617, 660 and 680 nm were observed; the fluorescence intensity of the irradiation group was higher than that of the control group, especially at 520, 560 and 617 nm. As a conclusion, diode laser irradiation can promote chloroplast growth of P. subcordiformis cells in the form of expanding area and the increasing content of protein, phospholipids and chlorophyll. LSCM, especially TPA imaging based on femtosecond laser excitation, provides a nondestructive, real-time and accurate method to study changes of living algal cells under laser irradiation and other environmental factors.     

A white light confocal microscope for spectrally resolved multidimensional imaging

Spectrofluorometric imaging microscopy is demonstrated in a confocal microscope using a supercontinuum laser as an excitation source and a custom‐built prism spectrometer for detection. This microscope system provides confocal imaging with spectrally resolved fluorescence excitation and detection from 450 to 700 nm. The supercontinuum laser provides a broad spectrum light source and is coupled with an acousto‐optic tunable filter to provide continuously tunable fluorescence excitation with a 1‐nm bandwidth. Eight different excitation wavelengths can be simultaneously selected. The prism spectrometer provides spectrally resolved detection with sensitivity comparable to a standard confocal system. This new microscope system enables optimal access to a multitude of fluorophores and provides fluorescence excitation and emission spectra for each location in a 3D confocal image. The speed of the spectral scans is suitable for spectrofluorometric imaging of live cells. Effects of chromatic aberration are modest and do not significantly limit the spatial resolution of the confocal measurements.     

The use of laser scanning confocal microscopy (LSCM) in materials science

Laser scanning confocal microscopes are essential and ubiquitous tools in the biological, biochemical and biomedical sciences, and play a similar role to scanning electron microscopes in materials science. However, modern laser scanning confocal microscopes have a number of advantages for the study of materials, in addition to their obvious uses for high resolution reflected and transmitted light optical microscopy. In this paper, we provide several examples that exploit the laser scanning confocal microscope's capabilities of pseudo-infinite depth of field imaging, topographic imaging, photo-stimulated luminescence imaging and Raman spectroscopic imaging.     

Use of a white light supercontinuum laser for confocal interference-reflection microscopy

Shortly after its development, the white light supercontinuum laser was applied to confocal scanning microscopy as a more versatile substitute for the multiple monochromatic lasers normally used for the excitation of fluorescence. This light source is now available coupled to commercial confocal fluorescence microscopes. We have evaluated a supercontinuum laser as a source for a different purpose: confocal interferometric imaging of living cells and artificial models by interference reflection. We used light in the range 460-700 nm where this source provides a reasonably flat spectrum, and obtained images free from fringe artefacts caused by the longer coherence length of conventional lasers. We have also obtained images of cytoskeletal detail that is difficult to see with a monochromatic laser.     

Surface investigations on HgBr2 single crystals by using confocal scanning laser microscopy

A confocal scanning laser microscope operating at 514 and 488 nm has been used to obtain two-dimensional (2-D) images of the mercuric bromide (HgBr2) crystal surface by photoluminescence, reflection, and transmission phenomena. Our measurements indicate that regions showing a strong photoluminescence may appear on the surface. By processing the 2-D images. we obtained the three-dimensional images, which offer a better possibility for the investigation. The analysis of spectral lines may be correlated with the presence of the Hg impurities.     

Novel lambda FRET spectral confocal microscopy imaging method

We report a highly specific, sensitive, and robust method for analyzing fluorescence resonance energy transfer (FRET) based on spectral laser scanning confocal microscopy imaging. The lambda FRET (lambdaFRET) algorithm comprises imaging of a FRET sample at multiple emission wavelengths rendering a FRET spectrum, which is separated into its donor and acceptor components to obtain a pixel-based calculation of FRET efficiency. The method uses a novel off-line precalibration procedure for spectral bleed-through correction based on the acquisition of reference reflection images, which simplifies the method and reduces variability. LambdaFRET method was validated using structurally characterized FRET standards with variable linker lengths and stoichiometries designed for this purpose. LambdaFRET performed better than other well-established methods, such as acceptor photobleaching and sensitized emission-based methods, in terms of specificity, reproducibility, and sensitivity to distance variations. Moreover, lambdaFRET analysis was unaffected by high fluorochrome spectral overlap and cellular autofluorescence. The lambdaFRET method demonstrated outstanding performance in intra- and intermolecular FRET analysis in both fixed and live cell imaging studies.     

Scanning microphotolysis: A new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging

The fluorescence photobleaching method has been widely used to study molecular transport in single living cells and other microsystems while confocal microscopy has opened new avenues to high-resolution, three-dimensional imaging. A new technique, scanning microphotolysis (Scamp), combines the potential of photobleaching, beam scanning and confocal imaging. A confocal scanning laser microscope was equipped with a sufficiently powerful laser and a novel device, the ‘Scamper’. This consisted essentially of a filter changer, an acousto-optical modulator (AOM) and a computer. The computer was programmed to activate the AOM during scanning according to a freely defined image mask. As a result almost any desired pattern could be bleached (‘written’) into fluorescent samples at high definition and then imaged (‘read’) at non-bleaching conditions, employing full confocal resolution. Furthermore, molecular transport could be followed by imaging the dissipation of bleach patterns. Experiments with living cells concerning dynamic processes in cytoskeletal filaments and the lateral mobility of membrane lipids suggest a wide range of potential biological applications. Thus, Scamp offers new possibilities for the optical manipulation and analysis of both technical and biological microsystems.     

Working with the confocal scanning UV-laser microscope: specific DNA localization at high sensitivity and multiple-parameter fluorescence

The use of fast-staining DNA-specific dyes such as DAPI or Hoechst 33342/33258 has been a major problem for confocal scanning laser microscopy (CSLM) studies of intranuclear chromatin organization. Moreover, the availability of a confocal ultraviolet scanning laser microscope configuration, which allows an excitation at wavelengths of 364 nm as well as 488, 514 and 543 nm, is a prerequisite for single as well as multiple fluorescence parameter studies, especially if these studies are concerned with the precise localization of intranuclear signals. Here we report the characteristics and application of a CSLM, which was adapted for UV-excitation and therefore enables comparison of the spatial distribution of several types of signals within one preparation. In addition to multiple-parameter studies, we have also investigated the sensitivity of the system with regard to the identification of the double-stranded DNA of lampbrush chromosome loops in germinal vesicles of amphibian oocytes.