An optical spectrometer for a confocal scanning laser microscope

An optical spectrometer for the visible range has been developed for the confocal scanning laser microscope (CSLM) Phoibos 1000. The spectrometer records information from a single point or a user-defined region within the microscope specimen. A prism disperses the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit cools the diode array, thereby reducing the detector dark current to a level, which allows integration times of up to 60 s. The spectral resolving power, λ/Δλ, ranges from 400 at λ = 375 nm to 100 at λ = 700 nm. Since the entrance aperture of the spectrometer has the same diameter as the detector aperture of the CSLM, the three-dimensional spatial resolution for spectrometer readings is equivalent to that of conventional confocal scanning, that is, down to 0.2 μm lateral and 0.8 μm axial resolution with an N.A.=1.3 objective.     

Enhanced scanning control in a confocal scanning laser microscope

A fast and flexible scanning unit, allowing scanning rates of more than 1 kHz over regions identified in a specimen, has been developed and evaluated. This scanning unit replaces the original scanning unit in the Phoibos confocal scanning laser microscope and features full backward compatibility, while at the same time allowing fast and flexible scanning modes, such as point scanning, line scanning, and scanning along user-selected closed curves. The scanning unit uses two galvanometer-mounted mirrors for scanning. A standard procedure for recordings with this scanning unit would be to scan an overview image with conventional raster scanning to identify a region of interest, mark a point, a line, or a closed curve over this region, and to start the scanner. An iterating algorithm then calculates the waveforms needed by the scanner to follow the identified curves with pixel precision. With this scanning unit and its controlling software, experiments demanding time-resolved recordings within the millisecond range can be performed. Repetition rates up to > 1 kHz for line scanning and curve scanning, and > 100 kHz for point scanning are obtainable. This allows time-resolved studies of fast reactions in living tissue to be performed with the spatial resolution and signal-to-noise ratio obtainable with a point scanning confocal microscope.     

Enhanced scanning control in a confocal scanning laser microscope

A fast and flexible scanning unit, allowing scanning rates of more than 1 kHz over regions identified in a specimen, has been developed and evaluated. This scanning unit replaces the original scanning unit in the Phoibos confocal scanning laser microscope and features full backward compatibility, while at the same time allowing fast and flexible scanning modes, such as point scanning, line scanning, and scanning along user-selected closed curves. The scanning unit uses two galvanometer-mounted mirrors for scanning. A standard procedure for recordings with this scanning unit would be to scan an overview image with conventional raster scanning to identify a region of interest, mark a point, a line, or a closed curve over this region, and to start the scanner. An iterating algorithm then calculates the waveforms needed by the scanner to follow the identified curves with pixel precision. With this scanning unit and its controlling software, experiments demanding time-resolved recordings within the millisecond range can be performed. Repetition rates up to >1 kHz for line scanning and curve scanning, and >100 kHz for point scanning are obtainable. This allows time-resolved studies of fast reactions in living tissue to be performed with the spatial resolution and signal-to-noise ratio obtainable with a point scanning confocal microscope.     

激光共焦扫描显微镜及其应用

介绍了共焦激光显微镜的基本光路、成像原理、关键技术及应用。     

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。     

Imaging 'intact' myofibrils with a near-field scanning optical microscope

Fluorescently labelled myofibrils were imaged in physiological salt solution by near-field scanning optical microscopy and shear-force microscopy. These myofibrils were imaged in vitro , naturally adhering to glass while retaining their ability to contract. The Z-line protein structure of the myofibrils was antibody labelled and easily identified in the near-field fluorescence images. The distinctive protein banding structure of the myofibril was also seen clearly in the shear-force images without any labelling requirement. With the microscope in the transmission mode, resolution of the fluorescence images was degraded significantly by excessive specimen thickness (>1 μm), whereas the shear-force images were less affected by specimen thickness and more affected by poor adherence to the substrate. Although the exact mechanism generating contrast in the shear-force images is still unknown, shear-force imaging appears to be a promising new imaging modality.     

Imaging of reactive oxygen species burst from mitochondria using laser scanning confocal microscopy

Objective: Although several methods have been used to detect the intracellular reactive oxygen species (ROS) generation, it is still difficult to determine where ROS generate from. This study aimed to demonstrate whether ROS generate from mitochondria during oxidative stress induced mitochondria damage in cardiac H9c2 cells by laser scanning confocal microscopy (LSCM). Methods: Cardiac H9c2 cells were exposed to H₂O₂ (1200μM) to induce mitochondrial oxidant damage. Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE); ROS generation was measured by staining cells with dichlorodihydrofluorescein diacetate (H₂DCFDA). Results: A rapid/transient ROS burst from mitochondria was induced in cardiac cells treated with H₂O₂ compared with the control group, suggesting that mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells. Meanwhile, the TMRE fluorescence intensity of mitochondria which had produced a great deal of ROS decreased significantly, indicating that the burst of ROS induces the loss of ΔΨm. In addition, the structure of mitochondria was damaged seriously after ROS burst. However, we also demonstrated that the TMRE fluorescence intensity might be affected by H₂DCFDA. Conclusions: Mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells and these findings provide a new method to observe whether ROS generate from mitochondria by LSCM. However, these observations also suggested that it is inaccurate to test the fluorescence intensities of cells stained with two or more different fluorescent dyes which should be paid more attention to. Microsc. Res. Tech. 76:612–617, 2013. © 2013 Wiley Periodicals, Inc.     

Optic fibre bundle contact imaging probe employing a laser scanning confocal microscope

A small diameter (600 µm) fused optic fibre imaging bundle was used as a probe to compare fluorescent specimens by direct contact imaging using both a conventional fluorescence microscope and a laser scanning confocal microscope (LSCM) system. Green fluorescent polyester fibres placed on a green fluorescent cardboard background were used to model biological tissue. Axial displacement curves support the hypothesis that pinhole size in the LSCM system reduces the contribution of non‐focal plane light. Qualitative comparison showed that the LSCM system produced superior image quality and contrast over the conventional system. The results indicate that the new LSCM–probe combination is an improvement over conventional fluorescence–probe systems. This study shows the feasibility of employing such a small diameter probe in the investigation of biological function in difficult to access areas.     

Fluorescence imaging with a laser trapping scanning near-field optical microscope

We investigated fluorescence imaging using a near-field scanning optical microscope which uses a laser-stabilized gold nanoparticle as a near-field probe. This microscope is suitable for observations of biological specimens in aqueous solutions because the probe particle is held by a noncontact force exerted by a laser beam. Theoretical calculations based on Mie scattering theory are presented to evaluate the near-field enhancement by a gold particle of 40 nm diameter. We also present fluorescence images of a single fluorescent bead and discuss the near-field contribution to the fluorescence image in this type of microscope.     

Studies of the microstructure of polymer-modified bitumen emulsions using confocal laser scanning microscopy

Polymer-modified bitumen emulsions present a safer and more environmentally friendly binder for enhancing the properties of roads. Cationic bitumen emulsion binders containing polymer latex were investigated using confocal laser scanning microscopy. The latex was incorporated into the bitumen emulsion by using four different addition methods and all emulsions were processed with a conventional colloid mill. The emulsion binder films were studied after evaporation of the emulsion aqueous phase. We show how the microstructure and distribution of the polymer varies within the bitumen binder depending on latex addition method, and that the microstructure of the binder remains intact when exposed to elevated temperature. It was found that a distinctly fine dispersion of polymer results when the polymer is blended into the bitumen before the emulsifying process (a monophase emulsion). In contrast, bi-phase emulsion binders produced by either post-adding the latex to the bitumen emulsion, or by adding the latex into the emulsifier solution phase before processing, or by comilling the latex with the bitumen, water and emulsifier all resulted in a network formation of bitumen particles surrounded by a continuous polymer film. The use of emulsified binders appears to result in a more evenly distributed polymer network compared to the use of hot polymer-modified binders, and they therefore have greater potential for consistent binder cohesion strength, stone retention and therefore improved pavement performance.     

共聚焦激光扫描显微系统光学设计

为了实现非接触式、快速高精度的光学检测,设计了一种共聚焦激光扫描显微光学系统。在保证设计指标的前提下,简化了各光组的结构,采用7片球面透镜并以K9玻璃作为透镜材料。使用Zemax软件对光学系统进行了设计和仿真。结果表明:物镜的数值孔径为0.49;系统的径向和轴向光学分辨率分别为0.400μm和0.772μm;显微聚焦系统聚焦弥散斑直径小于2μm;照明系统聚焦弥散斑直径小于10μm;探测系统的聚焦光斑直径小于20μm;根据仿真结果确定了针孔1和针孔2的尺寸均为20μm,且厚度不超过0.1mm;各子系统的MTF曲线均接近衍射极限,具有很高的光学传输效率。     

激光扫描共聚焦光谱成像系统

在激光扫描共聚焦显微成像技术基础上引入了光谱成像技术以便区分生物组织中的不同荧光成分。采用分光棱镜对荧光进行光谱展开,在光谱谱面处设置两个可移动缝片形成出射狭缝,两个步进电机带动安装其上的两个缝片设置系统在整个工作波长（400~700 nm）内的光谱带宽,其最小光谱带宽优于5 nm。用488 nm激光和低压汞灯实际测量了几条谱线对应的狭缝位置并和理论值做了比较,结果显示实际狭缝位置和理论值的差值均小于0.1 mm。在全光谱和50 μm出射狭缝（对应2.5 nm光谱带宽）对老鼠肾脏组织进行了共聚焦光谱成像实验,获得了老鼠肾脏组织中DAPI标定的细胞核图像和Alexa Fluor^®488标定的肾脏小球曲管图像,实现了对老鼠肾脏组织不同成分的区分。实验结果表明：提出的系统能够进行共聚焦光谱成像,扩大了共聚焦显微镜的适用范围。     

激光共聚焦扫描显微镜的光学设计

为获得较高分辨率的细胞图像,设计了激光共聚焦光学系统。通过较复杂结构物镜实现了照明光路系统和发射光路系统的设计。用Zemax对照明光路和发射光路进行了设计,仿真过程中照明光路的聚焦弥散斑直径小于1μm,照明针孔处的聚焦弥散斑直径小于20μm,发射光路的聚焦弥散斑直径小于20μm,同时照明光路和发射光路的MTF曲线接近衍射极限,达到较理想的情况。     

激光扫描共聚焦光谱系统设计

为提高激光扫描共聚焦显微镜光谱模块的分光性能,设计了双Amici棱镜光谱结构.根据棱镜色散特性,计算得到Amici棱镜和双Amici棱镜的线性与非线性数学模型,给出直视型棱镜结构参数.比较相同色散条件下Amici和双Amici棱镜结构,得到双Amici棱镜组能提供更好的色散线性度.在光谱波段400～700 nm,中心波...     

Scanning and detection techniques used in a confocal scanning laser microscope

A two-mirror scanning mechanism for confocal microscopy is described. No optical components, in addition to the scanning mirrors, are used. Design criteria and performance of the scanner are discussed. The photometric linearity of a detector unit incorporating a photomultiplier tube is reported, and a dual detector unit with tunable split wavelength is described.     

An optical sectioning programmable array microscope implemented with a digital micromirror device

The defining feature of a programmable array microscope (PAM) is the presence of a spatial light modulator in the image plane. A spatial light modulator used singly or as a matched pair for both illumination and detection can be used to generate an optical section. Under most conditions, the basic optical properties of an optically sectioning PAM are similar to those of rotating Nipkow discs. The method of pattern generation, however, is fundamentally different and allows arbitrary illumination patterns to be generated under programmable control, and sectioning strategies to be changed rapidly in response to specific experimental conditions. We report the features of a PAM incorporating a digital micromirror device, including the axial sectioning response to fluorescent thin films and the imaging of biological specimens. Three axial sectioning strategies were compared: line scans, dot lattice scans and pseudo-random sequence scans. The three strategies varied widely in light throughput, sectioning strength and robustness when used on real biological samples. The axial response to thin fluorescent films demonstrated a consistent decrease in the full width at half maximum (FWHM), accompanied by an increase in offset, as the unit cells defining the patterns grew smaller. Experimental axial response curves represent the sum of the response from a given point of illumination and cross-talk from neighbouring points. Cross-talk is minimized in the plane of best focus and when measured together with the single point response produces a decrease in FWHM. In patterns having constant throughput, there appears to be tradeoff between the FWHM and the size of the offset. The PAM was compared to a confocal laser scanning microscope using biological samples. The PAM demonstrated higher signal levels and dynamic range despite a shorter acquisition time. It also revealed more structures in x - z sections and less intensity drop-off with scanning depth.     

A scanning near-field optical microscope having scanning electron tunnelling microscope capability using a single metallic probe tip

A new microscope system that has the combined capabilities of a scanning near-field optical microscope (SNOM) and a scanning tunnelling microscope (STM) is described. This is achieved with the use of a single metallic probe tip. The distance between the probe tip and the sample surface is regulated by keeping the tunnelling current constant. In this mode of operation, information about the optical properties of the sample, such as its refractive index distribution and absorption characteristics, can be disassociated from the information describing its surface structure. Details of the surface structure can be studied at resolutions smaller than the illumination wavelength. The performance of the microscope is evaluated by analysing a grating sample that was made by coating a glass substrate with gold. The results are then compared with the corresponding SNOM and STM images of the grating.     

基于数字微镜的共焦显微系统的光路设计

详细叙述了共焦技术中的横向扫描技术,介绍了基于数字微镜(DMD)的共焦显微镜结构与原理,建立了基于DMD的并行检测系统,并且进行了光路的优化设计.实验结果表明,采用传统共焦显微镜光路时,光线出射分光棱镜时存在棱镜内表面反射问题,导致DMD上同一像素块在CCD上成两个像.在此分析了上述现象产生的原理,给出了解决此问题的方...     

Systematic evaluation of FRAP experiments performed in a confocal laser scanning microscope

The diffusion coefficient as well as the dimensionality of the diffusion process can be determined by straightforward and facile data analysis, when fluorescence recovery after photobleaching (FRAP) is measured as a function of time and space by means of confocal laser scanning microscopy. Experiments representing one-dimensional diffusion from a plane source or two-dimensional diffusion from a line source are readily realized. In the data analysis, the deviations of the actual initial conditions from ideal models are consistently taken into account, so that no calibration measurements are needed. The method is applied to FRAP experiments on solutions of Rhodamine B in glycerol and aqueous suspensions of polymethyl methacrylate microspheres.