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.     

A 2D MEMS mirror with sidewall electrodes applied for confocal MACROscope imaging

This paper presents microelectromechanical system micromirrors with sidewall electrodes applied for use as a Confocal MACROscope for biomedical imaging. The MACROscope is a fluorescence and brightfield confocal laser scanning microscope with a very large field of view. In this paper, a microelectromechanical system mirror with sidewall electrodes replaces the galvo-scanner and XYZ-stage to improve the confocal MACROscope design and obtain an image. Two micromirror-based optical configurations are developed and tested to optimize the optical design through scanning angle, field of view and numerical aperture improvement. Meanwhile, the scanning frequency and control waveform of the micromirror are tested. Analysing the scan frequency and waveform becomes a key factor to optimize the micromirror-based confocal MACROscope. When the micromirror is integrated into the MACROscope and works at 40 Hz, the micromirror with open-loop control possesses good repeatability, so that the synchronization among the scanner, XYZ-stage and image acquisition can be realized. A laser scanning microscope system based on the micromirror with 2 μm width torsion bars was built and a 2D image was obtained as well. This work forms the experimental basis for building a practical confocal MACROscope.     

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.     

Low-cost image-capture system for a scanning electron microscope

We describe a PC-based active-capture system for recording digital images from a scanning electron microscope. The system is based on a National Instruments data-acquisition board and a Pentium computer, controlled by software that we have written in Visual Basic.     

A flexible instrument control and image acquisition system for a scanning electron microscope

We have developed an instrument control and image acquisition system for use with scanning electron microscopes. By making the system flexible over a wide range of operating voltages, scan generation and image acquisition modes can be easily accommodated to a wide range of instruments. We show the implementation of this system for use with a custom‐built low‐voltage scanning electron microscope. We then explore the simple modifications that are required for control of two instruments intended for use as free electron lasers.     

激光共聚焦扫描显微镜在微机电系统中的应用

在微机电系统中,三维微结构分析是对微加工工艺进行表征的一种重要手段。随着微机电系统研究的深入和产业化的需求,其微结构分析在微机电系统中的重要性日益凸现。激光共聚焦扫描显微镜因其高分辨率、非接触、数据结构分析快等优点,在微结构分析中得到了大量的应用。本文介绍激光共聚焦扫描显微镜的成像原理,重点介绍激光共聚焦显微镜在大角度测量和形貌分析中的应用。同时,与台阶仪、扫描电子显微镜和白光干涉仪相比较,指出激光共聚焦扫描显微镜在微结构分析中的优点和局限性。     

Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope

High-speed beam scanning and data acquisition in a laser scanning confocal microscope system are normally implemented with a resonant galvanometer scanner and a frame grabber. However, the nonlinear scanning speed of a resonant galvanometer can generate nonuniform photobleaching in a fluorescence sample as well as image distortion near the edges of a galvanometer scanned fluorescence image. Besides, incompatibility of signal format between a frame grabber and a point detector can lead to digitization error during data acquisition. In this article, we introduce a masked illumination scheme which can effectively decrease drawbacks in fluorescence images taken by a laser scanning confocal microscope with a resonant galvanometer and a frame grabber. We have demonstrated that the difference of photobleaching between the center and the edge of a fluorescence image can be reduced from 26 to 5% in our confocal laser scanning microscope with a square illumination mask. Another advantage of our masked illumination scheme is that the zero level or the lowest input level of an analog signal in a frame grabber can be accurately set by the dark area of a mask in our masked illumination scheme. We have experimentally demonstrated the advantages of our masked illumination method in detail.     

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 simple method for overcoming some problems when observing thick reflective biological samples with a confocal scanning laser microscope

A simple device is described, which allows the range of depth of scanning to be reduced when observing thick reflecting biological samples with a confocal scanning laser microscope (CSLM). Thick histological sections of human skin and rat brain stem were mounted between two coverslips (‘sandwich’ style) and the optical tomography was performed from both sides by turning the ‘sandwich’ upside-down. The samples were impregnated using standard Golgi–Cox, ‘rapid Golgi’ or other silver methods. The ability to turn the ‘sandwich’ upside-down is particularly useful when the reflective structure inspected is deep inside the section, i.e. near the lower surface of the specimen, or when it is opaque to the laser beam or excessively reflective.     

Application of confocal scanning laser microscopy for an automated nuclear grading of prostate lesions in three dimensions

Confocal scanning laser microscopy provides the opportunity to obtain three-dimensional (3-D) images by piling up consecutive confocal planes. This technique was applied to capture 3-D images from 100-μm-thick tissue blocks from prostate lesions (hyperplasia, dysplasia, adenocarcinomas). Automated methods were implemented to perform a nuclear grading of 3-D cell nuclei from these specimens. Special attention was focused on the development of a new approach to 3-D chromatin texture analysis. This method uses mathematical morphology operations to tessellate the chromatin into homogeneous domains. The nuclear features (volume, shape, texture) were subjected to a discriminant analysis. Using a set of five features, the classification of cell nuclei yielded an accuracy of 963%. The results indicate the potential of 3-D imaging and analysis techniques for an automated nuclear grading of prostate lesions.     

Accurate measure of laser irradiance threshold for near-infrared photo-oxidation with a modified confocal microscope

Femtosecond mode‐locked lasers are now being used routinely in multiphoton fluorescence and autofluorescence spectroscopy, are just beginning to be used in refractive surgery, and may be used in the future diagnosis of skin cancer. Pulses from these lasers induce non‐linear effects in resultant tissue interactions. Using a modified confocal microscope with dispersion compensation and accurate measurements of beam diameter, a very low threshold was measured for photochemical oxidation in cultured cells. The measured threshold showed non‐linear photo‐oxidation at a peak irradiance and photon‐flux density of 8.4 × 10⁸ W cm^?2 and 3.4 × 10²⁷ photons cm^?2 s^?1, respectively (90‐fs pulse). The impact of these findings is significant to those using ultrashort lasers because they provide a tangible reference point (microscope‐independent) for the generation of photo‐oxidative stress in laser‐exposed tissues, and because they highlight the importance of dispersion compensation in minimizing collateral tissue damage.     

A simple method for checking the illumination profile in a laser scanning microscope and the dependence of resolution on this profile

One of the conditions for a laser scanning microscope to reach its optimal performance is for it to operate at its full numerical aperture (NA). In most commonly used systems, the illumination intensity at the back focal plane of the objective lens is apodized. This paper presents a simple method using a photodiode for checking the actual illumination intensity profile. We show as an example the measured profiles of a laser beam when working with two high-NA immersion objectives in two different confocal systems, and also show that in theoretical studies of the point-spread function, the assumption of a flat compared with a truncated Gaussian beam profile gives rise to severe discrepancies. The measured profiles also serve as an indication of the necessity of a realignment of the optical system.     

The use of Lucifer yellow,bodipy, FITC,TRITC, RITC and Texas red for dual immunofluorescence visualized with a confocal scanning laser microscope

A new method of comparing the relative merits of different fluorophores that undergo relatively rapid irreversible photo-inactivation is described. This method showed that the levels of fluorescent emission seen with both fluorescein isothiocyanate (FITC) and bodipy fl conjugated to streptavidin were similar when examined under conditions where they exhibited equal rates of irreversible photo-inactivation. Bodipy fl and FITC give lower levels of cross-talk into images of cells immunofluorescently stained with either rhodamine isothiocyanate (RITC) or tetramethyl rhodamine isothiocyanate (TRITC) than into images of cells stained with Texas red, under conditions where the three red fluorophores exhibited an equal level of sensitivity. Furthermore, bodipy fl gave much lower levels of cross-talk into images of RITC-stained cells than either FITC or Lucifer yellow. TRITC, but not RITC or Texas red, gave significant levels of cross-talk into the green band-pass filters used to visualize FITC and bodipy fl. From these results it seems that a combination of bodipy fl and RITC provides the best contrast when visualizing dual immunofluorescence with a confocal scanning laser microscope if the 488-nm line of an argon ion laser is used as the excitation source.     

A new continuous sliding mode control approach with actuator saturation for control of 2-DOF helicopter system

The 2-degree of freedom (DOF) helicopter system is a typical higher-order, multi-variable, nonlinear and strong coupled control system. The helicopter dynamics also includes parametric uncertainties and is subject to unknown external disturbances. Such complicated system requires designing a sophisticated control algorithm that can handle these difficulties. This paper presents a new robust control algorithm which is a combination of two continuous control techniques, composite nonlinear feedback (CNF) and super-twisting control (STC) methods. In the existing integral sliding mode (ISM) based CNF control law, the discontinuous term exhibits chattering which is not desirable for many practical applications. As the continuity of well known STC reduces chattering in the system, the proposed strategy is beneficial over the current ISM based CNF control law which has a discontinuous term. Two controllers with integral sliding surface are designed to control the position of the pitch and the yaw angles of the 2- DOF helicopter. The adequacy of this specific combination has been exhibited through general analysis, simulation and experimental results of 2-DOF helicopter setup. The acquired results demonstrate the good execution of the proposed controller regarding stabilization, following reference input without overshoot against actuator saturation and robustness concerning to the limited matched disturbances.