Multidie LED combined with homogenizing optics to improve frequency response and intensity for FLIM applications

Application of light‐emitting diodes (LEDs) in frequency‐domain fluorescence lifetime imaging microscopy (FLIM) has been limited by the trade‐off between modulation frequency and illumination intensity of LEDs, which affects the signal‐to‐noise ratio in fluorescence lifetime measurements. To increase modulation frequency without sacrificing output power of LEDs, we propose to use LEDs with multiple dice connected in series. The LED capacitance was reduced with series connection; therefore, the frequency response of multidie LED was significantly increased. LEDs in visible light, including blue, green, amber and red, were all applicable in FLIM. We also present a homogenizing optics design, so that multidie LEDs produced uniform illumination on the same focal spot. When the homogenizing optics was combined with multicolour emitters, it provides multiple colour selection in a compact and convenient design.     

Wavelength considerations in confocal microscopy of botanical specimens

We have used a multiple-laser confocal microscope with lines at 325, 442, 488, 514 and 633 nm to investigate optical sectioning of botanical specimens over a wide range of wavelengths. The 442-nm line allowed efficient excitation of Chromomycin A3, with minimal background autofluorescence, to visualize GC-rich heterochromatin as an aid to chromosome identification. Sequential excitation with 442- and 488-nm light enabled ratio imaging of cytosolic pH using BCECF. The red HeNe laser penetrated deep into intact plant tissues, being less prone to scattering than shorter blue lines, and was also used to image fluorescent samples in reflection, prior to fluorescence measurements, to reduce photobleaching. Chromatic corrections are more important in confocal microscope optics than in conventional microscopy. Measured focus differences between blue, green and red wavelengths, for commonly used objectives, were up to half the optical section thickness for both our multi-laser system and a multi-line single-laser instrument. This limited high-resolution sectioning at visible wavelengths caused a loss in signal. For ultraviolet excitation the focus shift was much larger and had to be corrected by pre-focusing the illumination. With this system we have imaged DAPI-stained nuclei, callose in pollen tubes using Aniline Blue and the calcium probe Indo-1.     

LED illumination for video-enhanced DIC imaging of single microtubules

In many applications high‐resolution video‐enhanced differential interference contrast microscopy is used to visualize and track the ends of single microtubules. We show that single ultrabright light emitting diodes from Luxeon can be used to replace conventional light sources for these kinds of applications without loss of function. We measured the signal‐to‐noise ratio of microtubules imaged with three different light emitting diode colours (blue, red, green). The blue light emitting diode performed best, and the signal‐to‐noise ratios were high enough to automatically track the ends of dynamic microtubules. Light emitting diodes as light sources for video‐enhanced differential interference contrast microscopy are high performing, low‐cost and easy to align alternatives to existing illumination solutions.     

Simultaneous confocal recording of multiple fluorescent labels with improved channel separation

Confocal microscopes are often used to study specimens labelled with fluorophores. A commonly used method for simultaneous recording of the distribution of multiple fluorophores is to divide the fluorescent light emitted by the specimen into different wavelength regions using dichroic and bandpass filters. These different wavelength regions are then distributed to multiple detectors. However, the broad and overlapping spectra of commonly used fluorophores often result in considerable crosstalk between channels. A new technique, intensity-modulated multiple-beam scanning (IMS) microfluorometry, can be used to reduce this cross-talk substantially. The IMS technique is implemented with two laser beams of different wavelengths, intensity-modulated at different frequencies, which illuminate the specimen simultaneously. The two laser wavelengths predominantly excite one fluorophore each. Fluorescent light from the specimen is divided into two wavelength regions (red and green) which are detected by two photomultiplier tubes. The output signals from the photomultiplier tubes are connected to lock-in amplifiers. The effect of using modulated laser beams, in combination with lock-in amplifiers, is strongly to reduce cross-talk between the channels. The performance of the IMS technique using various types of specimen is compared with the results obtained using the conventional multi-detector method.     

RGB颜色格雷码结构光三维测量技术研究

提出一种基于RGB颜色格雷码的结构光编码方法。该方法是将红、蓝条纹按二进制格雷码编码方式进行编排，并在红、蓝条纹之间用一个像素的绿条纹作为分界。由于投射光强相同，基于CCD的摄像机拍得的条纹图像中绿条纹向红、蓝区域扩散程度等同，提取绿条纹的中心即可获得条纹的准确定位，再依据三角法原理实现三维物体测量。文中具体介绍了该方法的编码、解码原理，分析了基于HSI空间的彩色图像分割方法，给出了基于3dsMAX和MATLAB环境下的重构仿真实验结果。     

Minimization of dose as a criterion for the selection of imaging modes in electron microscopy of amorphous specimens.

A fundamental limitation in electron microscopy of organic specimens is radiation damage by the electron beam. To minimize damage it is necessary to have maximum information collection for a given dose. Various modes of operation of conventional and scanning transmission microscopes are compared with respect to their ability to detect small changes in specimen thickness or density with a given signal to noise ratio. Incoherent imaging is assumed, and this is expected to apply to amorphous specimens under a variety of microscope conditions. For either very thin or very thick specimens, the scanning transmission microscope is found to require nearly 10 times less dose than a conventional microscope for the same signal to noise ratio in the image. For specimens of intermediate thickness, scanning and conventional transmission electron microscopes are roughly equivalent.     

Use of a night vision intensifier for direct visualization by eye of far-red and near-infrared fluorescence through an optical microscope

We describe the design, construction and testing of a prototype device that allows the direct visualization by eye of far‐red and near‐infrared (NIR) fluorescence through an optical microscope. The device incorporates a gallium arsenide (GaAs) image intensifier, typically utilized in low‐light or ‘night vision’ applications. The intensifier converts far‐red and NIR light into electrons and then into green light, which is visible to the human eye. The prototype makes possible the direct, real‐time viewing by eye of normally invisible far‐red and NIR fluorescence from a wide variety of fluorophores, using the full field of view of the microscope to which it is applied. The high sensitivity of the image intensifier facilitates the viewing of a wide variety of photosensitive specimens, including live cells and embryos, at vastly reduced illumination levels in both fluorescence and bright‐field microscopy. Modifications to the microscope are not required in order to use the prototype, which is fully compatible with all current fluorescence techniques. Refined versions of the prototype device will have broad research and clinical applications.     

Even illumination in total internal reflection fluorescence microscopy using laser light

In modern fluorescence microscopy, lasers are a widely used source of light, both for imaging in total internal reflection and epi-illumination modes. In wide-field imaging, scattering of highly coherent laser light due to imperfections in the light path typically leads to nonuniform illumination of the specimen, compromising image analysis. We report the design and construction of an objective-launch total internal reflection fluorescence microscopy system with excellent evenness of specimen illumination achieved by azimuthal rotation of the incoming illuminating laser beam. The system allows quick and precise changes of the incidence angle of the laser beam and thus can also be used in an epifluorescence mode.     

Digital stain separation for histological images

It is often desirable to perform digital image analyses on sections prepared for human interpretation, e.g. nuclear chromatin texture analysis or three-dimensional reconstructions using sections requiring human delineation of structures of interest. Unfortunately such analyses are often more effective using stains with less complex contrast. Here an automated selective 'de-staining' method for digital images is presented. The method separates an image into its red, green and blue and hue, saturation and intensity components. A mask of stained tissue is prepared by automatic percentile thresholding. A single weighted inverted colour channel is then added to each of the three primary colour channels separately by an iterative algorithm that adjusts the weights to give minimum variance within the mask. The modified red, green and blue channels are then recombined. This method is automatic requiring no pre-definition of stain colours or special hardware. The method is demonstrated to 'de-stain' nuclei in haematoxylin and eosin (H&E) sections (and a separate haematoxylin image can be derived from this). An image of isolated brown reaction product is produced with immunoperoxidase preparations counterstained with haematoxylin. Furthermore trichrome (haematoxylin van Gieson, picrosirius red) and other common stains may be separated into their components with modifications of the same algorithm. Although other methods for colour separation do exist (e.g. spectral pathology and colour deconvolution) these require special apparatus or precise calibration and foreknowledge of pure dye colour spectra. The present method of digital stain separation is fully automatic with no such prerequisites.     

Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics

In this paper, differential phase imaging (DPC) with transmitted light is implemented by adding a suitable detection system to a standard commercially available scanning confocal microscope. DPC, a long‐established method in scanning optical microscopy, depends on detecting the intensity difference between opposite halves or quadrants of a split photodiode detector placed in an aperture plane. Here, DPC is compared with scanned differential interference contrast (DIC) using a variety of biological specimens and objective lenses of high numerical aperture. While DPC and DIC images are generally similar, DPC seems to have a greater depth of field. DPC has several advantages over DIC. These include low cost (no polarizing or strain‐free optics are required), absence of a double scanning spot, electronically variable direction of shading and the ability to image specimens in plastic dishes where birefringence prevents the use of DIC. DPC is also here found to need 20 times less laser power at the specimen than DIC.     

双光束扫描激光电视系统设计

阐述了双光束扫描激光电视系统的设计,系统采用两束激光(每束都由红、绿、蓝三基色配比而成)通过一个扫描头同时扫描,扫描头的行偏转采用多面转镜、场偏转采用电流计偏转实现,与单束扫描方式相比较有如下优点:1.多面转镜的转速可降至1/2,提高了扫描装置的性能,同时降低了光学扫描系统制作难度;其转镜镜面尺寸和出射光束直径可以相应加大,扫描光斑在屏幕上聚焦更好,图像显示更清晰;2.两组调制器同时工作,屏幕水平分辨率可提高2倍;3.在相同的屏幕亮度下,单点功率密度相应降低,提高了安全性。     

Specimen-induced distortions in light microscopy

Specimen‐induced aberrations affect the imaging properties in optical 3D microscopy, especially when high numerical aperture lenses are used. Studies on aberrations are often properly concerned with the degradation of image quality such as compromised resolution or reduced signal intensity. Apart from these, aberration effects can also introduce geometric image distortions. The effects, discussed here are particularly strong when thick biological specimens are investigated. Using a high numerical aperture interferometer, we measured wavefront aberrations in transmission mode and quantified geometric distortions associated with specimen‐induced aberrations. This assessment for a range of biological specimens allows estimation of the accuracy of spatial measurements. The results show that high‐resolution spatial measurements can be significantly compromised by specimen‐induced aberrations.     

Experimental study of the nonlinear effects generated in a concrete structure with damaged integrity

The paper deals with nonlinear interaction between elastic waves and structural defects in concrete specimens. Three groups of concrete specimens which differed from each other in the structure quality as a consequence of different ripening conditions were performed. Nonstandard concrete ripening conditions resulted in the development of microcracks in the specimen structure. The nonlinear elastic properties of the structure were tested by using the analysis of response frequency spectrum generated from a continuous wave transmission through the concrete specimens. It was tested whether or not the microcracks in the structure gave rise to parasitic components which were able to generate nonlinear phenomena in the signal transmission. A single harmonic ultrasonic signal method was applied to the specimens and evaluation, especially of the second and third harmonic components as indicators of nonlinearity was carried out. Verification measurements which have been carried out in parallel with the ultrasonic ones, give evidence of the specimen structure integrity deteriorations and confirm the correlation between the nonlinear effects on the transfer characteristics with the existence of defects in the specimen internal structure.     

Variable bright-darkfield-contrast, a new illumination technique for improved visualizations of complex structured transparent specimens

Variable bright-darkfield contrast (VBDC) is a new technique in light microscopy which promises significant improvements in imaging of transparent colorless specimens especially when characterized by a high regional thickness and a complex three-dimensional architecture. By a particular light pathway, two brightfield- and darkfield-like partial images are simultaneously superimposed so that the brightfield-like absorption image based on the principal zeroth order maximum interferes with the darkfield-like reflection image which is based on the secondary maxima. The background brightness and character of the resulting image can be continuously modulated from a brightfield-dominated to a darkfield-dominated appearance. When the weighting of the dark- and brightfield components is balanced, medium background brightness will result showing the specimen in a phase- or interference contrast-like manner. Specimens can either be illuminated axially/concentrically or obliquely/eccentrically. In oblique illumination, the angle of incidence and grade of eccentricity can be continuously changed. The condenser aperture diaphragm can be used for improvements of the image quality in the same manner as usual in standard brightfield illumination. By this means, the illumination can be optimally adjusted to the specific properties of the specimen. In VBDC, the image contrast is higher than in normal brightfield illumination, blooming and scattering are lower than in standard darkfield examinations, and any haloing is significantly reduced or absent. Although axial resolution and depth of field are higher than in concurrent standard techniques, the lateral resolution is not visibly reduced. Three dimensional structures, reliefs and fine textures can be perceived in superior clarity.     

Accelerating fabrication speed in two-laser beam stereolithography system using adaptive crosshatch technique

The aim of this research is to develop a novel two-laser beam stereolithography system. The wavelengths of the two semiconductor laser beams are determined to be 405 nm (blue light) and 532 nm (green light), respectively, according to the relative absorbance rate of used visible light curable resin. The blue light laser is suitable for scanning contour of objects because of its fast absorbance, thus giving a narrow cured depth. The green light laser is better suited for scanning the internal crosshatch for condensing the fabrication time because its high power results in a wide cured width and deep penetration. The influence of the photoabsorber, carbon powder with average diameter of 0.1 µm, is discussed and an optimal weight percentage of 1.5 is specified for controlling cured thickness. An adaptive crosshatch technique is introduced and applied to the fabrication process. In addition, a turbine is fabricated using the proposed approach for comparison with the E-DARTS system in terms of fabrication capability. Results show that the developed system can fabricate objects quickly with high accuracy.     

A simple technique of image analysis for specific nuclear immunolocalization of proteins

Colocalization of fluorescent signals in confocal microscopy is usually evaluated by inspecting merged images from different colour channels or by using commercially available software packages. We describe in this paper a simple method for assessment of nuclear localization of proteins in tissue sections through confocal immunolocalization, propidium iodide counterstaining and image analysis. Through a macro command developed for the public domain, Java‐based software imagej , red, green, blue (RGB) images are automatically split in the red and green channels and a new image composed of the nonblack pixels coincident in both channels is created and inverted for better visualization. This method renders images devoid of both, extranuclear staining and background, thus emphasizing the nuclear signal. The resulting images can easily be used for comparison or quantification of the results. Given the simplicity of the technique and the worldwide diffusion of the software utilized, we think that this method could be useful in order to define standards of colocalization in confocal microscopy.     

Digital image processing: a path to better pictures

Digital image processing can be used to provide enhanced performance in scanning electron column instruments. Improved visualization, reduced specimen damage, and quantization can be achieved. The system described here provides hardware for digitization and storage of multiple images simultaneously, and multimode scan generation. System software provides easy scan control, image digitization, automatic prescale adjust on acquisition, grey scale histogram generation, grey scale compression or expansion, image filtering, smoothing, and random color assignment to grey levels. Image digitization and processing was done using the EG&G ORTEC Image Master Microanalyzer in conjunction with a JEOL 100CX TEMSCAN and a JEOL JSM 35. Examples of image processing of bright and dark field STEM images of biological specimens are shown. An example of X-ray image processing using a two-dimensional filter function to reduce image noise is also shown.     

Contrast and resolution from biological objects examined in the electron microscope with particular reference to negatively stained specimens

For the practical biologist applying electron microscopy to the study of biological macromolecules, there are serious problems in obtaining high resolution images showing detail below 2.5–3.0 nm. The limitation in resolution from biological specimens can be attributed to support film thickness and granularity, specimen preparation, irradiation damage, focusing effects and possible contamination in the electron beam. Specimens possessing repeating features can be analysed and averaged by optical diffraction and image reconstruction methods which offer some improvement to the signal to noise ratio. The above problems, with particular reference to irradiation damage, still impose the basic limitation for high resolution applications. When considered together they offer formidable difficulties in practical terms in attempting to make full use of the potential resolving power of modern electron microscopes.     

Curing Characteristics of the Photopolymer Used in the Solid Laser-Diode Plotter RP System

Most stereolithography (SL) processes employ ultraviolet (UV) or diode pumped solid state lasers to cure the associated photopolymer to build a 3D part. A UV or diode pumped solid state laser is more expensive in initial cost and maintenance than a semiconductor laser. A semiconductor laser is used in the solid laser-diode plotter (SLP) rapid prototyping system to cure a specific photopolymer (DF2000). The DF2000 photo-polymer is formulated from a based resin, hardening agent, and powder. The wavelength of the semiconductor laser used in the SLP system is 680 nm and is in the range of red colour light. In this work, the curing effect of the visible wavelength spectrums generated by a lamp on the DF2000 photopolymer was investigated, using differential scanning photo calorimetry (DSPC). The DSPC result indicates that the curing degree of the photopolymer using blue light is better than that when using red light. The result also seems showing that the shorter the wavelength, the better the curing effect of the photopolymer. Hence, a blue semiconductor laser (405 nm) or a shorter wavelength laser or even a higher-power lamp, instead of a red laser, can be employed to increase the curing degree of the photopolymer used in the SLP system. The quantitative effect of the SLP processing parameters on the curing degree of DF2000 photopolymer and the mechanical property of the SLP test specimen was studied using the Taguchi method. The results show that the mechanical property of a fabricated part is proportional to the curing degree of the photopolymer. The results also indicate that the scanning pitch of processing parameters is the most sensitive parameter of the SLP system affecting the curing degree and, hence, also affecting the mechanical property. Therefore, the curing characteristics of the photopolymer used in SLP system could be used as an index of the mechanical property of a fabricated part. Also, the employment of a shorter wavelength laser rather than red laser can further improve the part mechanical property and fabrication speed of the part.