Use of astronomy filters in fluorescence microscopy

Monochrome astronomy filters are well suited for use as excitation or suppression filters in fluorescence microscopy. Because of their particular optical design, such filters can be combined with standard halogen light sources for excitation in many fluorescent probes. In this "low energy excitation," photobleaching (fading) or other irritations of native specimens are avoided. Photomicrographs can be taken from living motile fluorescent specimens also with a flash so that fluorescence images can be created free from indistinctness caused by movement. Special filter cubes or dichroic mirrors are not needed for our method. By use of suitable astronomy filters, fluorescence microscopy can be carried out with standard laboratory microscopes equipped with condensers for bright-field (BF) and dark-field (DF) illumination in transmitted light. In BF excitation, the background brightness can be modulated in tiny steps up to dark or black. Moreover, standard industry microscopes fitted with a vertical illuminator for examinations of opaque probes in DF or BF illumination based on incident light (wafer inspections, for instance) can also be used for excitation in epi-illumination when adequate astronomy filters are inserted as excitatory and suppression filters in the illuminating and imaging light path. In all variants, transmission bands can be modulated by transmission shift.     

构建多模式全光谱暗场显微镜用于纳米单颗粒局域表面等离子共振实时动力学研究

金属纳米颗粒由于其局域表面等离子共振(LSPR),能显示出独特的光吸收和散射特性,常被应用于物理、化学和生物领域的分析检测。这类探针具有高强度、高稳定性,以及可以长时间成像观察等优势。对于单个金属纳米颗粒的LSPR光谱研究通常采用暗场显微镜(DFM)与光谱仪来观察。但是,现有的暗场显微镜-光谱仪联用装置受限于自带照明光源的强度与光谱范围等原因,造成对散射信号较弱的样品光谱采集时间长、采集范围窄,例如,无法做到对粒径在30 nm以下的小颗粒纳米金进行实时观察。本文针对这一问题使用超连续激光器作为光源,使对单个金属纳米颗粒的光谱采集时间可以缩短至1 ms。此外,针对细胞功能成像的需求,增加了光片成像模式,通过切换滤块,能够实现荧光成像与暗场成像的共定位。     

Multispectral optical imaging combined in situ with XPS or ToFSIMS and principal component analysis

All X‐ray photoelectron spectroscopy (XPS) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) instruments have optical cameras to image the specimen under analysis, and often to image the sample holder as it enters the system too. These cameras help the user find the appropriate points for analysis of specimens. However they seldom give as good images as stand‐alone bench optical microscopes, because of the limited geometry, source/analyser solid angle and ultra‐high‐vacuum (UHV) design compromises. This often means that the images displayed to the user necessarily have low contrast, low resolution and poor depth‐of‐field. To help identify the different regions of the samples present we have found it useful to perform multispectral imaging by illuminating the sample with narrow‐wavelength‐range light emitting diodes (LEDs). By taking an image under the illumination of these LEDs in turn, each at a successively longer wavelength, one can build up a set of registered images that contain more information than a simple Red–Green–Blue image under white‐light illumination. We show that this type of multispectral imaging is easy and inexpensive to fit to common XPS and ToF‐SIMS instruments, using LEDs that are widely available. In our system we typically use 14 LEDs including one emitting in the ultraviolet (so as to allow fluorescent imaging) and three in the near infra‐red. The design considerations of this system are discussed in detail, including the design of the drive and control electronics, and three practical examples are presented where this multispectral imaging was extremely useful. Copyright © 2016 The Authors Surface and Interface Analysis Published by John Wiley & Sons Ltd.     

Low-energy STEM of multilayers and dopant profiles.

A conventional scanning electron microscope equipped with a LaB6 source has been modified to operate in a scanning transmission mode. Two detection strategies have been considered, one based on the direct collection of transmitted electrons, the other on the collection of secondary electrons resulting from the conversion of the transmitted ones. Two types of specimens have been mainly investigated: semiconductor multilayers and dopant profiles in As-implanted Si. The results show that the contrast obeys the rules of mass-thickness contrast whereas the resolution is always defined by the probe size independently of specimen thickness and beam broadening. The detection strategy may affect the bright field (light regions look brighter) or dark field (heavy regions look brighter) appearance of the image. Using a direct collection of the transmitted electrons, the contrast can be deduced from the angular distribution of transmitted electrons and their collection angles. When collecting the secondary electrons to explain the image contrast, it is also necessary to take into account the secondary yield dependence on the incidence angle of the transmitted electrons.     

Total Internal Reflection‐Based Extinction Spectroscopy of Single Nanoparticles

Herein we report a reflection‐mode total internal reflection microscopy (TIRM) to measure the extinction spectrum of individual dielectric, plasmonic, or light‐absorbing nanoparticles, and to differentiate absorption and scattering components from the total optical output. These capabilities were enabled via illuminating the sample with evanescent wave of which the lightpath length was comparable with the size of single nanoparticles, leading to a dramatically improved reflectance change (ΔI/I₀) up to tens of percent. It was further found that scattering and absorption of light contributed to bright and dark centroids, respectively, in the optical patterns of single nanoparticles, allowing to distinguish scattering and absorption components from the extinction spectrum by the use of an appropriate image processing method. In addition, wide‐field feature of TIRM enabled the studies on tens of nanoparticles simultaneously with gentle illumination.     

大数值孔径光学镜约束多级衍射机理

针对基于激光照明的离轴全息显微成像系统存在散斑和寄生条纹噪声,以及基于部分相干光照明的离轴数字全息显微技术存在相干条纹对比度差的问题,本文提出了一种基于单色LED照明的衍射相位显微成像系统。该系统利用大数值孔径物镜及光栅对物光进行多级衍射,并采用4f系统和空间滤波器分离出0级和+1级信息,分别作为参考光和物光,最终两束光在CCD阵面上干涉产生离轴全息图,从而形成共光路全息成像结构。通过理论分析和计算,对实验用到的光学元器件进行选型,确保衍射光频谱信息能够分开且满足抽样条件。最后与传统激光离轴数字全息显微成像检测结果进行对比,实验结果表明,本文提出的系统能够获得较高的成像准确度和信噪比。     

Combined reflection and transmission microscope for telemedicine applications in field settings

We demonstrate a field-portable upright and inverted microscope that can image specimens in both reflection and transmission modes. This compact and cost-effective dual-mode microscope weighs only ～135 grams (<4.8 ounces) and utilizes a simple light emitting diode (LED) to illuminate the sample of interest using a beam-splitter cube that is positioned above the object plane. This LED illumination is then partially reflected from the sample to be collected by two lenses, creating a reflection image of the specimen onto an opto-electronic sensor-array that is positioned above the beam-splitter cube. In addition to this, the illumination beam is also partially transmitted through the same specimen, which then casts lensfree in-line holograms of the same objects onto a second opto-electronic sensor-array that is positioned underneath the beam-splitter cube. By rapid digital reconstruction of the acquired lensfree holograms, transmission images (both phase and amplitude) of the same specimen are also created. We tested the performance of this field-portable microscope by imaging various micro-particles, blood smears as well as a histopathology slide corresponding to skin tissue. Being compact, light-weight and cost-effective, this combined reflection and transmission microscope might especially be useful for telemedicine applications in resource limited settings.     

Simulations of photomodulated solute transport in doubly illuminated liquid membranes containing photoactive carriers

A steady-state model describing photofacilitated transport in liquid membranes under double illumination is presented. The model allows for the exploration of the effects of a wide range of thermodynamic and kinetic carrier properties on the control of photoinduced transport rates of solutes, called photomodulation. Most previous experimental and theoretical studies have explored the illumination of only the feed or sweep side of the membrane, while this study examines the effects of illuminating both sides simultaneously. Under double illumination, solute transport rates can be as much as five times greater than those measured in the dark and 2.5 times greater than rates obtained under single illumination. Carriers that are predominantly in the weakly binding form in the dark generally provide slightly better performance at lower light intensities than do carriers that are predominantly in the strongly binding form in the dark. The greatest enhancement in solute transport under double illumination is seen for carriers with very slow interconversion rate constants between the strongly and weakly binding forms. These results provide guidelines to help those studying photofacilitated membranes select or design photoactive molecules that will act as optimal carriers in liquid membranes under double illumination.     

Investigation of the partially coherent effects in a 2D Talbot interferometer

The recent use of a one-dimensional (1D) X-ray Talbot interferometer has triggered great interest in X-ray differential phase contrast imaging. As an improved version of a 1D interferometer, the development of two-dimensional (2D) grating interferometry strongly stimulated applications of grating-based imaging. In the framework of Fresnel diffraction theory, we investigated the self-image of 2D-phase gratings under partially coherent illumination. The fringe visibility of the self-image has been analyzed as a function of the spatial coherence length. From the viewpoint of self-image visibility, it is possible to find the optimal 2D grid for 2D X-ray grating interferometer imaging. Numerical simulations have been also carried out for quantitative evaluation. Results, in good agreement with theoretical analysis, indicate the spatial coherence requirements of the radiation illuminating a 2D grating interferometer. Moreover, our results can be used to optimize performances of a 2D grating interferometer and for further theoretical and experimental research on grating-based imaging systems.     

Light induced shifts of ferroelectric mesophase transitions

Several series of recently synthesized chiral azobenzene liquid crystals exhibit transitions driven by both illumination and temperature: some compounds can be 'melted' from the SmC* phase to the isotropic phase by increasing illumination only; according to the phase sequences, SmA, TGBA, N*, and blue phases are also involved in such behaviour. The observed phenomena are reversible and transition temperatures are reproducible in identical light conditions. Some specific transitions have been studied. Their temperatures can be lowered to 10 degrees below the dark transition values by increasing the illumination of samples. Under illumination, those transitions exhibit first order behaviour.     

Light induced shifts of ferroelectric mesophase transitions

Several series of recently synthesized chiral azobenzene liquid crystals exhibit transitions driven by both illumination and temperature: some compounds can be 'melted' from the SmC* phase to the isotropic phase by increasing illumination only; according to the phase sequences, SmA, TGBA, N*, and blue phases are also involved in such behaviour. The observed phenomena are reversible and transition temperatures are reproducible in identical light conditions. Some specific transitions have been studied. Their temperatures can be lowered to 10 degrees below the dark transition values by increasing the illumination of samples. Under illumination, those transitions exhibit first order behaviour.     

Off-axis STEM or TEM holography combined with four-dimensional diffraction imaging.

Ultrahigh-resolution imaging may be achieved using modifications of the off-axis holography scheme in a scanning transmission electron microscopy (STEM) instrument equipped with one or more electrostatic biprisms in the illuminating system. The resolution is governed by the diameter of a reference beam, reduced by channeling through a line of atoms in an atomic-focuser crystal. Alternatively, the off-axis holography may be combined with the Rodenburg method in which a four-dimensional data set is obtained by recording a nanodiffraction pattern from each point of the specimen as the incident beams are scanned. An ultrahigh-resolution image is derived by computer processing to give a particular two-dimensional section of this data set. The large amount of data recording and data processing involved with this method may be avoided if the two-dimensional section is derived by recording the hologram while the four beams produced by two perpendicular biprisms are scanned in opposing directions across the specimen by varying the voltages on the biprisms. An equivalent scheme for conventional TEM is also possible. In each case, the complex transmission function of the specimen may be derived and resolutions of about 0.05 nm may be expected.     

On the Possibility of Evanescent Wave Excitation Distal from a Solid-Liquid Interface Using Light Quenching

Abstract— Evanescent wave illumination with total internal reflection is often used to provide excitation near a quartz-water interface. We now show that evanescent illumination at one wavelength and incident angle, coupled with light quenching at a second wavelength and incident angle, can be used for selective excitation of fluorophores located up to 5000 Å into the aqueous phase. The displacement of the fluorophore population from the solid-liquid interface depends on the angles of incidence of the excitation and quenching beams and the optical power of the quenching beam. Light quenching with an evanescent wave was demonstrated to be experimentally possible using Pyridinez and a light-quenching wavelength of 736 nm. The use of combined evanescent wave excitation and evanescent wave quenching could provide selective excitation of fluorophores in the cytoplasmic region of cells and may provide improved response times for optical sensors based on evanescent excitation.     

Photopolarization and depolarization characteristics of poly(N-vinyl carbazole)

The photoelectret properties of poly(N-vinyl carbazole) sandwiched between a conducting glass electrode and an aluminum electrode have been studied. Photoelectret polarization was carried out by simultaneous application of an electric field and ultraviolet illumination. The polarization exhibits a linear dependence on the polarizing voltage, polarization time, and illumination intensity, with saturation effects for large values of these parameters. The depolarization current decay is quite fast, which indicates the absence of deep-trap levels. The ratio of photoelectret charge to the dark polarization charge is found to be 100, which determines the image contrast if the polymer film is used in persistent internal polarization (PIP) electrophotography.     

Hypericin-mediated photocytotoxic effect on HT-29 adenocarcinoma cells is reduced by light fractionation with longer dark pause between two unequal light doses

The present study demonstrates the in vitro effect of hypericin-mediated PDT with fractionated light delivery. Cells were photosensitized with unequal light fractions separated by dark intervals (1 or 6 h). We compared the changes in viability, cell number, survival, apoptosis and cell cycle on HT-29 cells irradiated with a single light dose (12 J/cm(2)) to the fractionated light delivery (1 + 11 J/cm(2)) 24 and 48 h after photodynamic treatment. We found that a fractionated light regime with a longer dark period resulted in a decrease of hypericin cytotoxicity. Both cell number and survival were higher after light sensitization with a 6-h dark interval. DNA fragmentation occurred after a single light-dose application, but in contrast no apoptotic DNA formation was detected with a 6-h dark pause. After fractionation the percentage of cells in the G1 phase of the cell cycle was increased, while the proportion of cells in the G2 phase decreased as compared to a single light-dose application, i.e. both percentage of cells in the G1 and G2 phase of the cell cycle were near control levels. We presume that the longer dark interval after the irradiation of cells by first light dose makes them resistant to the effect of the second illumination. These findings confirm that the light application scheme together with other photodynamic protocol components is crucial for the photocytotoxicity of hypericin.     

A systematic experimental study of moiré patterns in the electron image of solution-grown polyethylene crystals

A systematic study of solution-grown polyethylene crystals in the electron microscope using dark-field imaging in several diffracted beams has warranted a detailed analysis of the moiré patterns recorded. Direct proof for the existence of tilt moiré patterns has been obtained for the first time, while parallel and tilt patterns have been positively distinguished. Consideration of the formation conditions and properties of moiré patterns in dark field has been extended to include interference between an electron beam diffracted in both participating crystals with one diffracted in only one of them. Examples of moiré patterns depending thus on double diffraction have been identified.     

NiO/TiO2异质结构纳米管阵列膜对不锈钢的光生阴极保护及其储能性能

本工作通过修饰TiO₂制备半导体复合膜,提高其光吸收和光电化学性能,以期应用于光生阴极保护。先采用阳极氧化法在Ti表面制备TiO₂纳米管阵列膜,再应用水热处理法在膜表面沉积NiO纳米颗粒,形成具有异质结构纳米管复合膜。利用扫描电子显微镜、X-射线衍射、X-射线光电子能谱、紫外-可见吸收光谱、光致发光谱和光电化学技术对制备的纳米膜进行表征。结果表明,与纯TiO₂纳米管膜比较,NiO/TiO₂纳米管复合膜的光吸收扩展到可见光区。白光照射下,其在0.5 mol·L^?1 KOH和1 mol·L^?1 CH3OH混合液中的光电流密度达到176μA·cm^?2,是纯TiO₂纳米管膜的2倍。复合膜具有良好的光生阴极保护作用,与0.5 mol·L^?1 NaCl溶液中的403不锈钢耦连后,可使其电极电位下降440 mV,在光照2.5 h再转为暗态后,因具有电荷储存能力还可继续提供约15.5 h的阴极保护效应。     

Thin dielectric film thickness determination by advanced transmission electron microscopy.

High-resolution transmission electron microscopy (HR-TEM) has been used as the ultimate method of thickness measurement for thin films. The appearance of phase contrast interference patterns in HR-TEM images has long been confused as the appearance of a crystal lattice by nonspecialists. Relatively easy to interpret crystal lattice images are now directly observed with the introduction of annular dark-field detectors for scanning TEM (STEM). With the recent development of reliable lattice image processing software that creates crystal structure images from phase contrast data, HR-TEM can also provide crystal lattice images. The resolution of both methods has been steadily improved reaching now into the sub-Angstrom region. Improvements in electron lens and image analysis software are increasing the spatial resolution of both methods. Optimum resolution for STEM requires that the probe beam be highly localized. In STEM, beam localization is enhanced by selection of the correct aperture. When STEM measurement is done using a highly localized probe beam, HR-TEM and STEM measurement of the thickness of silicon oxynitride films agree within experimental error. In this article, the optimum conditions for HR-TEM and STEM measurement are discussed along with a method for repeatable film thickness determination. The impact of sample thickness is also discussed. The key result in this article is the proposal of a reproducible method for film thickness determination.     

Fractionated illumination after topical application of 5-aminolevulinic acid on normal skin of hairless mice: the influence of the dark interval

We have previously shown that light fractionation during topical aminolevulinic acid based photodynamic therapy (ALA-PDT) with a dark interval of 2h leads to a significant increase in efficacy in both pre-clinical and clinical PDT. However this fractionated illumination scheme required an extended overall treatment time. Therefore we investigated the relationship between the dark interval and PDT response with the aim of reducing the overall treatment time without reducing the efficacy. Five groups of mice were treated with ALA-PDT using a single light fraction or the two-fold illumination scheme with a dark interval of 30 min, 1, 1.5 and 2h. Protoporphyrin IX fluorescence kinetics were monitored during illumination. Visual skin response was monitored in the first seven days after PDT and assessed as PDT response. The PDT response decreases with decreasing length of the dark interval. Only the dark interval of 2h showed significantly more damage compared to all the other dark intervals investigated (P<0.05 compared to 1.5h and P<0.01 compared to 1h, 30 min and a single illumination). No relationship could be shown between the utilized PpIX fluorescence during the two-fold illumination and the PDT response. The rate of photobleaching was comparable for the first and the second light fraction and not dependent of the length of dark interval used. We conclude that in the skin of the hairless mouse the dark interval cannot be reduced below 2h without a significant reduction in PDT efficacy.     

A DUAL-BEAM SOURCE OF ACTINIC LIGHT FOR PHOTOSYNTHESIS RESEARCH

Abstract— A dual-beam source for providing illumination of white or nearly monochromatic light for photosynthesis studies is presented. The device provides programmed, repetitive sample illumination with variable intensity, pulse duration and sequence and a means of monitoring that program. It utilizes a lamp with an integral heat rejecting reflector, and a fiber optic bundle which efficiently transmits the light, diffusely and evenly illuminating the sample.