Axial Phase-Darkfield-Contrast (APDC), a new technique for variable optical contrasting in light microscopy

Axial phase-darkfield-contrast (APDC) has been developed as an illumination technique in light microscopy which promises significant improvements and a higher variability in imaging of several transparent 'problem specimens'. With this method, a phase contrast image is optically superimposed on an axial darkfield image so that a partial image based on the principal zeroth order maximum (phase contrast) interferes with an image, which is based on the secondary maxima (axial darkfield). The background brightness and character of the resulting image can be continuously modulated from a phase contrast-dominated to a darkfield-dominated character. In order to achieve this illumination mode, normal objectives for phase contrast have to be fitted with an additional central light stopper needed for axial (central) darkfield illumination. In corresponding condenser light masks, a small perforation has to be added in the centre of the phase contrast providing light annulus. These light modulating elements are properly aligned when the central perforation is congruent with the objective's light stop and the light annulus is conjugate with the phase ring. The breadth of the condenser light annulus and thus the intensity of the phase contrast partial image can be regulated with the aperture diaphragm. Additional contrast effects can be achieved when both illuminating light components are filtered at different colours. In this technique, the axial resolution (depth of field) is significantly enhanced and the specimen's three-dimensional appearance is accentuated with improved clarity as well as fine details at the given resolution limit. Typical artefacts associated with phase contrast and darkfield illumination are reduced in our methods.     

Phase contrast without phase plates and phase rings—optical solutions for improved imaging of phase structures

Using the optical methods described, phase specimens can be observed with a modified light microscope in enhanced clarity, purified from typical artifacts which are apparent in standard phase contrast illumination. In particular, haloing and shade‐off are absent, lateral and vertical resolution are maximized and the image quality remains constant even in problematic preparations which cannot be well examined in normal phase contrast, such as specimens beyond a critical thickness or covered by obliquely situated cover slips. The background brightness and thus the range of contrast can be continuously modulated and specimens can be illuminated in concentric‐peripheral, axial or paraxial light. Additional contrast effects can be achieved by spectral color separation. Normal glass or mirror lenses can be used; they do not need to be fitted with a phase plate or a phase ring. The methods described should be of general interest for all disciplines using phase microscopy. Microsc. Res. Tech., 76:1050–1056, 2013. © 2013 Wiley Periodicals, Inc.     

Variable multimodal light microscopy with interference contrast and phase contrast; dark or bright field

Using the optical methods described, specimens can be observed with modified multimodal light microscopes based on interference contrast combined with phase contrast, dark‐ or bright‐field illumination. Thus, the particular visual information associated with interference and phase contrast, dark‐ and bright‐field illumination is joined in real‐time composite images appearing in enhanced clarity and purified from typical artefacts, which are apparent in standard phase contrast and dark‐field illumination. In particular, haloing and shade‐off are absent or significantly reduced as well as marginal blooming and scattering. The background brightness and thus the range of contrast can be continuously modulated and variable transitions can be achieved between interference contrast and complementary illumination techniques. The methods reported should be of general interest for all disciplines using phase and interference contrast microscopy, especially in biology and medicine, and also in material sciences when implemented in vertical illuminators.     

An electron-optical lens effect as a possible source of contrast in biological preparations

Heavy-metal stain aggregates on the surface of thin sections of biological material have higher contrast than those embedded within the sections and both have greater contrast than can be accounted for by the amplitude image. Disturbances of the incident illumination by a specimen in both light- and electron-optical systems and their possible contribution to image contrast are considered. The hypothesis is proposed that a lens effect produced by the stain aggregates may account for their contrast in the electron microscope in a similar manner to the contrast of glass beads in the light microscope with a low numerical aperture.     

Spatial calibration of structured illumination fluorescence microscopy using capillary tissue phantoms

Quantitative assessment of microvascular structure is relevant to the investigations of ischemic injury, reparative angiogenesis and tumor revascularization. In light microscopy applications, thick tissue specimens are necessary to characterize microvascular networks; however, thick tissue leads to image distortions due to out-of-focus light. Structured illumination confocal microscopy is an optical sectioning technique that improves contrast and resolution by using a grid pattern to identify the plane-of-focus within the specimen. Because structured illumination can be applied to wide-field (nonscanning) microscopes, the microcirculation can be studied by sequential intravital and confocal microscopy. To assess the application of structured illumination confocal microscopy to microvessel imaging, we studied cell-sized microspheres and fused silica microcapillary tissue phantoms. As expected, structured illumination produced highly accurate images in the lateral (X-Y) plane, but demonstrated a loss of resolution in the Z-Y plane. Because the magnitude of Z-axis distortion was variable in complex tissues, the silica microcapillaries were used as spatial calibration standards. Morphometric parameters, such as shape factor, were used to empirically optimize Z-axis software compression. We conclude that the silica microcapillaries provide a useful tissue phantom for in vitro studies as well as spatial calibration standard for in vivo morphometry of the microcirculation.     

Evaluating performance in three-dimensional fluorescence microscopy

In biological fluorescence microscopy, image contrast is often degraded by a high background arising from out of focus regions of the specimen. This background can be greatly reduced or eliminated by several modes of thick specimen microscopy, including techniques such as 3-D deconvolution and confocal. There has been a great deal of interest and some confusion about which of these methods is 'better', in principle or in practice. The motivation for the experiments reported here is to establish some rough guidelines for choosing the most appropriate method of microscopy for a given biological specimen. The approach is to compare the efficiency of photon collection, the image contrast and the signal-to-noise ratio achieved by the different methods at equivalent illumination, using a specimen in which the amount of out of focus background is adjustable over the range encountered with biological samples. We compared spot scanning confocal, spinning disk confocal and wide-field/deconvolution (WFD) microscopes and find that the ratio of out of focus background to in-focus signal can be used to predict which method of microscopy will provide the most useful image. We also find that the precision of measurements of net fluorescence yield is very much lower than expected for all modes of microscopy. Our analysis enabled a clear, quantitative delineation of the appropriate use of different imaging modes relative to the ratio of out-of-focus background to in-focus signal, and defines an upper limit to the useful range of the three most common modes of imaging.     

Testing mounting media to eliminate background noise in confocal microscope 3-D images of insect genitalia

Recently, the confocal laser scanning microscope (CLSM) has been used to image and generate three-dimensional reconstructions of miniscule insect tissues and cuticular structures. These three-dimensional reconstructions provide the investigator with key information concerning the spatial relationship among structures and substructures. Unfortunately, there can be high levels of background "noise" which can obscure the specimen in these three-dimensional reconstructions. This background "noise" might be a result of the mounting media either autofluorescing or reflecting and scattering the imaged specimen's fluorescence. The standard nonpermanent mounting medium is glycerine jelly (a 1:17:17 ratio of porcine gelatin to glycerine to water). In this study, the organic molecule (lipid, protein, or carbohydrate) added to the glycerine water mixture was varied. The relative background to specimen signal (the mean voxel brightness reading in ImageJ freeware) was compared across mountants. The mounting media tested are ranked from best (least background noise) to worst (most background noise) as follows: agarose, agar, pectin, gelatin (the standard), petroleum jelly. A 1% agarose mountant (1:50:50 ratio of agarose to glycerine to water) is recommended because it causes little to no background noise, provides consistent high quality contrast between specimen and background with increasing depth, and is easy to handle.     

钢轨磨损视觉测量的轮廓精确快速提取

为保证钢轨磨损动态视觉测量的高精度,综合图像获取和图像处理技术,实现了清晰光条图像获取和光条中心点亚像素坐标精确提取。根据光条与背景环境亮度的高对比度,提出一种依据光条亮度的相机自动曝光法,用于获取清晰的光条图像;分析图像光条法线方向的亮度衰变特征,采用动态阈值分割法初步提取光条,滤除图像背景的同时保留光条法线方向的亮度衰减信息;根据图像过度曝光信息确定光条中心点像素大致位置,再对分割的光条图像相对应像素位置点计算Hessian矩阵,获取光条中心点的亚像素坐标。采用MFC编写应用程序进行试验,在不同光照环境和背景物的干扰下,该方法可精确地提取光条中心,与经典Steger算法相比提取精度偏差为0.05pixel,运算时间节约40%。试验结果验证了该方法稳定性好,有较强的抗干扰能力,较好地满足钢轨磨损测量的现场要求。     

Video-enhanced microscopy with a computer frame memory

Video-enhanced microscopy combined with the use of a computer frame memory extends considerably the useful range of our video enhanced contrast (AVEC) methods for polarizing, double-beam interference and differential interference contrast microscopy. Increased visual contrast is achieved by two stages of amplifications: the first optical, by using high bias retardation settings, and the second electronic. These steps are followed by a reduction of background brightness by means of a clamp voltage applied to a DC restoration circuit of the video camera. One of the limitations of the AVEC method alone is the inevitable appearance under high gain conditions of a pattern of mottle due to inaccessible dirt and defects in the lenses even of high quality. This limitation has been circumvented by storing the mottle pattern in the frame memory (frame store) and continuously subtracting it from each succeeding frame to clear the image. A major gain in image quality has resulted. In polarizing microscopy, the frame memory can be used also to subtract the image at one compensator setting from that at the equivalent setting of opposite sign, thus removing from the final image not only most of the mottle pattern but also the contrast due to the bright-field contrast. In the polarizing microscope, these manipulations of the raw video image make it possible to observe and measure the birefringence of various organelles and elements such as microtubules, intermediate filaments and bundles of as few as a half dozen actin filaments. Since scattered light is also removed from the image, features hidden from view in the unprocessed image become visible. In differential interference microscopy, the AVEC method makes visible (i.e. detectable) many linear elements and particles that are an order of magnitude smaller than the resolution limit and not visible in the optical image. Such features are inflated by diffraction, however, to Airy disk size.     

The modulation contrast microscope: principles and performance

The modulation contrast microscope produces an image of high contrast and resolution. The image has a three-dimensional appearance wherein a rounded object appears dark on one side, bright on the other with grey in between against a grey background. The performance features are optical sectioning, directionality, high resolution and control of contrast and coherence. A bright field microscope is converted to the modulation contrast microscope by adding the modulator, a special amplitude filter, in the objective. A slit aperture part of which is polarized is placed before the condenser. Below this is a rotatable polarizer. The modulator processes light from opposite gradients oppositely, that is brighter for one and darker for the other; thereby preserving the sign. The diffraction theory has been extended to show that gradient image intensity is the intensity of the zero order and when modified by the modulator creates a high contrast image. The modulation contrast microscope is simple and easy to adjust. It is useful in reflected and transmitted light systems, with plastic and glass vessels as well as in combination with fluorescence systems and polarization techniques. There is virtually no limit to the type of specimen that can be studied.     

A new light microscopic method for the synchronous bidirectional illumination and viewing of living cells in different contrast modes, and/or at different focal levels or magnifications.

A new method of light microscopy for the analysis of the behaviour of living cells in vitro exploits two objects for simultaneous image formation, each serving the other as a condenser. Simultaneous viewing from opposite sides allows the specimen to be examined at: (a) two different magnifications, permitting the locomotion of whole cell (groups) to be studied at a low magnification and details of interaction of colliding surfaces at a high magnification; (b) two different focal levels, permitting, for example, details near the substrate surface to be recorded at the same time as information concerning the behaviour of the free, dorsal surface; and (c) two different contrast modes, such as negative and positive phase contrast, and dark and bright field illuminations. These possibilities can be combined, for example, to contrast a high magnification view in negative phase contrast at one focal level with a low magnification image in ordinary brightfield at another focal level in the same living cells.     

Methods for compensation of the light attenuation with depth of images captured by a confocal microscope

A confocal laser scanning microscope (CLSM) enables us to capture images from a biological specimen in different depths and obtain a series of precisely registered fluorescent images. However, images captured from deep layers of the specimen may be darker than images from the topmost layers because of light loss distortions. This effect causes difficulties in subsequent analysis of biological objects. We propose a solution using two approaches: either an online method working already during image acquisition or an offline method assisting as a postprocessing step. In the online method, the gain value of a photomultiplier tube of a CLSM is controlled according to the difference of mean image intensities between the reference and currently acquired image. The offline method consists of two stages. In the first stage, a standard histogram maintaining relative frequencies of gray levels and improving brightness and contrast is created from all images in the series. In the second stage, individual image histograms are warped according to this standard histogram. The methods were tested on real confocal image data captured from human placenta and rat skeletal muscle specimens. It was shown that both approaches diminish the light attenuation in images captured from deep layers of the specimen.     

Quantitative theory of ideal phase-contrast microscopy,taking object width into account

In ‘ideal’ phase-contrast microscopy all the direct light and none of the diffracted light is influenced by the phase plate in the back focal plane of the objective. Contrary to almost all previous work, it appears that the intensity of an ideal phase-contrast image is affected not only by the transmittance and retardation of the object and of the phase plate, but also by the width of the specimen (or total width of multiple specimens) relative to the microscopic field. Equations and computer code are presented with which the intensity of such images can be calculated. Previously published equations are special cases, and implicitly or explictly assume either that the object is of negligible width, or occupies precisely half the microscopic field. The absolute brightness of an image in ideal central dark-field microscopy is a function of the object retardation, but the intensity of the image relative to the background is a function only of the width of the object(s) relative to the field. The equations give results for ideal phase-contrast microscopy identical with those of a computer program simulating microscopic imaging. The program can in addition take into account non-ideal factors including a finite width of phase plate, finite objective aperture, deviations from best focus, glare, primary spherical aberration and obliquity of the coherent illumination.     

Condenser‐free contrast methods for transmitted‐light microscopy

Phase contrast microscopy allows the study of highly transparent yet detail‐rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser‐free yet highly effective method of obtaining phase contrast in transmitted‐light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light‐path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser‐free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser‐free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour‐contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser‐free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next‐generation transmitted‐light microscopy designs. The condenser‐free illumination method, using rings of independent or radially‐scanned emitters, may be exploited in future in other electromagnetic wavebands, including X‐rays or the infrared.     

多图像融合Retinex用于弱光图像增强

为解决弱光图像增强过程中对比度增强和自然度保持问题,本文提出一种基于Retinex的多图像自适应加权最小二乘滤波算法。首先,在图像的每个像素的R,G,B三通道中找到最大亮度值作为该像素的初始照明估计,根据Retinex理论生成反射图像,并通过形态学闭合方式调整反射图;接着,在初始照明图基础上,通过Gamma变换和双对数变换方法分别生成全局对比度增强图和局部自然度保持照明图;随后,设计一种自适应加权最小二乘滤波融合策略将三幅照明图融合成最终照明估计图;最后合成上述的最终照明图和调整反射图以获得弱光增强后的图像。实验结果表明,本文所提出算法的亮度顺序差(LOE)及盲图像质量评价(NIQE)值更低,可同时降低到4.12和3.25,较其他方法表现出更好的增强效果。证明了本文算法能有效地增强弱光图像对比度,同时保持图像自然度。     

The influence of specimen refractive index,detector signal integration,and non-uniform scan speed on the imaging properties in confocal microscopy

Refraction of light in a specimen volume may cause aberrations that influence the imaging properties in confocal microscopy. In this paper the influence on three-dimensional resolution and geometry is experimentally investigated for a uniform specimen volume. It is found that the depth resolution is more severely affected than the lateral resolution. This is unfortunate, because even under ideal conditions the depth resolution is lower than the lateral resolution. Lateral image geometry is little affected by the specimen refractive index, whereas the depth scale can be considerably elongated or compressed. The influence of a finite detector integration time is also considered. This can give a noticeable, but not particularly severe effect on the image resolution in the line-scan direction. Because the integration time can be accurately controlled, a shorter integration time can be used when maximum resolution is essential, albeit at the price of a higher noise level. In scanning fluorescence microscopy a non-uniform scan speed may give large variations in bleaching over the specimen surface. Experiments illustrate how serious such non-uniform bleaching effects can be when a specimen area is repeatedly scanned, for example when recording optical serial sections.     

Use of spot-scan procedure for recording low-dose micrographs of beam-sensitive specimens

Electron micrographs of monolayer crystals of paraffin have been recorded with a spot-scan mode of imaging which uses a small 50 nm diameter moving beam. In comparison with normal stationary beam images using 5 μm illuminating beams, the spot-scan micrographs show a higher and more consistent contrast from the 3.8–4.2 Å hydrocarbon chain spacings. On average the improvement in contrast is twofold, but this still leaves scope for further improvement: the best spot-scan images still do not quite reach the level of contrast calculated theoretically from electron diffraction. We believe that the cause of the low contrast in paraffin images must be specimen motion caused by radiation damage rather than a charging effect, for two reasons. First, it does not occur in control images of vermiculite, a non-beam-sensitive mineral, when treated identically. Secondly, although charging might still be a problem with paraffin, when images are taken with the objective aperture in the column, a procedure which is normally expected to reduce charging, no improvement in contrast is found. Thus we think that the use of the small beam minimizes the effect of specimen motion on image contrast by minimizing the specimen area exposed at each instant and therefore the resultant image blurring. Further improvements with even smaller illumination beam diameters might be expected.     

Recording and controlling the 4D light field in a microscope using microlens arrays

By inserting a microlens array at the intermediate image plane of an optical microscope, one can record four-dimensional light fields of biological specimens in a single snapshot. Unlike a conventional photograph, light fields permit manipulation of viewpoint and focus after the snapshot has been taken, subject to the resolution of the camera and the diffraction limit of the optical system. By inserting a second microlens array and video projector into the microscope's illumination path, one can control the incident light field falling on the specimen in a similar way. In this paper, we describe a prototype system we have built that implements these ideas, and we demonstrate two applications for it: simulating exotic microscope illumination modalities and correcting for optical aberrations digitally.     

Image contrast of dielectric specimens in transmission mode near-field scanning optical microscopy: imaging properties and tip artefacts

Near-field scanning optical microscopy (NSOM) is a scanned probe technique utilizing a subwavelength-sized light source for high-resolution imaging of surfaces. Although NSOM has the potential to exploit and extend the experimental utility of the modern light microscope, the interpretation of image contrast is not straightforward. In near-field microscopy the illumination intensity of the source (probe) is not a constant value, rather it is a function of the probe–sample electronic environment. A number of dielectric specimens have been studied by NSOM to elucidate the contrast role of specimen type, topography and crystallinity; a summary of metallic specimen observations is presented for comparative purposes. Near-field image contrast is found to be a result of lateral changes in optical density and edge scattering for specimens with little sample topography. For surfaces with considerable topography the contributions of topographic (Z) axis contrast to lateral (X,Y) changes in optical density have been characterized. Selected near-field probes have also been shown to exhibit a variety of unusual contrast artefacts. Thorough study of polarization contrast, optical edge (scattering) contrast, as well as molecular orientation in crystalline specimens, can be used to distinguish lateral contrast from topographic components. In a few cases Fourier filtering can be successfully applied to separate the topographic and lateral contrast components.     

Shack-Hartmann wave front measurements in cortical tissue for deconvolution of large three-dimensional mosaic transmitted light brightfield micrographs

We present a novel approach for deconvolution of 3D image stacks of cortical tissue taken by mosaic/optical‐sectioning technology, using a transmitted light brightfield microscope. Mosaic/optical‐sectioning offers the possibility of imaging large volumes (e.g. from cortical sections) on a millimetre scale at sub‐micrometre resolution. However, a blurred contribution from out‐of‐focus light results in an image quality that usually prohibits 3D quantitative analysis. Such quantitative analysis is only possible after deblurring by deconvolution. The resulting image quality is strongly dependent on how accurate the point spread function used for deconvolution resembles the properties of the imaging system. Since direct measurement of the true point spread function is laborious and modelled point spread functions usually deviate from measured ones, we present a method of optimizing the microscope until it meets almost ideal imaging conditions. These conditions are validated by measuring the aberration function of the microscope and tissue using a Shack‐Hartmann sensor. The analysis shows that cortical tissue from rat brains embedded in Mowiol and imaged by an oil‐immersion objective can be regarded as having a homogeneous index of refraction. In addition, the amount of spherical aberration that is caused by the optics or the specimen is relatively low. Consequently the image formation is simplified to refraction between the embedding and immersion medium and to 3D diffraction at the finite entrance pupil of the objective. The resulting model point spread function is applied to the image stacks by linear or iterative deconvolution algorithms. For the presented dataset of large 3D images the linear approach proves to be superior. The linear deconvolution yields a significant improvement in signal‐to‐noise ratio and resolution. This novel approach allows a quantitative analysis of the cortical image stacks such as the reconstruction of biocytin‐stained neuronal dendrites and axons.