A focusing algorithm for high magnification cell imaging

An algorithm to produce a uniformly focused image in digital acquisition of high magnification light microscopy images is presented. In very high magnification microscopic imaging the specimen surface cannot be considered ideally flat so that capturing a single image frame is usually not sufficient to capture an image that is focused everywhere. An image formation model for light microscopic images is presented, and based on this model an algorithm to construct a uniformly focused image is presented. The algorithm requires that multiple frames of the image at different focal planes be processed to combine their information to obtain an estimated of the desired image which is more completely focused than any of the individual frames. Experimental results show that the proposed algorithm is very effective in approximating the desired image in high magnification microscopic imaging and highly robust comparing to the gradient method.     

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.     

Contrast matching in a sequence of images by histogram normalization

Variations in contrast between digital light microscopy images of a video sequence can be filtered out by matching the contrast of each individual image in the sequence to a reference image using the principle of histogram matching. This ensures the reliability of grey-scale threshold values in each frame within an image sequence. Here we describe examples of digital image-to-image contrast matching as applied to video light microscopy.     

A simple computer program to obtain digital pseudo-colored high resolution electron microscopy images

Computer programs which allow one to simulate high resolution electron microscopy images generally deliver the output on a line printer with the overprinting technique. Although this method suffers from poor resolution and limited contrast dynamics, it is still in widespread use. A more effective way is to represent the images in digital form on a color video display, and several solutions have been proposed recently, which often, however, make necessary use of rather expensive pictorial video terminals. In this work we describe a simple program which allows one to represent the output of a multislice program (projected potentials, lattice images, and diffraction patterns) on the display of an inexpensive color video terminal. Useful operations on the image, such as color selection over a wide range, image magnification, and filtering (to enhance specific details of interest) have been implemented. The displayed image can be either photographed from the screen or recorded on a good quality hard copier.     

Virtual electron microscopy in cell biology

Virtual microscopy of histological glass slides can emulate conventional light microscopy. Up till now, such a digital simulation does not exist for ultrathin electron microscopic slides. Because of the relative inaccessibility of electron microscopy, evaluation of subcellular structures by (bio)medical students is performed with the aid of photographic prints. In this article, the generation and evaluation of virtual electron microscopic slides is discussed. A T‐lymphoblastic cell was used as an example. Electron microscopic pictures were taken at two magnifications (25,000 and 50,000), processed in an analogue or digital way and stitched to reconstruct the image of the total cell. This image is viewed with a webviewer equipped with pan and zoom functions. The possibility of distinguishing the trilaminar structure of cellular membranes was the requisite. Virtual images obtained at an original magnification of 25,000, scanned at a resolution of 800 ppi could compete with pictures developed directly from negatives obtained by electron microscopy. It is possible to navigate and zoom into details in a way emulating electron microscopy. Virtual electron microscopy is innovative and offers new perspectives to interpret cytological pictures and to teach cell biology in an interactive and unique way. Microsc. Res. Tech., 2010. © 2010 Wiley‐Liss, Inc.     

Application of ion etching to immunoscanning electron microscopy

A method for achieving both the light and electron microscopic observations of the same immunolabeled semithin section is described. Mild ion etching (IE) was performed on the semithin LR white resin sections of rat pancreas to evaluate conditions for scanning electron microscopic secondary electron image observations. Before immunocytochemical staining, very mild, rapid etching was conducted as follows: ionization voltage 300 V, operating vacuum 35 Pa, and etching time 1 min, employing an ion coater above sections on glass slides. The sections were immunohistochemically stained with anti-insulin and immunogold in association with silver enhancement techniques for light microscopic observation, in which B cells in pancreatic islets were positively stained brown. Subsequently, essential mild IE was performed over the stained section as follows: 350 V, 38 Pa, 29 min. The samples were coated with platinum for scanning electron microscopic secondary electron images, in which the cores of secretory granules of the B cells were positively labeled with gold-silver particles. The present method is suitable for detection of substances involving immunogold labeling. It enables us to obtain high-resolution images at low magnification that can be correlated with light microscopic observations. Middle to high magnifications are applicable for detailed observations with secondary electron imaging scanning electron microscopy.     

Digital image tiles: a method for the processing of large sections

Segmentation of large areas of light microscopic slides into N by N fields, and each of these fields into M digital image tiles, allows the scanning, storage and digital processing of large images. Any of the original N² fields or composites of M adjacent tiles can be recalled to the video display for analysis. Developed procedures for use on a microscope equipped with a precision scanning stage allow registration of the image coordinates (X-Y) for any original or composite field and the alignment of one of these fields along the depth (Z) axis by means of external, machined fiducial marks in serial sections. To facilitate work whenever unavoidable, we have incorporated methods for digital image panning and zooming (changes of magnification) and discuss their use and implications.     

An automated system for whole microscopic image acquisition and analysis

The field of anatomic pathology has experienced major changes over the last decade. Virtual microscopy (VM) systems have allowed experts in pathology and other biomedical areas to work in a safer and more collaborative way. VMs are automated systems capable of digitizing microscopic samples that were traditionally examined one by one. The possibility of having digital copies reduces the risk of damaging original samples, and also makes it easier to distribute copies among other pathologists. This article describes the development of an automated high‐resolution whole slide imaging (WSI) system tailored to the needs and problems encountered in digital imaging for pathology, from hardware control to the full digitization of samples. The system has been built with an additional digital monochromatic camera together with the color camera by default and LED transmitted illumination (RGB). Monochrome cameras are the preferred method of acquisition for fluorescence microscopy. The system is able to digitize correctly and form large high resolution microscope images for both brightfield and fluorescence. The quality of the digital images has been quantified using three metrics based on sharpness, contrast and focus. It has been proved on 150 tissue samples of brain autopsies, prostate biopsies and lung cytologies, at five magnifications: 2.5×, 10×, 20×, 40×, and 63×. The article is focused on the hardware set‐up and the acquisition software, although results of the implemented image processing techniques included in the software and applied to the different tissue samples are also presented. Microsc. Res. Tech. 77:697–713, 2014. © 2014 Wiley Periodicals, Inc.     

Multidirectional focus measure for accurate three-dimensional shape recovery of microscopic objects

Shape from focus (SFF) is a technique to recover the shape of an object from multiple images taken at various focus settings. Most of conventional SFF techniques compute focus value of a pixel by applying one of focus measure operators on neighboring pixels on the same image frame. However, in the optics with limited depth of field, neighboring pixels of an image have different degree of focus for curved objects, thus the computed focus value does not reflect the accurate focus level of the pixel. Ideally, an accurate focus value of a pixel needs to be measured from the neighboring pixels lying on tangential plane of the pixel in image space. In this article, a tangential plane on each pixel location (i, j) in image sensor is searched by selecting one of five candidate planes based on the assumption that the maximum variance of focus values along the optical axis is achieved from the neighborhood lying on tangential plane of the pixel (i, j). Then, a focus measure operator is applied on neighboring pixels lying on the searched plane. The experimental results on both the synthetic and real microscopic objects show the proposed method produces more accurate three-dimensional shape in comparison to conventional SFF method that applies focus measures on original image planes.     

Computed tomography of cryogenic biological specimens based on X-ray microscopic images

Soft X-ray microscopy employs the photoelectric absorption contrast between water and protein in the 2.34-4.38 nm wavelength region to visualize protein structures down to 30 nm size without any staining methods. Due to the large depth of focus of the Fresnel zone plates used as X-ray objectives, computed tomography based on the X-ray microscopic images can be used to reconstruct the local linear absorption coefficient inside the three-dimensional specimen volume. High-resolution X-ray images require a high specimen radiation dose, and a series of images taken at different viewing angles is needed for computed tomography. Therefore, cryo microscopy is necessary to preserve the structural integrity of hydrated biological specimens during image acquisition. The cryo transmission X-ray microscope at the electron storage ring BESSY I (Berlin) was used to obtain a tilt series of images of the frozen-hydrated green alga Chlamydomonas reinhardtii. The living specimens were inserted into borosilicate glass capillaries and, in this first experiment, rapidly cooled by plunging into liquid nitrogen. The capillary specimen holders allow image acquisition over the full angular range of 180 degrees. The reconstruction shows for the first time details down to 60 nm size inside a frozen-hydrated biological specimen and conveys a clear impression of the internal structures. This technique is expected to be applicable to a wide range of biological specimens, such as the cell nucleus. It offers the possibility of imaging the three-dimensional structure of hydrated biological specimens close to their natural living state.     

Contrast definition and contour detection for logarithmic images

Logarithmic images, such as images obtained by transmitted light or those produced by the human visual system, differ from linear images. Their processing and analysis require consequently specific laws and structures. The latter have been developed in the concept of a logarithmic image processing (LIP) model (Jourlin & Pinoli, 1987, 1988; Pinoli, 1987a). This model permits the introduction of a well-justified contrast definition: from a physical point of view, it is closely linked with logarithmic images and from a mathematical point of view, it is set up in an algebraic structure. The applications presented at the end of this paper concern image preprocessing and segmentation. In particular, in the case of microscopic images, the proposed method of segmentation gives good results with transmitted light (thin foils in biology or transmitted electronic microscopy). However, images obtained by reflected light microscopy are not within the scope of this model.     

Investigations on the leaf surface ultrastructure in grapevine (Vitis vinifera L.) by scanning microscopy

Several Scanning microscopy techniques were used to investigate the leaf surface ultrastructure in the local “Razegui” grapevine cultivar (Vitis vinifera L.). Conventional scanning electron microscopy performed on glutaraldehyde‐fixed samples allowed observation of well‐preserved epidermal cells with an overlaying waxy layer. At a high magnification, the waxy layer exhibited crystalline projections in the form of horizontal and vertical platelets. Also, to avoid eventual ultrastructural alterations inherent in the use of solvents during sample preparation, fresh leaf blade samples were directly observed by environmental scanning electron microscopy. A classical image of convex living epidermal cells was observed. At 2400× magnification, epicuticular waxes exhibited a granular structure. However, high‐magnification images were not obtained with this device. The atomic force microscopy (AFM) performed on fresh leaf blade samples allowed observation of a textured surface and heterogeneous profiles attributed to epicuticular wax deposits. AFM topography images confirmed further, the presence of irregular crystalloid wax projections as multishaped platelets on the adaxial surface of grapevine leaf. SCANNING 31: 127–131, 2009. © 2009 Wiley Periodicals, Inc.     

Capturing and displaying microscopic images used in medical diagnostics and forensic science using 4K video resolution – an application in higher education

To analyze, interpret and evaluate microscopic images, used in medical diagnostics and forensic science, video images for educational purposes were made with a very high resolution of 4096 × 2160 pixels (4K), which is four times as many pixels as High‐Definition Video (1920 × 1080 pixels). The unprecedented high resolution makes it possible to see details that remain invisible to any other video format. The images of the specimens (blood cells, tissue sections, hair, fibre, etc.) are recorded using a 4K video camera which is attached to a light microscope. After processing, this resulted in very sharp and highly detailed images. This material was then used in education for classroom discussion. Spoken explanation by experts in the field of medical diagnostics and forensic science was also added to the high‐resolution video images to make it suitable for self‐study.     

Automatic setae segmentation from Chaetoceros microscopic images

A novel image processing model Grayscale Surface Direction Angle Model (GSDAM) is presented and the algorithm based on GSDAM is developed to segment setae from Chaetoceros microscopic images. The proposed model combines the setae characteristics of the microscopic images with the spatial analysis of image grayscale surface to detect and segment the direction thin and long setae from the low contrast background as well as noise which may make the commonly used segmentation methods invalid. The experimental results show that our algorithm based on GSDAM outperforms the boundary‐based and region‐based segmentation methods Canny edge detector, iterative threshold selection, Otsu's thresholding, minimum error thresholding, K‐means clustering, and marker‐controlled watershed on the setae segmentation more accurately and completely. Microsc. Res. Tech. 77:684–690, 2014. © 2014 Wiley Periodicals, Inc.     

Restoration of distorted colour microscopic images from transverse chromatic aberration of imperfect lenses

An algorithm is presented for restoration of colour microscopic images with distortions from imperfect microscope lenses having transverse chromatic aberrations, resulting in a magnification that slightly varies with wavelengths or colours. The differential of each colour component image is computed as the difference between the component image and its slightly magnified version. The absolute values in the differential component images are generally higher at the edges where greater discontinuities occur. The two cross-correlation functions of the absolute differentials between red and green colours and between red and blue colours are then computed. The maximum in the two cross-correlation functions were sought, respectively, and the cross-correlation delays were then calculated. The two cross-correlation delays were used to determine dispersions and to realign the three colour components. Results of real microscopic images are provided. The restored image and the original are compared both visually and quantitatively in terms of the estimated entropies measured for the degree of concentrations using vector distributions.     

An improved method for collecting and staining microorganisms for enumeration by fluorescence light microscopy

A rapid, robust method for the enumeration of total and viable microorganisms is described. A method using specific stains for viable and total cells and fluorescence light microscopy on membrane filters had been previously developed, but was sub-optimal in that some non-specific staining of the filters occurred and the filters were not flat enough for automatic image analysis methods to be employed, because not all cells in a field of view were in focus simultaneously. A new membrane filter has recently become available: the Anopore? membrane was described by the manufacturers as having a number of properties which would overcome these limitations. These include inorganic construction (giving resistance to solvents), high porosity (giving high flow rates), low surface adsorption (giving low background staining) and inherent hydrophilicity (simplifying wetting with aqueous solutions). Anopore membrane filters were found to produce very high contrast images of bacteria stained with ethidium bromide. Even with a relatively low power (magnification = 40) dry objective, these images could be easily thresholded for image analysis using only grey-level information. The methods developed here are considered to be a suitable basis for a fully automated procedure for the enumeration of total microbial populations.     

The imaging mechanism of single-walled carbon nanotubes on Si/SiO₂ wafer in scanning electron microscopy

Scanning electron microscopy imaging of both suspended single‐walled carbon nanotubes (SWNTs) and contacted SWNTs with Si/SiO₂ substrate has been studied in this paper. The voltage contrast has been investigated by supplying external electric field around the samples. The results show that the image contrast of SWNTs attributes to both voltage contrast from the area surrounding SWNTs (tens of nanometres in both sides of the SWNTs) and electron beam induced emission from SWNTs themselves under low primary beam energy. Under high primary beam energy, however, EBIE dominates the image contrast due to the fact that the voltage contrast caused by implanted charges of the SiO₂ layer is weakened. Imaging under the primary beam energy lower than 1 keV offers widened diameter of SWNTs, which promises that the SWNTs are observable at very low magnification (lower than 100×). At a larger magnification, however, imaging under the primary beam energy higher than 10 keV can display more realistic images of the SWNTs. In addition, an appropriate external electric field can improve the images.     

Image deformation in field ion microscopy of faceted crystals

We perform detailed numerical simulations of field ion microscopy images of faceted crystals and compare them with experimental observations. In contrast to the case of crystals with a smooth surface, for a faceted topography we find extreme deformations of the ion image. Local magnification is highly inhomogeneous and may vary by an order of magnitude: from 0.64 to 6.7. Moreover, the anisotropy of the magnification at a point located on the facet edge may reach a factor of 10.     

全视场在线图像可视铁谱磨粒显微成像特性分析

为了评估全视场在线图像可视铁谱磨粒显微成像特性,提出了一种反射光显微成像模型。首先,基于朗伯余弦与小角度散射理论建立了全视场(OLVF)的反射光辐照度模型,实现了磨粒显微成像清晰度定量评价。然后,仿真计算磨粒显微成像的反差透视比,确定了最优光学倍率和适用于全视场OLVF探测的油液衰减系数范围,明确了光学参数对磨粒显微成像质量的影响规律。结果显示：光学倍率为2.0×且油液衰减系数≤2.0条件下,磨粒沉积于物方视场的主光轴附近,全视场OLVF可获得最佳磨粒显微成像清晰度。最后,开展了磨粒显微成像实验测试,结果表明：全视场OLVF能够从油液衰减系数小于2.0的在用液压油和齿轮油中获取反射光谱片图像,并提取磨粒视觉信息进行磨损在线监测。