A model for predicting pathologist's velocity profiles when navigating virtual slides

Navigation through large microscopic images is a potential benefit for histology or pathology teaching, for improving the quality of diagnosis in pathology, or for communicating pathologists in some telemedicine applications. However, the size of this kind of images is prohibitive for navigation with conventional techniques. This article presents a soft computing model, which permits to anticipate the pathologist trajectories in diagnosis tasks when exploring virtual slides. The Bayesian strategy combines an offline model of a baseline pathologist knowledge (the prior) and a prediction online module (the likelihood) that captures a particular pathologist navigation pattern. While optimal parameters for the biologically inspired offline model are calculated using an Expectation‐Maximization strategy, prediction is carried out by a particle filter. Parameters are estimated from several series of actual navigations performed by several pathologists in different virtual slides. The present approach is compared with other conventional prediction methods and decreases the calculated MSE in about a 50% for the entire group of pathologists. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

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.     

基于虚拟仪器的组合导航系统测试平台的设计

提出利用虚拟仪器技术开发测试平台的设计方法,详细介绍了某型组合导航系统测试平台的软硬件设计,重点分析了设计中难点问题及其解决方法.通过实际测试和用户使用,证实该测试平台具有系统性能稳定、可靠,操作方便,界面友好等特点.     

Gated‐sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching

The spatial resolution of a stimulated emission depletion (STED) microscope is theoretically unlimited and practically determined by the signal‐to‐noise ratio. Typically, an increase of the STED beam's power leads to an improvement of the effective resolution. However, this improvement may vanish because an increased STED beam's power is often accompanied by an increased photobleaching, which worsen the effective resolution by reducing the signal strength. A way to lower the photobleaching in pulsed STED (P‐STED) implementations is to reduce the peak intensity lengthening the pulses duration (for a given average STED beam's power). This also leads to a reduction of the fluorophores quenching, thus a reduction of the effective resolution, but the time‐gated detection was proved to be successful in recovering these reductions. Here we demonstrated that a subnanosecond fiber laser beam (pulse width ∼600 ps) reduces the photobleaching with respect to a traditional stretched hundreds picosecond (∼200 ps) beam provided by a Ti:Sapphire laser, without any effective spatial resolution lost.     

基于虚拟仪器技术的捷联惯性/ GNSS组合导航系统自动测试系统设计

捷联惯性/GNSS组合导航系统是飞机定位和导航的重要机载电子设备;但是开发具有模块化、柔性、通用性和远程控制和测试功能的通用自动测试平台,却是一个难题.文中在简要介绍虚拟仪器技术的基础上,提出用虚拟仪器技术开发通用自动测试系统的设计方法;详细介绍了捷联惯性/GNSS组合导航系统测试系统的软硬件设计,重点分析了设计中难点问题及其解决方法.通过实际测试和评估,该综合测试系统性能稳定、可靠,操作方便,界面友好.     

Pipeline for illumination correction of images for high‐throughput microscopy

The presence of systematic noise in images in high‐throughput microscopy experiments can significantly impact the accuracy of downstream results. Among the most common sources of systematic noise is non‐homogeneous illumination across the image field. This often adds an unacceptable level of noise, obscures true quantitative differences and precludes biological experiments that rely on accurate fluorescence intensity measurements. In this paper, we seek to quantify the improvement in the quality of high‐content screen readouts due to software‐based illumination correction. We present a straightforward illumination correction pipeline that has been used by our group across many experiments. We test the pipeline on real‐world high‐throughput image sets and evaluate the performance of the pipeline at two levels: (a) Z′‐factor to evaluate the effect of the image correction on a univariate readout, representative of a typical high‐content screen, and (b) classification accuracy on phenotypic signatures derived from the images, representative of an experiment involving more complex data mining. We find that applying the proposed post‐hoc correction method improves performance in both experiments, even when illumination correction has already been applied using software associated with the instrument. To facilitate the ready application and future development of illumination correction methods, we have made our complete test data sets as well as open‐source image analysis pipelines publicly available. This software‐based solution has the potential to improve outcomes for a wide‐variety of image‐based HTS experiments.     

Super‐resolution optical microscopy based on scannable cantilever‐combined microsphere

We report an ingenious method of super‐resolution optical microscopy utilizing scannable cantilever‐combined microsphere. By scanning the microsphere over the sample surface in a cantilever‐combined microsphere‐sample contact state, super‐resolution images can be acquired at arbitrary sample regions through near‐field information collection by the microsphere. In addition, such a state can effectively reduce the possibility of breaking the cantilever and damaging the microsphere or sample surface. This work has developed a new method and technique of sub‐diffraction‐limit optical microscopy, and can be practically applied in various fields of micro/nanoscopy. Microsc. Res. Tech. 78:1128–1132, 2015. © 2015 Wiley Periodicals, Inc.     

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.     

Cryo‐scanning x‐ray diffraction microscopy of frozen‐hydrated yeast

We developed cryo‐scanning x‐ray diffraction microscopy, utilizing hard x‐ray ptychography at cryogenic temperature, for the noninvasive, high‐resolution imaging of wet, extended biological samples and report its first frozen‐hydrated imaging. Utilizing phase contrast at hard x‐rays, cryo‐scanning x‐ray diffraction microscopy provides the penetration power suitable for thick samples while retaining sensitivity to minute density changes within unstained samples. It is dose‐efficient and further minimizes radiation damage by keeping the wet samples at cryogenic temperature. We demonstrate these capabilities in two dimensions by imaging unstained frozen‐hydrated budding yeast cells, achieving a spatial resolution of 85 nm with a phase sensitivity of 0.0053 radians. The current work presents the feasibility of cryo‐scanning x‐ray diffraction microscopy for quantitative, high‐resolution imaging of unmodified biological samples extending to tens of micrometres.     

Sub‐100‐nanometre resolution in total internal reflection fluorescence microscopy

Combining total internal reflection fluorescence microscopy with structured illumination allows optical wide‐field imaging with sub‐100‐nanometre resolution. We present a novel objective‐launch set‐up for standing wave illumination that takes advantage of a tunable transmission diffraction grating and transparent phase shifters actuated by electro‐active polymers to control the excitation pattern in three dimensions. Image acquisition is completed in less than 1 s. To reconstruct the extended image spectrum, we apply a new apodization function that results in a lateral resolution of 89 nm for green emission wavelength.     

Compact non‐contact total emission detection for in vivo multiphoton excitation microscopy

We describe a compact, non‐contact design for a total emission detection (c‐TED) system for intra‐vital multiphoton imaging. To conform to a standard upright two‐photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non‐contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c‐TED device compared to heavily optimized objective‐based emission collection. The best light collection enhancement was seen from murine fat (5×–2× gains as a function of depth), whereas murine skeletal muscle and rat kidney showed gains of over two and just under twofold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a twofold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers, enabled by greater light collection efficiency, yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo‐damage, as well as the applicability of this device to other multiphoton imaging methods is discussed.     

Second‐harmonic imaging microscopy for identifying colorectal intraepithelial neoplasia

In this study, second‐harmonic imaging microscopy was used to monitor precancerous colorectal lesions at different stages. It was found that the morphology of glands and lamina propria in mucosa changes with the progression of colorectal diseases from normal to low‐grade intraepithelial neoplasia to high‐grade intraepithelial neoplasia and this microscopy has the ability of direct visualization of these warning symptoms. Furthermore, two morphologic variables were quantified to determine the changes of glands and collagen in lamina propria during the development of colorectal intraepithelial neoplasia. These results suggest that second‐harmonic imaging microscopy has the potential in label‐freely and effectively distinguishing between normal and precancerous colorectal tissues, and will be helpful for early diagnosis and treatment of colorectal diseases.     

Preserving elemental content in adherent mammalian cells for analysis by synchrotron‐based x‐ray fluorescence microscopy

Trace metals play important roles in biological function, and x‐ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side‐by‐side comparison shows that plunge‐freezing‐based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. When chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x‐ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.     

Exploring the use of soft X‐ray microscopy for imaging subcellular structures of the inner ear1

The soft X‐ray microscope at the Lawrence Berkeley National Laboratory was developed for visualization of biological tissue. Soft X‐ray microscopy provides high‐resolution visualization of hydrated, non‐embedded and non‐sectioned cells and is thus potentially an alternative to transmission electron microscopy. Here we show for the first time soft X‐ray micrographs of structures isolated from the guinea‐pig inner ear. Sensory outer hair cells and supporting pillar cells are readily visualized. In the hair cells, individual stereocilia can easily be identified within the apical hair bundle. The underlying cuticular plate is, however, too densely composed or too thick to be clearly visualized, and thus appears very dark. The cytoplasmic structures protruding from the cuticular plates as well as the fibrillar material surrounding and projecting from the cell nuclei can be seen. In the pillar cells the images reveal individual microtubule bundles. Soft X‐ray images of the acellular tectorial membrane and thin two‐layered Reissner's membrane display a level of resolution comparable to low‐power electron microscopy.     

Micro‐CT scouting for transmission electron microscopy of human tissue specimens

Transmission electron microscopy (TEM) provides sub‐nanometre‐scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. We describe micro‐CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench‐top micro‐CT scanner with 10 μm resolution was used to determine the location of patches of the mucous membrane in osmium‐stained human nasal scraping samples. Once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra‐thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation.     

High‐contrast optical microscopy of graphene sheets

In the way of making graphene an industry‐friendly material, it must be mass‐produced with high‐quality and reduced cost over large areas. Assisted by machine‐learning techniques, rapid, nondestructive and accurate determination of large graphene sheets on SiO₂/Si substrates has been made possible in recent years by the optical microscopy method. Optimization of the substrate to achieve the maximum contrast can further extend the application of the optical microscopy method for quality control of the mass‐produced graphene. Graphene/n₂/n₃three‐layer structures, where n₂ and n₃ are refractive indices, are routinely used for identifying the number of graphene layers by optical reflection microscopy. In this paper, two analytical equations are derived that can be easily used for high‐contrast optical imaging of graphene sheets without any need to resort to the cumbersome numerical methods. One of the equations is derived for choosing the best material with refractive index n₂ that when coated on a substrate with refractive index n₃, maximizes the optical contrast. The other equation is derived for finding the best thickness of the SiO₂ layer in graphene/SiO₂/Si structures, which are in common use for fabrication of graphene‐based devices. The results are implemented in a MATLAB GUI, see Supporting Information, to assist the users in using the equations.