3‐D PSF fitting for fluorescence microscopy: implementation and localization application

Localization microscopy relies on computationally efficient Gaussian approximations of the point spread function for the calculation of fluorophore positions. Theoretical predictions show that under specific experimental conditions, localization accuracy is significantly improved when the localization is performed using a more realistic model. Here, we show how this can be achieved by considering three‐dimensional (3‐D) point spread function models for the wide field microscope. We introduce a least‐squares point spread function fitting framework that utilizes the Gibson and Lanni model and propose a computationally efficient way for evaluating its derivative functions. We demonstrate the usefulness of the proposed approach with algorithms for particle localization and defocus estimation, both implemented as plugins for ImageJ.     

Using cross‐correlation for automated stitching of two‐dimensional multi‐tile electron backscatter diffraction data

A method for automatically aligning consecutive data sets of large, two‐dimensional multi‐tile electron backscatter diffraction (EBSD) scans with high accuracy was developed. The method involved first locating grain and phase boundaries within search regions containing overlapping data in adjacent scan tiles, and subsequently using cross‐correlation algorithms to determine the relative position of the individual scan tiles which maximizes the fraction of overlapping boundaries. Savitzky‐Golay filtering in two dimensions was used to estimate the background, which was then subtracted from the cross‐correlation to enhance the peak signal in samples with a high density of interfaces. The technique was demonstrated on data sets with a range of interface densities. The equations were implemented as enhancements to a recently published open source code for stitching of multi‐tile EBSD data sets.     

Configurations of the Re‐scan Confocal Microscope (RCM) for biomedical applications

The new high‐sensitive and high‐resolution technique, Re‐scan Confocal Microscopy (RCM), is based on a standard confocal microscope extended with a re‐scan detection unit. The re‐scan unit includes a pair of re‐scanning mirrors that project the emission light onto a camera in a scanning manner. The signal‐to‐noise ratio of Re‐scan Confocal Microscopy is improved by a factor of 4 compared to standard confocal microscopy and the lateral resolution of Re‐scan Confocal Microscopy is 170 nm (compared to 240 nm for diffraction limited resolution, 488 nm excitation, 1.49 NA). Apart from improved sensitivity and resolution, the optical setup of Re‐scan Confocal Microscopy is flexible in its configuration in terms of control of the mirrors, lasers and filters. Because of this flexibility, the Re‐scan Confocal Microscopy can be configured to address specific biological applications. In this paper, we explore a number of possible configurations of Re‐scan Confocal Microscopy for specific biomedical applications such as multicolour, FRET, ratio‐metric (e.g. pH and intracellular Ca²⁺ measurements) and FRAP imaging.     

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.     

A 340/380 nm light‐emitting diode illuminator for Fura‐2 AM ratiometric Ca2+ imaging of live cells with better than 5 nM precision

We report the first demonstration of a fast wavelength‐switchable 340/380 nm light‐emitting diode (LED) illuminator for Fura‐2 ratiometric Ca²⁺ imaging of live cells. The LEDs closely match the excitation peaks of bound and free Fura‐2 and enables the precise detection of cytosolic Ca²⁺ concentrations, which is only limited by the Ca²⁺ response of Fura‐2. Using this illuminator, we have shown that Fura‐2 acetoxymethyl ester (AM) concentrations as low as 250 nM can be used to detect induced Ca²⁺ events in tsA‐201 cells and while utilising the 150 s switching speeds available, it was possible to image spontaneous Ca²⁺ transients in hippocampal neurons at a rate of 24.39 Hz that were blunted or absent at typical 0.5 Hz acquisition rates. Overall, the sensitivity and acquisition speeds available using this LED illuminator significantly improves the temporal resolution that can be obtained in comparison to current systems and supports optical imaging of fast Ca²⁺ events using Fura‐2.     

Development of fan‐shaped tracker for single particle tracking

With the development of super‐resolution fluorescence microscopy, complex dynamic processes in living cells can be observed and recorded with unprecedented temporal and spatial resolution. Single particle tracking (SPT) is the most important step to explore the relationship between the spatio‐temporal dynamics of subcellular molecules and their functions. Although previous studies have developed SPT algorithms to quantitatively analyze particle dynamics in cell, traditional tracking methods have poor performance when dealing with intersecting trajectories. This can be attributed to two main reasons: (a) they do not have point compensation process for overlapping objects; (b) they use inefficient motion prediction models. In this paper, we present a novel fan‐shaped tracker (FsT) algorithm to reconstruct the trajectories of subcellular vesicles in living cells. We proposed a customized point compensation method for overlapping objects based on the fan‐shaped motion trend of the particles. Furthermore, we validated the performance of the FsT in both simulated time‐lapse movies with variable imaging quality and in real vesicle moving images. Meanwhile, we compared the performance of FsT with other five state‐of‐the‐art tracking algorithms by using commonly defined measures. The results showed that our FsT achieves better performance in high signal‐to‐noise ratio conditions and in tracking of overlapping objects. We anticipate that our FsT method will have vast applications in tracking of moving objects in cell.     

不规则多孔零件的通用匹配算法研究

汽车工业存在大量形状各异的带孔零件,针对不规则多孔零件的模板匹配,提出了一种基于特征三角形的快速模板匹配算法。利用面积特征和距离特征选取匹配孔,采用限定的最小二乘法圆拟合计算匹配孔的精准圆心构建特征三角形;根据三角形的旋转不变性和平移不变性,利用特征三角形提取匹配中心和匹配角进行仿射变换,实现零件的模板匹配和配准。实验结果表明,该模板匹配算法适用于不同孔数的零件,对零件变形的宽容度较高,且不易受匹配孔部分缺失、粘连等缺陷的影响,匹配速度快,可以达到工业零件缺陷检测的要求。     

Advanced spinning disk‐TIRF microscopy for faster imaging of the cell interior and the plasma membrane

Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high‐resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high‐resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD‐TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD‐TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup.     

Out‐of‐focus background subtraction for fast structured illumination super‐resolution microscopy of optically thick samples

We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three‐dimensional (3D) samples that allows the use of two‐dimensional (2D) data processing. Indeed, obtaining super‐resolution images of thick samples is a difficult task if low spatial frequencies are present in the in‐focus section of the sample, as these frequencies have to be distinguished from the out‐of‐focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high‐resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out‐of‐focus content from the raw images. After this cleaning step, we can obtain super‐resolution images of optical sections in thick samples using a two‐beam harmonic illumination pattern and a limited number of raw images. This two‐step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two‐dimensional.     

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.     

Application of an advanced maximum likelihood estimation restoration method for enhanced‐resolution and contrast in second‐harmonic generation microscopy

Second‐harmonic generation (SHG) microscopy has gained popularity because of its ability to perform submicron, label‐free imaging of noncentrosymmetric biological structures, such as fibrillar collagen in the extracellular matrix environment of various organs with high contrast and specificity. Because SHG is a two‐photon coherent scattering process, it is difficult to define a point spread function (PSF) for this modality. Hence, compared to incoherent two‐photon processes like two‐photon fluorescence, it is challenging to apply the various PSF‐engineering methods to improve the spatial resolution to be close to the diffraction limit. Using a synthetic PSF and application of an advanced maximum likelihood estimation (AdvMLE) deconvolution algorithm, we demonstrate restoration of the spatial resolution in SHG images to that closer to the theoretical diffraction limit. The AdvMLE algorithm adaptively and iteratively develops a PSF for the supplied image and succeeds in improving the signal to noise ratio (SNR) for images where the SHG signals are derived from various sources such as collagen in tendon and myosin in heart sarcomere. Approximately 3.5 times improvement in SNR is observed for tissue images at depths of up to ～480 nm, which helps in revealing the underlying helical structures in collagen fibres with an ～26% improvement in the amplitude contrast in a fibre pitch. Our approach could be adapted to noisy and low resolution modalities such as micro‐nano CT and MRI, impacting precision of diagnosis and treatment of human diseases.     

Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium‐tin‐oxide

Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field‐of‐view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold‐labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium‐tin‐oxide was deposited by ion‐sputtering on gold‐decorated HeLa cells and neurons. Indium‐tin‐oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold‐conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. © 2015 Wiley Periodicals, Inc.     

Application of sensitive,high‐resolution imaging at a commercial lab‐based X‐ray micro‐CT system using propagation‐based phase retrieval

Several dedicated commercial lab‐based micro‐computed tomography (μCT) systems exist, which provide high‐resolution images of samples, with the capability to also deliver in‐line phase contrast. X‐ray phase contrast is particularly beneficial when visualizing very small features and weakly absorbing samples. The raw measured projections will include both phase and absorption effects. Extending our previous work that addressed the optimization of experimental conditions at the commercial ZEISS Xradia 500 Versa system, single‐distance phase‐contrast imaging is demonstrated on complex biological and material samples. From data captured at this system, we demonstrate extraction of the phase signal or the correction of the mixed image for the phase shift, and show how this procedure increases the contrast and removes artefacts. These high‐quality images, measured without the use of a synchrotron X‐ray source, demonstrate that highly sensitive, micrometre‐resolution imaging of 3D volumes is widely accessible using commercially advanced laboratory devices.     

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.     

Image matching between experimental and simulated high‐resolution electron micrographs of sapphire on the orientation

The effects of imaging parameters have been studied on their roles of the severe mismatches between experimental and simulated high‐resolution transmission electron micrographs of sapphire along the direction. Image simulation and convergent‐beam electron diffraction techniques have been performed on misalignments of the electron beam and the crystal specimen. Based on this study, we have introduced an approach to achieve reliable simulation for experimental images of sapphire on the projection by the use of iterative digital image matching.     

Depth enhancement of 3D microscopic living‐cell image using incoherent fluorescent digital holography

Multilayer images of living cells are typically obtained using confocal or multiphoton microscopy. However, limitations on the distance between consecutive scan layers hinder high‐resolution three‐dimensional reconstruction, and scattering strongly degrades images of living cell components. Consequently, when overlapping information from different layers is focused on a specific point in the camera, this causes uncertainty in the depiction of the cell components. We propose a method that combines the Fresnel incoherent correlation holography and a depth‐of‐focus reduction algorithm to enhance the depth information of three‐dimensional cell images. The proposed method eliminates overlap between light elements in the different layers inside living cells and limitations on the interlayer distance, and also enhances the contrast of the reconstructed holograms of living cells.     

3D reconstruction of histological sections: Application to mammary gland tissue

In this article, we present a novel method for the automatic 3D reconstruction of thick tissue blocks from 2D histological sections. The algorithm completes a high‐content (multiscale, multifeature) imaging system for simultaneous morphological and molecular analysis of thick tissue samples. This computer‐based system integrates image acquisition, annotation, registration, and three‐dimensional reconstruction. We present an experimental validation of this tool using both synthetic and real data. In particular, we present the 3D reconstruction of an entire mouse mammary gland and demonstrate the integration of high‐resolution molecular data. Microsc. Res. Tech. 73:1019–1029, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Orientation and phase mapping in the transmission electron microscope using precession‐assisted diffraction spot recognition: state‐of‐the‐art results

A recently developed technique based on the transmission electron microscope, which makes use of electron beam precession together with spot diffraction pattern recognition now offers the possibility to acquire reliable orientation/phase maps with a spatial resolution down to 2 nm on a field emission gun transmission electron microscope. The technique may be described as precession‐assisted crystal orientation mapping in the transmission electron microscope, precession‐assisted crystal orientation mapping technique–transmission electron microscope, also known by its product name, ASTAR, and consists in scanning the precessed electron beam in nanoprobe mode over the specimen area, thus producing a collection of precession electron diffraction spot patterns, to be thereafter indexed automatically through template matching. We present a review on several application examples relative to the characterization of microstructure/microtexture of nanocrystalline metals, ceramics, nanoparticles, minerals and organics. The strengths and limitations of the technique are also discussed using several application examples.