Comparative evaluation of performance measures for shading correction in time‐lapse fluorescence microscopy

Time‐lapse fluorescence microscopy is a valuable technology in cell biology, but it suffers from the inherent problem of intensity inhomogeneity due to uneven illumination or camera nonlinearity, known as shading artefacts. This will lead to inaccurate estimates of single‐cell features such as average and total intensity. Numerous shading correction methods have been proposed to remove this effect. In order to compare the performance of different methods, many quantitative performance measures have been developed. However, there is little discussion about which performance measure should be generally applied for evaluation on real data, where the ground truth is absent. In this paper, the state‐of‐the‐art shading correction methods and performance evaluation methods are reviewed. We implement 10 popular shading correction methods on two artificial datasets and four real ones. In order to make an objective comparison between those methods, we employ a number of quantitative performance measures. Extensive validation demonstrates that the coefficient of joint variation (CJV) is the most applicable measure in time‐lapse fluorescence images. Based on this measure, we have proposed a novel shading correction method that performs better compared to well‐established methods for a range of real data tested.     

Combined ultramicrotomy for AFM and TEM using a novel sample holder

A new sample holder that allows combined microtomy for atomic force microscopy (AFM) and transmission electron microscopy (TEM) is described. The main feature of this sample holder is a small central part holding the sample. This central part fits into the head of an atomic force microscope. AFM measurements can be performed with a sample mounted in this central part of the sample holder. This makes the alignment of a microtomed bulk sample unnecessary, and offers the opportunity of an easy and fast combined sample preparation for AFM and TEM.     

High‐quality imaging in environmental scanning electron microscopy – optimizing the pressure limiting system and the secondary electron detection of a commercially available ESEM

In environmental scanning electron microscopy applications in the kPa regime are of increasing interest for the investigation of wet and biological samples, because neither sample preparation nor extensive cooling are necessary. Unfortunately, the applications are limited by poor image quality. In this work the image quality at high pressures of a FEI Quanta 600 (field emission gun) and a FEI Quanta 200 (thermionic gun) is greatly improved by optimizing the pressure limiting system and the secondary electron (SE) detection system. The scattering of the primary electron beam strongly increases with pressure and thus the image quality vanishes. The key to high‐image quality at high pressures is to reduce scattering as far as possible while maintaining ideal operation conditions for the SE‐detector. The amount of scattering is reduced by reducing both the additional stagnation gas thickness (aSGT) and the environmental distance (ED). A new aperture holder is presented that significantly reduces the aSGT while maintaining the same field‐of‐view (FOV) as the original design. With this aperture holder it is also possible to make the aSGT even smaller at the expense of a smaller FOV. A new blade‐shaped SE‐detector is presented yielding better image quality than usual flat SE‐detectors. The electrode of the new SE detector is positioned on the sample table, which allows the SE‐detector to operate at ideal conditions regardless of pressure and ED.     

Recent progress in freeze-fracturing of high-pressure frozen samples

Pancreatic tissue, bacteria and lipid vesicles were high‐pressure frozen and freeze‐fractured. In addition to the normal holder, a new type of high‐pressure freezing holder was used that is particularly suitable for suspensions. This holder can take up an EM grid that has been dipped in the suspension and clamped in between two low‐mass copper platelets, as used for propane‐jet freezing. Both the standard and the new suspension holder allowed us to make cryo‐fractures without visible ice crystal damage. High‐pressure frozen rat pancreas tissue samples were cryo‐fractured and cryo‐sectioned with a new type diamond knife in the microtome of a freeze‐etching device. The bulk fracture faces and blockfaces were investigated in the frozen‐hydrated state by use of a cryo‐stage in an in‐lens SEM. Additional structures can be made visible by controlled sublimation of ice (‘etching’), leading to a better understanding of the three‐dimensional organization of organelles, such as the endoplasmic reticulum. With this approach, relevant biological structures can be investigated with a few nanometre resolution in a near life‐like state, preventing the artefacts associated with conventional fixation techniques.     

Time‐lapse microscopy using smartphone with augmented reality markers

A prototype system that replaces the conventional time‐lapse imaging in microscopic inspection for use with smartphones is presented. Existing time‐lapse imaging requires a video data feed between a microscope and a computer that varies depending on the type of image grabber. Even with proper hardware setups, a series of tedious and repetitive tasks is still required to relocate to the region‐of‐interest (ROI) of the specimens. In order to simplify the system and improve the efficiency of time‐lapse imaging tasks, a smartphone‐based platform utilizing microscopic augmented reality (μ‐AR) markers is proposed. To evaluate the feasibility and efficiency of the proposed system, a user test was designed and performed, measuring the elapse time for a trial of the task starting from the execution of the application software to the completion of restoring and imaging of an ROI saved in advance. The results of the user test showed that the average elapse time was 65.3 ± 15.2 s with 6.86 ± 3.61 μm of position error and 0.08 ± 0.40 degrees of angle error. This indicates that the time‐lapse imaging task was accomplished rapidly with a high level of accuracy. Thus, simplification of both the system and the task was achieved via our proposed system. Microsc. Res. Tech. 77:243–249, 2014. © 2014 Wiley Periodicals, Inc.     

Signal analysis of total internal reflection fluorescent speckle microscopy (TIR-FSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells

Fluorescent speckle microscopy (FSM) uses low levels of fluorescent proteins to create fluorescent speckles on cytoskeletal polymers in high‐resolution fluorescence images of living cells. The dynamics of speckles over time encode subunit turnover and motion of the cytoskeletal polymers. We sought to improve on current FSM technology by first expanding it to study the dynamics of a non‐polymeric macromolecular assembly, using focal adhesions as a test case, and second, to exploit for FSM the high contrast afforded by total internal reflection fluorescence microscopy (TIR‐FM). Here, we first demonstrate that low levels of expression of a green fluorescent protein (GFP) conjugate of the focal adhesion protein, vinculin, results in clusters of fluorescent vinculin speckles on the ventral cell surface, which by immunofluorescence labelling of total vinculin correspond to sparse labelling of dense focal adhesion structures. This demonstrates that the FSM principle can be applied to study focal adhesions. We then use both GFP‐vinculin expression and microinjected fluorescently labelled purified actin to compare quantitatively the speckle signal in FSM images of focal adhesions and the actin cytoskeleton in living cells by TIR‐FM and wide‐field epifluorescence microscopy. We use quantitative FSM image analysis software to define two new parameters for analysing FSM signal features that we can extract automatically: speckle modulation and speckle detectability. Our analysis shows that TIR‐FSM affords major improvements in these parameters compared with wide‐field epifluorescence FSM. Finally, we find that use of a crippled eukaryotic expression promoter for driving low‐level GFP‐fusion protein expression is a useful tool for FSM imaging. When used in time‐lapse mode, TIR‐FSM of actin and GFP‐conjugated focal adhesion proteins will allow quantification of molecular dynamics within interesting macromolecular assemblies at the ventral surface of living cells.     

High temperature controlled atmosphere experiments for catalysts in a transmission electron microscope

A new transmission electron microscopy (TEM) specimen preparation procedure for high temperature experiments using a controlled atmosphere specimen holder (HTCASH) has been developed. It is designed for studying the microstructure of catalyst specimens before and after treatments in various gases. The procedure involved (1) finding a new formula for the embedding material, (2) devising a new method of making specimen supports, and (3) developing a method for removing the embedding material after the specimen has been microtomed. These techniques were then brought together to produce the ideal specimens for the HTCASH experiments. As an extra benefit, this procedure is also suitable for preparing specimens for ultrahigh resolution imaging experiments. The application of the new procedure in HTCASH experiments is illustrated through a high temperature reduction of a Co/SiO₂-923 catalyst.     

An improved specimen loader for cryo-scanning electron microscopy

Cryo-electron microscopy of cryofixed samples is a well-established and accepted technique for imaging liquid-containing specimens without removing water and other volatile components. There are many steps between cryofixation and cryo-observation in the microscope, during which the sample and sample holder need to be handled. One such major step is the loading of the specimen onto the sample holder and the fixing of the sample holder onto the transfer mechanism. During this handling, the specimen is often exposed (mostly inadvertently) to moisture in the atmosphere, which results in frost deposition. The new specimen loader described here is designed to overcome the traditional tedious handling and to achieve ease in specimen loading. The modifications made are mainly towards allowing movement of the liquid freon cup, eliminating the need for a lock-screw and improving the shape of the stage holder, which makes the mounting of the specimen holder easy, thereby permitting smooth specimen loading without too much handling and with consequent reduced frost deposition.     

New application of variable‐pressure/environmental microscopy to semiconductor inspection and metrology

Variable‐pressure/environmental scanning electron microscopy has been used for successful investigation binary and phase‐shifting chromium on quartz optical photomasks. This methodology was also applied to patterned 193 nm photoresist structures. The application of this methodology to semiconductor metrology is new because of the recent availability of variable‐pressure scanning electron microscopy (SEM) instrumentation equipped with high‐resolution, high‐signal, thermally assisted field emission technology in conjunction with large chamber and sample transfer capabilities. The variable‐pressure SEM methodology employs a gaseous environment around the sample to help diminish the charge build‐up that occurs under irradiation with the electron beam. Although very desirable for the charge reduction in many biological, pharmaceutical, and food applications, this methodology has not been employed for semiconductor photomask or wafer metrology until now. This is a new application of this technology to this area, and it shows great promise in inspection, imaging, and metrology in a charge‐free operational mode. For accurate metrology, variable‐pressure SEM methodology also affords a path that minimizes, if not eliminates, the need for charge modeling. This paper presents some of the early results in the variable‐pressure SEM metrology of photomask and photoresist structures.     

An environmental sample chamber for X-ray microscopy

The manufacture, construction and performance of a special specimen holder for biological samples suspended in aqueous solution is described. The holder was designed for use in a scanning transmission X-ray microscope. Using it, we have successfully obtained high-resolution images of fresh cellular and subcellular specimens in suspension and have been able to vary the sample environment during viewing in the microscope.     

Sample holder for axial rotation of specimens in 3D microscopy

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three‐dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three‐dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three‐dimensional specimens is essential. Fluorescence z‐projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results.     

High-resolution compact X-ray microscopy

We demonstrate compact full‐field soft X‐ray transmission microscopy with sub 60‐nm resolution operating at λ= 2.48 nm. The microscope is based on a 100‐Hz regenerative liquid‐nitrogen‐jet laser‐plasma source in combination with a condenser zone plate and a micro‐zone plate objective for high‐resolution imaging onto a 2048 × 2048 pixel CCD detector. The sample holder is mounted in a helium atmosphere and allows imaging of both dry and wet specimens. The microscope design enables fast sample switching and the sample can be pre‐aligned using a visible‐light microscope. High‐quality images can be acquired with exposure times of less than 5 min. We demonstrate the performance of the microscope using both dry and wet samples.     

A novel sample holder allowing atomic force microscopy on transmission electron microscopy specimen grids: repetitive, direct correlation between AFM and TEM images

A novel sample holder that allows atomic force microscopy (AFM) to be performed on transmission electron microscope (TEM) grids is described. Consequently, AFM and TEM images were repeatedly obtained on exactly the same sample area. For both techniques, a thin carbon film was used as the imaging substrate. Although these techniques have been previously used in conjunction, AFM and TEM images on exactly the same area have not been repeatedly obtained for any system. Correlation of AFM and TEM images is useful for work where the three‐dimensional topographical information provided by the AFM could be used to better interpret the two‐dimensional images provided by the TEM and vice versa. To demonstrate the applicability of such correlation, new results pertaining to a fibrillar collagen system are summarized.     

A protocol for registration and correction of multicolour STED superresolution images

Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut‐shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co‐localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross‐correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co‐staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user‐friendly criteria, which – if fulfilled – support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co‐localisation analyses. Although the reference protocol is discussed exemplarily for two‐colour STED imaging, it can be readily expanded to three or more colours and STED channels.     

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.     

Automatic detection and analysis of cell motility in phase‐contrast time‐lapse images using a combination of maximally stable extremal regions and Kalman filter approaches

Phase‐contrast illumination is simple and most commonly used microscopic method to observe nonstained living cells. Automatic cell segmentation and motion analysis provide tools to analyze single cell motility in large cell populations. However, the challenge is to find a sophisticated method that is sufficiently accurate to generate reliable results, robust to function under the wide range of illumination conditions encountered in phase‐contrast microscopy, and also computationally light for efficient analysis of large number of cells and image frames. To develop better automatic tools for analysis of low magnification phase‐contrast images in time‐lapse cell migration movies, we investigated the performance of cell segmentation method that is based on the intrinsic properties of maximally stable extremal regions (MSER). MSER was found to be reliable and effective in a wide range of experimental conditions. When compared to the commonly used segmentation approaches, MSER required negligible preoptimization steps thus dramatically reducing the computation time. To analyze cell migration characteristics in time‐lapse movies, the MSER‐based automatic cell detection was accompanied by a Kalman filter multiobject tracker that efficiently tracked individual cells even in confluent cell populations. This allowed quantitative cell motion analysis resulting in accurate measurements of the migration magnitude and direction of individual cells, as well as characteristics of collective migration of cell groups. Our results demonstrate that MSER accompanied by temporal data association is a powerful tool for accurate and reliable analysis of the dynamic behaviour of cells in phase‐contrast image sequences. These techniques tolerate varying and nonoptimal imaging conditions and due to their relatively light computational requirements they should help to resolve problems in computationally demanding and often time‐consuming large‐scale dynamical analysis of cultured cells.     

Sample preparation method for visualization of nanoparticulate captured on mixed cellulose ester filter media by enhanced darkfield microscopy and hyperspectral imaging

A significant hurdle in conducting effective health and safety hazard analysis and risk assessment for the nanotechnology workforce is the lack of a rapid method for the direct visualization and analysis of filter media used to sample nanomaterials from work environments that represent potential worker exposure. Current best‐known methods include transmission electron microscopy (TEM) coupled with energy dispersive x‐ray spectroscopy (EDS) for elemental identification. TEM‐EDS is considerably time‐, cost‐, and resource‐intensive, which may prevent timely health and safety recommendations and corrective actions. A rapid screening method is currently being explored using enhanced darkfield microscopy with hyperspectral imaging (EDFM‐HSI). For this approach to be effective, rapid, and easy, sample preparation that is amenable to the analytical technique is needed. Here, we compare the sample preparation steps for mixed cellulose ester (MCE) filter media specified in NIOSH Method 7400—Asbestos and Other Fibers by Phase Contrast Microscopy (PCM)—against a new method, which involves saturation of the filter media with acetone. NIOSH Method 7400 was chosen as a starting point since it is an established technique for preparing transparent MCE filters for optical microscopy. Limitations in this method led to the development and comparison of a new method. The new method was faster, easier, and rendered filters more transparent, resulting in improved visualization and analysis of nanomaterials via EDFM‐HSI. This new method is suitable for a rapid screening protocol due to its speed, ease of use, and the improvement in image acquisition and analysis.     

A rapid microbiopsy system to improve the preservation of biological samples prior to high-pressure freezing

A microbiopsy system for fast excision and transfer of biological specimens from donor to high‐pressure freezer was developed. With a modified, commercially available, Promag 1.2 biopsy gun, tissue samples can be excised with a size small enough (0.6 mm × 1.2 mm × 0.3 mm) to be easily transferred into a newly designed specimen platelet. A self‐made transfer unit allows fast transfer of the specimen from the needle into the specimen platelet. The platelet is then fixed in a commercially available specimen holder of a high‐pressure freezing machine (EM PACT, Leica Microsystems, Vienna, Austria) and frozen therein. The time required by a well‐instructed (but not experienced) person to execute all steps is in the range of half a minute. This period is considered short enough to maintain the excised tissue pieces close to their native state. We show that a range of animal tissues (liver, brain, kidney and muscle) are well preserved. To prove the quality of freezing achieved with the system, we show vitrified ivy leaves high‐pressure frozen in the new specimen platelet.     

A flexible microrobotic platform for handling microscale specimens of fibrous materials for microscopic studies

One of the most challenging issues faced in handling specimens for microscopy, is avoiding artefacts and structural changes in the samples caused by human errors. In addition, specimen handling is a laborious and time‐consuming task and requires skilful and experienced personnel. This paper introduces a flexible microrobotic platform for the handling of microscale specimens of fibrous materials for various microscopic studies such as scanning electron microscopy and nanotomography. The platform is capable of handling various fibres with diameters ranging from 10 to 1000 μm and lengths of 100 μm–15 mm, and mounting them on different types of specimen holders without damaging them. This tele‐operated microrobotic platform minimizes human interaction with the samples, which is one of the main sources contributory to introducing artefacts into the specimens. The platform also grants a higher throughput and an improved success rate of specimen handling, when compared to the manual processes. The operator does not need extensive experience of microscale manipulation and only a short training period is sufficient to operate the platform. The requirement of easy configurability for various samples and sample holders is typical in the research and development of materials in this field. Therefore, one of the main criteria for the design of the microrobotic platform was the ability to adapt the platform to different specimen handling methods required for microscopic studies. To demonstrate this, three experiments are carried out using the microrobotic platform. In the first experiment, individual paper fibres are mounted successfully on scanning electron microscopy specimen holders for the in situ scanning electron microscopy diagonal compression test of paper fibres. The performance of the microrobotic platform is compared with a skilled laboratory worker performing the same experiment. In the second experiment, a strand of human hair and an individual paper fibre bond are mounted on a specimen holder for nanotomography studies. In the third experiment, individual paper fibre bonds with controlled crossing and vertical angles are made using the microrobotic platform. If an industrial application requires less flexibility but a higher speed when handling one type of sample to a specific holder, then the platform can be automated in the future.     

Ultrafast photon counting applied to resonant scanning STED microscopy

To take full advantage of fast resonant scanning in super‐resolution stimulated emission depletion (STED) microscopy, we have developed an ultrafast photon counting system based on a multigiga sample per second analogue‐to‐digital conversion chip that delivers an unprecedented 450 MHz pixel clock (2.2 ns pixel dwell time in each scan). The system achieves a large field of view (～50 × 50 μm) with fast scanning that reduces photobleaching, and advances the time‐gated continuous wave STED technology to the usage of resonant scanning with hardware‐based time‐gating. The assembled system provides superb signal‐to‐noise ratio and highly linear quantification of light that result in superior image quality. Also, the system design allows great flexibility in processing photon signals to further improve the dynamic range. In conclusion, we have constructed a frontier photon counting image acquisition system with ultrafast readout rate, excellent counting linearity, and with the capacity of realizing resonant‐scanning continuous wave STED microscopy with online time‐gated detection.