Acquisition speed comparison of microscope software programs

Reliable software is a prerequisite for successful operation of a modern wide field fluorescence microscope. When used for live cell imaging, acquisition speed is of particular interest. This is both because biological processes can be highly-dynamic, and to avoid unnecessary photobleaching and phototoxicity of living samples. This article shows that besides the hardware (microscope) components themselves, the acquisition control software is an important influencing factor of speed performance. We tested and compared the speed performance of five different generic applications (Image-Pro Plus, MetaMorph, Micro-Manager, SlideBook, and Volocity) using typical experimental setups involving a single specific state-of-the-art fluorescence microscope configuration. The test measurements included multichannel experiments, z-stacking, burst acquisition, as well as combinations of these measurements with time-lapse acquisitions. The measured data provided values for guiding the testing and analysis of other microscope systems with similar configurations. Despite the identical hardware settings, significant and surprisingly large speed differences were evident among the various software applications. Additionally, no application was identifiable as the fastest in all tests. Our work pinpoints the importance of the control software in determining a system's "real" maximal imaging speed. The study could serve as basis for further tests, eventually influencing the system selection criteria for speed-sensitive applications.     

An automated image alignment system for the scanning electron microscope

We have developed an automated image alignment system for the scanning electron microscope (SEM). This system enables specific locations on a sample to be located and automatically aligned with submicron accuracy. The system comprises a sample stage motorization and control unit together with dedicated imaging electronics and image processing software. The standard SEM sample stage is motorized in the X and Y axes with stepping motors which are fitted with rotary optical encoders. The imaging electronics are interfaced to beam deflection electronics of the SEM and provide the image data for the image processing software. The system initially moves the motorized sample stage to the area of interest and acquires an image. This image is compared with a reference image to determine the required adjustments to the stage position or beam deflection. This procedure is repeated until the area imaged by the SEM matches the reference image. A hierarchical image correlation technique is used to achieve submicron alignment accuracy in a few seconds. The ability to control the SEM beam deflection enables the images to be aligned with an accuracy far exceeding the positioning ability of the SEM stage. The alignment system may be used on a variety of samples without the need for registration or alignment marks since the features in the SEM image are used for alignment. This system has been used for the automatic inspection of devices on semiconductor wafers, and has also enabled the SEM to be used for direct write self-aligned electron beam lithography.     

A 2D MEMS mirror with sidewall electrodes applied for confocal MACROscope imaging

This paper presents microelectromechanical system micromirrors with sidewall electrodes applied for use as a Confocal MACROscope for biomedical imaging. The MACROscope is a fluorescence and brightfield confocal laser scanning microscope with a very large field of view. In this paper, a microelectromechanical system mirror with sidewall electrodes replaces the galvo-scanner and XYZ-stage to improve the confocal MACROscope design and obtain an image. Two micromirror-based optical configurations are developed and tested to optimize the optical design through scanning angle, field of view and numerical aperture improvement. Meanwhile, the scanning frequency and control waveform of the micromirror are tested. Analysing the scan frequency and waveform becomes a key factor to optimize the micromirror-based confocal MACROscope. When the micromirror is integrated into the MACROscope and works at 40 Hz, the micromirror with open-loop control possesses good repeatability, so that the synchronization among the scanner, XYZ-stage and image acquisition can be realized. A laser scanning microscope system based on the micromirror with 2 μm width torsion bars was built and a 2D image was obtained as well. This work forms the experimental basis for building a practical confocal MACROscope.     

A low-wavenumber-extended confocal Raman microscope with very high laser excitation line discrimination

We describe the simple modification of a confocal Raman imaging microscope to incorporate two ultra-narrow holographic notch filters. The modified microscope rejects the laser excitation line (Rayleigh peak) by a discrimination factor of ～10(11) and allows simultaneous measurements of Stokes/anti-Stokes Raman shifts as close as ～10/20 cm(-1) to the Rayleigh line. The extremely high rejection ratio of the Rayleigh peak results in its intensity becoming comparable to typical Raman scattering signals. This is essential for micro-Raman spectroscopy and imaging in the low-wavenumber region. We illustrate the resulting performance with measurements on silicon/silica, sapphire, sulfur, L-cystine, as well as on single-walled carbon nanotubes (SWNTs). We find that both aggregated (bulk) and individual (deposited on substrate) SWNTs demonstrate strong and broad characteristic Raman features below ～100 cm(-1)-in a region which has remained essentially unexplored in measurements of bulk SWNT samples and which has so far been inaccessible for Raman spectroscopy of individual SWNTs.     

Progress and perspectives for atomic-resolution electron microscopy

The transmission electron microscope (TEM) has evolved into a highly sophisticated instrument that is ideally suited to the characterization of advanced materials. Atomic-level information is routinely accessible using both fixed-beam and scanning TEMs. This report briefly considers developments in the field of atomic-resolution electron microscopy. Recent activities include renewed attention to on-line microscope control ('autotuning'), and assessment and correction of aberrations. Aberration-corrected electron microscopy has developed rapidly in several forms although more work needs to be done to identify standard imaging conditions and to explore novel operating modes. Preparation of samples and image interpretation have also become more demanding. Ongoing problems include discrepancies between measured and simulated image contrast, concerns about radiation damage, and inversion of electron scattering.     

Prerequisites for a Cc/Cs-corrected ultrahigh-resolution TEM

After the introduction of a corrector to compensate for the spherical aberration of a TEM and the acceptance of this new instrumentation for high-resolution CTEM (conventional transmission electron microscope) and STEM (scanning transmission electron microscope) by the electron microscopy community, a demand for even higher resolution far below 1A has emerged. As a consequence several projects around the world have been launched to make these new instruments available and to further push the resolution limits down toward fractions of 1A. For this purpose the so-called TEAM (transmission electron aberration-corrected microscope) has been initiated and is currently under development. With the present paper we give a detailed assessment of the stability required for the base instrument and the electric stability, the manufacturing precision, and feasible semi-automatic alignment procedures for a novel C(c)/C(s)-corrector in order to achieve aberration-free imaging with an information limit of 0.5A at an acceleration voltage of 200 kV according to the goals for the first TEAM instrument. This new aberration corrector, a so-called Achroplanat, in combination with a very stable high-resolution TEM leads to an imaging device with unprecedented resolving power and imaging properties.     

Inclined selective plane illumination microscopy adaptor for conventional microscopes

Driven by the biological sciences, there is an increased need for imaging modalities capable of live cell imaging with high spatial and temporal resolution. To achieve this goal in a comprehensive manner, three‐dimensional acquisitions are necessary. Ideal features of a modern microscope system should include high imaging speed, high contrast ratio, low photo‐bleaching and photo‐toxicity, good resolution in a 3D context, and mosaic acquisition for large samples. Given the importance of collecting data in live sample further increases the technical challenges required to solve these issues. This work presents a practical version of a microscopy method, Selective Plane Illumination Microscopy re‐introduced by Huisken et al. (Science 2004 ,305,1007–1009). This method is gaining importance in the biomedical field, but its use is limited by difficulties associated with unconventional microscope design which employs two objectives and a particular kind of sample preparation needed to insert the sample between the objectives. Based on the selective plane illumination principle but with a design similar to the Total Internal Reflection Fluorescence microscope, Dunsby (Dunsby, Opt Express 2008 ,16,20306–20316) demonstrated the oblique plane microscope (OPM) using a single objective which uses conventional sample preparation protocols. However, the Dunsby instrument was not intended to be part of a commercial microscope. In this work, we describe a system with the advantages of OPM and that can be used as an adaptor to commonly used microscopes, such as IX‐71 Olympus, simplifying the construction of the OPM and increasing performance of a conventional microscope. We named our design inclined selective plane illumination microscope (iSPIM). Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Time-domain whole-field fluorescence lifetime imaging with optical sectioning

A whole-field time-domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode-locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time-gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time-gated images, 2-D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real-world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all-solid-state diode-pumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.     

A white light confocal microscope for spectrally resolved multidimensional imaging

Spectrofluorometric imaging microscopy is demonstrated in a confocal microscope using a supercontinuum laser as an excitation source and a custom‐built prism spectrometer for detection. This microscope system provides confocal imaging with spectrally resolved fluorescence excitation and detection from 450 to 700 nm. The supercontinuum laser provides a broad spectrum light source and is coupled with an acousto‐optic tunable filter to provide continuously tunable fluorescence excitation with a 1‐nm bandwidth. Eight different excitation wavelengths can be simultaneously selected. The prism spectrometer provides spectrally resolved detection with sensitivity comparable to a standard confocal system. This new microscope system enables optimal access to a multitude of fluorophores and provides fluorescence excitation and emission spectra for each location in a 3D confocal image. The speed of the spectral scans is suitable for spectrofluorometric imaging of live cells. Effects of chromatic aberration are modest and do not significantly limit the spatial resolution of the confocal measurements.     

Study of dislocation structures near fatigue cracks using electron channelling contrast imaging technique (ECCI)

The fatigue of copper single crystals, orientated for single slip, has been studied using electron channelling contrast imaging in a scanning electron microscope. With the incident beam set at the Bragg condition, changes in the backscattered electron intensity occur as the beam is scanned over dislocations that cause a local tilting of the diffraction planes. This technique allows the evolution of dislocation structures over large areas to be followed through different stages of the fatigue life. Furthermore, it enables direct imaging of dislocation configurations at crack tips. The technique is compared with transmission electron microscopy and electron backscatter diffraction in its application to fatigue studies.     

Time-resolved momentum imaging system for molecular dynamics studies using a tabletop ultrafast extreme-ultraviolet light source

We describe a momentum imaging setup for direct time-resolved studies of ionization-induced molecular dynamics. This system uses a tabletop ultrafast extreme-ultraviolet (EUV) light source based on high harmonic upconversion of a femtosecond laser. The high photon energy (around 42 eV) allows access to inner-valence states of a variety of small molecules via single photon excitation, while the sub--10-fs pulse duration makes it possible to follow the resulting dynamics in real time. To obtain a complete picture of molecular dynamics following EUV induced photofragmentation, we apply the versatile cold target recoil ion momentum spectroscopy reaction microscope technique, which makes use of coincident three-dimensional momentum imaging of fragments resulting from photoexcitation. This system is capable of pump-probe spectroscopy by using a combination of EUV and IR laser pulses with either beam as a pump or probe pulse. We report several experiments performed using this system.     

Correction of image drift and distortion in a scanning electron microscopy

Continuous research on small‐scale mechanical structures and systems has attracted strong demand for ultrafine deformation and strain measurements. Conventional optical microscope cannot meet such requirements owing to its lower spatial resolution. Therefore, high‐resolution scanning electron microscope has become the preferred system for high spatial resolution imaging and measurements. However, scanning electron microscope usually is contaminated by distortion and drift aberrations which cause serious errors to precise imaging and measurements of tiny structures. This paper develops a new method to correct drift and distortion aberrations of scanning electron microscope images, and evaluates the effect of correction by comparing corrected images with scanning electron microscope image of a standard sample. The drift correction is based on the interpolation scheme, where a series of images are captured at one location of the sample and perform image correlation between the first image and the consequent images to interpolate the drift–time relationship of scanning electron microscope images. The distortion correction employs the axial symmetry model of charged particle imaging theory to two images sharing with the same location of one object under different imaging fields of view. The difference apart from rigid displacement between the mentioned two images will give distortion parameters. Three‐order precision is considered in the model and experiment shows that one pixel maximum correction is obtained for the employed high‐resolution electron microscopic system.     

Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements

A modified fluorescence correlation microscope (FCM) was built on a commercial confocal laser scanning microscope (CLSM) by adding two sensitive detectors to perform fluorescence correlation spectroscopy (FCS). A single pinhole for both imaging and spectroscopy and a simple slider switch between the two modes thus facilitate the accurate positioning of the FCS observation volume after the confocal image acquisition. Due to the use of a single pinhole for CLSM and FCS the identity of imaged and spectroscopically observed positions is guaranteed. The presented FCM system has the capability to position the FCS observation volume at any point within the inner 30% of the field of view without loss in performance and in the inner 60% of the field of view with changes of FCS parameters of less than 10%. A single pinhole scheme for spatial fluorescence cross correlation spectroscopy performed on the FCM system is proposed to determine microfluidic flow angles. To show the applicability and versatility of the system, we measured the translational diffusion coefficients on the upper and lower membranes of Chinese hamster ovary cells. Two-photon excitation FCS was also realized by coupling a pulsed Ti: sapphire laser into the microscope and used for flow direction characterization in microchannels.     

Specialized scanning ion-conductance microscope for imaging of living cells

A specialized scanning ion conductance microscope (SICM) for imaging living cells has been developed from a conventional patch-clamp apparatus, which uses a glass micropipette as the sensitive probe. In contrast with other types of scanning probe microscope, the SICM probe has significant advantages for imaging living cells: it is most suitable for imaging samples immersed in water solutions; and since the probe senses ion current and does not need physical contact with the sample during the scan, any preliminary preparation of cells (fixation or adherence to a substrate) is unnecessary. We have successfully imaged murine melanocytes in growth medium. The microscope images the highly convoluted surface structures without damaging or deforming them, and reveals the true, three-dimensional relief of the cells. This instrument has considerable ability to operate, potentially simultaneously, in applications as diverse as real-time microscopy, electrophysiology, micromanipulation and drug delivery.     

Near field optical microscopy in aqueous solution: implementation and characterization of a vibrating probe

Near field optical microscopy (NSOM) is one of the possible solutions to circumvent the diffraction limit, but the control of the optical probe in solution has been a technical challenge for practical applications. Most recently, it has been shown that the pipette used in the scanning ion conductance microscope can be modified to form a high resolution near field optical probe. When combined with a novel distance modulation mechanism, a robust near field microscope can be constructed for operation in aqueous solution. In this paper, we present technical details of this design and a further characterization of the NSOM system for imaging in solution. Fundamental limitations of this approach in comparison to other systems are also discussed. Based on the current technology, it is concluded that better than 50 nm resolution should be achievable with this technique for fluorescence, as well as fluorescence resonance energy transfer, imaging of biological specimens.     

A universal fluid cell for the imaging of biological specimens in the atomic force microscope

Recently, atomic force microscope (AFM) manufacturers have begun producing instruments specifically designed to image biological specimens. In most instances, they are integrated with an inverted optical microscope, which permits concurrent optical and AFM imaging. An important component of the set‐up is the imaging chamber, whose design determines the nature of the experiments that can be conducted. Many different imaging chamber designs are available, usually designed to optimize a single parameter, such as the dimensions of the substrate or the volume of fluid that can be used throughout the experiment. In this report, we present a universal fluid cell, which simultaneously optimizes all of the parameters that are important for the imaging of biological specimens in the AFM. This novel imaging chamber has been successfully tested using mammalian, plant, and microbial cells. Microsc. Res. Tech. 76:357–363, 2013. © 2013 Wiley Periodicals, Inc.     

Resonant control of an atomic force microscope micro-cantilever for active Q control

Active Q control may be used to modify the effective quality (Q) factor of an atomic force microscope (AFM) micro-cantilever when operating in tapping mode. The control system uses velocity feedback to obtain an effective cantilever Q factor to achieve optimal scan speed and image resolution for the imaging environment and sample type. Time delay of the cantilever displacement signal is the most common method of cantilever velocity estimation. Spill-over effects from unmodeled dynamics may degrade the closed loop system performance, possibly resulting in system instability, when time delay velocity estimation is used. A resonant controller is proposed in this work as an alternate method of velocity estimation. This new controller has guaranteed closed loop stability, is easy to tune, and may be fitted into existing commercial AFMs with minimal modification. Images of a calibration grating are obtained using this controller to demonstrate its effectiveness.     

Atomic imaging in aberration-corrected high-resolution transmission electron microscopy

In recent years, the successful implementation of a spherical-aberration corrector in a Philips CM 200 FEG ST microscope achieved by Haider et al. has attracted a great deal of attention. However, thus far extensive applications of this novel high-resolution transmission electron microscope (HRTEM) to materials research have been hampered by the problems concerning optimum imaging conditions and image interpretation. In this paper, we present our points of view concerning atomic imaging in an aberration-corrected HRTEM. Since atomic resolution images can also be obtained with other techniques such as through-focus exit-wave function reconstruction (TF-EWR), we have to emphasis that the strength of the aberration-corrected HRTEM particularly lies on its ability to resolve the atomic structure in real time. However, for this purpose it is mandatory that the image contrast be related in a one-to-one function with the projected structure of the object. We analyzed the atomic imaging conditions in much detail and we come to the following conclusion: this novel facility is no doubt a powerful and advanced HRTEM instrument in achieving atomic images with its highest resolution (information limit). We furthermore demonstrate that the combination of the new microscope and TF-EWR will yield optimal results.     

Zero-loss energy filtering under low-dose conditions using a post-column energy filter

Electron cryomicroscopy combined with energy filtering can be performed under low-dose conditions using a post-column energy filter. The microscope combined with the filter is set up such that it can be used with similar ease as a conventional microscope, the main difference being that all filter and microscope control is performed through a central computer and images are recorded with a cooled slow-scan CCD camera. The microscope can also still be used for regular imaging on film as without the filter. Owing to the 18 times post-magnification of the filter, the microscope normally has to be operated at a small magnification, e.g. 3000×, and the beam has to be contracted to a small spot, e.g. 5 mm, in the plane of the microscope viewing screen. Computer control allows one to perform a variety of tasks automatically, such as autofocusing, thickness measurements, most-probable-loss imaging, CCD spot-scanning and tomography. The gain in contrast due to zero-loss energy filtering is analysed using visual inspection, power spectra and Fourier ring correlation. The thickness range for ice-embedded specimens in which a filter at 120 kV is most useful appears to be between 100 and 300 nm.     

On the complex three-dimensional amplitude point spread function of lenses and microscope objectives: theoretical aspects, simulations and measurements by digital holography

The point spread function is widely used to characterize the three‐dimensional imaging capabilities of an optical system. Usually, attention is paid only to the intensity point spread function, whereas the phase point spread function is most often neglected because the phase information is not retrieved in noninterferometric imaging systems. However, phase point spread functions are needed to evaluate phase‐sensitive imaging systems and we believe that phase data can play an essential role in the full aberrations' characterization. In this paper, standard diffraction models have been used for the computation of the complex amplitude point spread function. In particular, the Debye vectorial model has been used to compute the amplitude point spread function of ×63/0.85 and ×100/1.3 microscope objectives, exemplifying the phase point spread function specific for each polarization component of the electromagnetic field. The effect of aberrations on the phase point spread function is then analyzed for a microscope objective used under nondesigned conditions, by developing the Gibson model ( Gibson & Lanni, 1991 ), modified to compute the three‐dimensional amplitude point spread function in amplitude and phase. The results have revealed a novel anomalous phase behaviour in the presence of spherical aberration, providing access to the quantification of the aberrations. This work mainly proposes a method to measure the complex three‐dimensional amplitude point spread function of an optical imaging system. The approach consists in measuring and interpreting the amplitude point spread function by evaluating in amplitude and phase the image of a single emitting point, a 60‐nm‐diameter tip of a Near Field Scanning Optical Microscopy fibre, with an original digital holographic experimental setup. A single hologram gives access to the transverse amplitude point spread function. The three‐dimensional amplitude point spread function is obtained by performing an axial scan of the Near Field Scanning Optical Microscopy fibre. The phase measurements accuracy is equivalent to λ/60 when the measurement is performed in air. The method capability is demonstrated on an Achroplan ×20 microscope objective with 0.4 numerical aperture. A more complete study on a ×100 microscope objective with 1.3 numerical aperture is also presented, in which measurements performed with our setup are compared with the prediction of an analytical aberrations model.