Compact,common path quantitative phase microscopic techniques for imaging cell dynamics

Microscopy using visible electromagnetic radiation can be used to investigate living cells in various environments. But bright field microscopy only provides two-dimensional (2D) intensity distribution at a single object plane. One of the ways to retrieve object height/thickness information is to employ quantitative phase microscopic (QPM) techniques. Interferometric QPM techniques are widely used for this. Digital holographic microscopy (DHM) is one of the state-of-the-art methods for quantitative three-dimensional (3D) imaging. Usually it is implemented in two-beam geometry, which is prone to mechanical vibrations. But to study dynamics of objects like red blood cells, one needs temporal stability much better than the fluctuations of the object, which the two-beam geometry fails to deliver. One way to overcome this hurdle is to use self-referencing techniques, in which a portion of the object beam will act as the reference beam. Here the development of self-referencing QPM techniques is described along with the results.     

Green's formulation for robust phase unwrapping in digital holography

We present, for the first time to the best of our knowledge, a phase unwrapping method based on an algorithm which makes use of Green's first identity. This method aims at recovering the correct phase information encoded in a fringe pattern after digital holography (DH) numerical processing. DH provides a quantitative measurement of the three-dimensional surface profile of objects. The information about the profile can be obtained in principle from the phase-map. However, the measured phase-map provides the actual phase values wrapped mod.2π so that an unwrapping process is required in order to reconstruct the object profile.     

Accurate measurement method of the thickness of a quartz pendulous reed by combining polarized reflectance and vision image

The detection of thickness provides important information on the production process of the quartz pendulous reed (QRP). Through real-time detection of QPR thickness, high-quality products can be produced, and defective products can be identified and removed. In order to improve detection accuracy and efficiency, a novel noncontact method for measuring the QPR thickness by combining the polarized reflectance and vision image is presented, which can be automated. Based on Snell’s law of fundamental optics, the image of the laser spot is obtained by a laser vision system. The selection criterion of the laser incident angle is analyzed. An improved edge detection algorithm is proposed to locate the subpixel edge of the laser spot, and the calculation method of the laser spot center is presented based on a set of subpixel edge-point data obtained by defining an error function which is to optimize the distribution of data points and minimize it. Experimental results show that the method proposed has good stability and high measurement accuracy, which can achieve noncontact accurate measurement of QPR thickness.     

载波相移全息干涉计量

本文报道了一项称为载波相移全息干涉计量的自动相位估算的新技术。该技术的主要特点是不同相移的条纹图案可用双曝光全息重视。这样即可用来对与时间相关的问题进行自动相位估算。     

Phase-shifting digital holography with burst imaging method

The application of a burst imaging method to digital holography (DH) with multiple image capturing is firstly demonstrated. Nobody has discussed this approach explicitly, although the burst imaging method is not so uncommon. The burst imaging method intermittently captures several images with a high repetition rate followed by a relatively long rest time. If the light wave from an object has a much smaller complex amplitude change in the frame interval of an image sensor than the precision of the DH system, the wave can be regarded as being in a static state. As a result, this solves a fatal problem in DH for an object that moves or deforms while capturing multiple interference images. In this research, the burst imaging method was applied to phase-shifting DH, and it is simply implemented by a high-speed image sensor and a continuous phase shifting which is good for high-speed phase shifting. The measurement error was analytically solved for the movement speed of an object. Finally, the proposed method was experimentally used to observe an evaporating droplet of ethanol on a glass substrate.

     

Automated microaxial tomography of cell nuclei after specific labelling by fluorescence in situ hybridisation

Microaxial tomography provides a good means for microscopic image acquisition of cells or sub-cellular components like cell nuclei with an improved resolution, because shortcomings of spatial resolution anisotropy in optical microscopy can be overcome. Thus, spatial information of the object can be obtained without the necessity of confocal imaging. Since the very early developments of microaxial tomography, a considerable drawback of this method was a complicated image acquisition and processing procedure that requires much operator time. In order to solve this problem the Heidelberg 2π-tilting device has been mounted on the Brno high-resolution cytometer as an attempt to bring together advanced microscopy and fast automated computer image acquisition and analysis. A special software module that drives all hardware components required for automated microaxial tomography and performs image acquisition and processing has been developed. First, a general image acquisition strategy is presented. Then the procedure for automation of axial tomography and the developed software module are described. The rotation precision has been experimentally proved followed by experiments with a specific biological example. For this application, also a method for the preparation of cell nuclei attached to glass fibres has been developed that allows for the first time imaging of three-dimensionally conserved, fluorescence in situ hybridisation-stained cell nuclei fixed to a glass fibre.     

Estimation of curl in paper using a combination of shape measurement and least-squares modelling

Curl is a quality problem that makes paper less suitable for printing. A paper sheet that has structural variations in its thickness such as gradients of fibre orientation, density and filler content, will curl and hence assume a cylindrical shape when its humidity content is changed. We propose a method to measure curl that can be used for automated analysis of the paper quality. The shape of the curled paper is measured using a stereoscopic camera system, which is capable of viewing a random pattern that is projected onto the specimen. The shape of the object is found by calculating the perspective difference in the camera set-up using digital image correlation. The quality parameters that are searched for are the magnitude of curl, which is defined as the inverse of the radius of curvature and the orientation of the curled paper. These parameters are estimated by performing a least-squares fit of a cylindrical shape to the three-dimensional measurement data. The least-squares model is non-linear and an iterative technique based on the Gauss–Newton algorithm is used.     

Spectral-domain optical coherence phase microscopy for quantitative phase-contrast imaging

We describe a novel microscopy technique for quantitative phase-contrast imaging of a transparent specimen. The technique is based on depth-resolved phase information provided by common path spectral-domain optical coherence tomography and can measure minute phase variations caused by changes in refractive index and thickness inside the specimen. We demonstrate subnanometer level path-length sensitivity and present images obtained on reflection from a known phase object and human epithelial cheek cells.     

Automated Technique for Identifying Experimental Vibrational–Rotational Molecular Spectra Based on Variational Calculations

We describe a technique for automated identification of experimental vibrational–rotational molecular spectra, which is based on variational calculations. The proposed technique is used to analyze the experimental spectra of triatomic molecules H₂O and HDO. This technique significantly accelerates processing and analysis of experimental data and drastically improves accuracy and quality of the results obtained. The possibility of applying this technique for analyzing spectra of other polyatomic molecules is discussed.     

The fine structure of coelomocytes in the sipunculid Phascolosoma esculenta

Ultrastructural characteristics of coelomocytes of the sipunculid Phascolosoma esculenta were studied by transmission electron microscopy. There are several cell types in the coelomic fluid, including three kinds of granulocytes, vesicular cells, germ cells, amoebocytes, phagocytes, and erythrocytes; there are also a new cell complex which is composed of podocytes and granular cells. And several other cell types (erythrocyte and different kinds of granulocytes) gathering together was discovered in the coelomic fluid of P. esculenta. Functional interpretations were provided for these cells using morphological evidence. The coelomocytes from different sipunculid genera and Annelida were compared. The structural diversity of coelomocytes provides both taxonomic characteristics for discriminative identification and phylogenetic markers in Phascolosoma and other sipunculid taxa.     

Application of acoustic reflection tomography to sonar imaging

Computer-aided tomography is a technique for providing a two-dimensional cross-sectional view of a three-dimensional object through the digital processing of many one-dimensional views (or projections) taken at different look directions. In acoustic reflection tomography, insonifying the object and then recording the backscattered signal provides the projection information for a given look direction (or aspect angle). Processing the projection information for all possible aspect angles enables an image to be reconstructed that represents the two-dimensional spatial distribution of the object's acoustic reflectivity function when projected on the imaging plane. The shape of an idealized object, which is an elliptical cylinder, is reconstructed by applying standard backprojection, Radon transform inversion (using both convolution and filtered backprojections), and direct Fourier inversion to simulated projection data. The relative merits of the various reconstruction algorithms are assessed and the resulting shape estimates compared. For bandpass sonar data, however, the wave number components of the acoustic reflectivity function that are outside the passband are absent. This leads to the consideration of image reconstruction for bandpass data. Tomographic image reconstruction is applied to real data collected with an ultra-wideband sonar transducer to form high-resolution acoustic images of various underwater objects when the sonar and object are widely separated.     

Automated collimation testing in Lau interferometry using phase shifting technique

We demonstrate an automated procedure for detection of collimation position of an incoherent beam in Lau interferometry using phase shifting technique. The experimental arrangement consists of a Lau-based interferometer in which light from a source grating illuminates a set of two coarse gratings separated by self-imaging distance. Phase shifting procedure is incorporated by translating the detector grating, corresponding to different positions of the collimating lens. The phase map is plotted corresponding to each position. The slope of the phase map is indicative of the collimation position of the optical beam. The technique is fully automatic and provides good accuracy and high precision.     

Object plane detection and phase-amplitude imaging based on transport of intensity equation

By using transport of intensity equation (TIE), phase distribution of an object is retrieved from through-focus intensity images. This technique allows simple and robust phase imaging compared with an interferometric approach. However, it is hard to measure phase distribution when a dynamic object moves in the direction of an optical axis. To clear this problem, autofocusing TIE which is based on local statistics is proposed. The proposed technique achieves the detection of the object plane and the retrieval of a focused object phase distribution simultaneously. In this approach, an object plane is determined by the focusing techniques based on local statistics such as variance, gradient, and Laplacian of amplitude distribution, after phase distribution in an image sensor plane is retrieved by the TIE. The performance of these three statistics is evaluated in numerical and optical experiments, and a suitable focus value is determined for precise phase imaging.     

Calibrating the X‐ray attenuation of liquid water and correcting sample movement artefacts during in operando synchrotron X‐ray radiographic imaging of polymer electrolyte membrane fuel cells

Synchrotron X‐ray radiography, due to its high temporal and spatial resolutions, provides a valuable means for understanding the in operando water transport behaviour in polymer electrolyte membrane fuel cells. The purpose of this study is to address the specific artefact of imaging sample movement, which poses a significant challenge to synchrotron‐based imaging for fuel cell diagnostics. Specifically, the impact of the micrometer‐scale movement of the sample was determined, and a correction methodology was developed. At a photon energy level of 20 keV, a maximum movement of 7.5 µm resulted in a false water thickness of 0.93 cm (9% higher than the maximum amount of water that the experimental apparatus could physically contain). This artefact was corrected by image translations based on the relationship between the false water thickness value and the distance moved by the sample. The implementation of this correction method led to a significant reduction in false water thickness (to ～0.04 cm). Furthermore, to account for inaccuracies in pixel intensities due to the scattering effect and higher harmonics, a calibration technique was introduced for the liquid water X‐ray attenuation coefficient, which was found to be 0.657 ± 0.023 cm^?1 at 20 keV. The work presented in this paper provides valuable tools for artefact compensation and accuracy improvements for dynamic synchrotron X‐ray imaging of fuel cells.     

Imaging Transparent Objects in a Turbid Medium Using a Femtosecond Optical Kerr Gate

A femtosecond optical Kerr gate time-gated ballistic imaging method is demonstrated to image a transparent object in a turbid medium. The shape features of the object are obtained by time-resolved selection of the ballistic photons with different optical path lengths, the thickness distribution of the object is mapped, and the maximum is less than 3.6%. This time-resolved ballistic imaging has potential applications in studying properties of the liquid core in the near field of the fuel spray.     

High resolution digital holographic microscopy for the study of aggregated natural cellulose nanowhisker fibers

In this paper, digital holographic (DH) microscopy demonstrates its ability to perform a full characterization of nanofibers. The high resolution and magnification of the presented method to study the nanofibers are tested using standard MIL-STD-150A 1951 USAF resolution test target. In this investigation, aggregated natural cellulose nanowhisker fibers are positioned in the front of the microscopic objective using a 3D translation stage in the object arm of DH setup. The recorded off-axis holograms are refocused using the angular spectrum method. The reconstructed complex field is used to calculate optical phase and intensity distributions of the object at different reconstruction depths. A simple algorithm is used to define the focused image with suitable accuracy. The dimensions and orientation of the fibers can be evaluated from the optical field at different depths. Then, the shape and textures along the aggregated natural cellulose nanowhisker fiber can be presented in a 3D space.     

Estimation of Nanoparticle Size Distributions by Image Analysis

Knowledge of the nanoparticle size distribution is important for the interpretation of experimental results in many studies of nanoparticle properties. An automated method is needed for accurate and robust estimation of particle size distribution from nanoparticle images with thousands of particles. In this paper, we present an automated image analysis technique based on a deformable ellipse model that can perform this task. Results of using this technique are shown for both nearly spherical particles and more irregularly shaped particles. The technique proves to be a very useful tool for nanoparticle research.     

Motion deblurring of infrared images from a microbolometer camera

The infrared image of a microbolometer camera can show significant blurring effects if the object is moving. The blurring mechanism of a microbolometer is different to that encountered with classical CCD and CMOS cameras. The electrical signal in the pixel of a microbolometer detector decays exponentially with a time constant of 10–15 ms; therefore, the moving object is mapped to more pixels, resulting in a blurred image. Because of this blurring effect, the recorded infrared radiation of the object is dispersed and the contrast of the object corrupted. In this paper it is shown how the image can be restored and the blurring eliminated. The point spread function of the microbolometer camera is determined and the impact of the blurring from objects of different sizes is investigated. In order to suppress the noise in the restoration, a Wiener filter is used and it is demonstrated how objects of different sizes can be restored. Examples are presented for objects moving linearly and/or rotating; furthermore, an application is described where this technique has been used for automated thermographical testing.     

Unsupervised cell identification on multidimensional X‐ray fluorescence datasets

A novel approach to locate, identify and refine positions and whole areas of cell structures based on elemental contents measured by X‐ray fluorescence microscopy is introduced. It is shown that, by initializing with only a handful of prototypical cell regions, this approach can obtain consistent identification of whole cells, even when cells are overlapping, without training by explicit annotation. It is robust both to different measurements on the same sample and to different initializations. This effort provides a versatile framework to identify targeted cellular structures from datasets too complex for manual analysis, like most X‐ray fluorescence microscopy data. Possible future extensions are also discussed.     

Semi-automated analysis of three-dimensional track images

In the past, three-dimensional (3-d) track images in solid state detectors were difficult to obtain. With the introduction of the confocal scanning laser microscope it is now possible to record 3-d track images in a non-destructive way. These 3-d track images can latter be used to measure typical track parameters. Preparing the detectors and recording the 3-d images however is only the first step. The second step in this process is enhancing the image quality by means of deconvolution techniques to obtain the maximum possible resolution. The third step is extracting the typical track parameters. This can be done on-screen by an experienced operator. For large sets of data however, this manual technique is not desirable. This paper will present some techniques to analyse 3-d track data in an automated way by means of image analysis routines. Advanced thresholding techniques guarantee stable results in different recording situations. By using pre-knowledge about the track shape, reliable object identification is obtained. In case of ambiguity, manual intervention is possible.