A comparison of deconvolution techniques for stress relaxation

Stress relaxation (or equivalently creep) allows a large range of the relaxation (retardation) spectrum of materials to be examined, particularly at lower frequencies. However, higher frequency components of the relaxation curves (typically of the order of Hertz) are attenuated due to the finite time taken to strain the specimen. This higher frequency information can be recovered by deconvolution of the stress and strain during the loading period. This paper examines the use of three separate deconvolution techniques: numerical (Fourier) deconvolution, semi-analytical deconvolution using a theoretical form of the strain, and deconvolution by a linear approximation method. Both theoretical data (where the exact form of the relaxation function is known) and experimental data were used to assess the accuracy and applicability of the deconvolution methods. All of the deconvolution techniques produced a consistent improvement in the higher frequency data up to the frequencies of the order of Hertz, with the linear approximation method showing better resolution in high-frequency analysis of the theoretical data. When the different deconvolution techniques were applied to experimental data, similar results were found for all three deconvolution techniques. Deconvolution of the stress and strain during loading is a simple and practical method for the recovery of higher frequency data from stress-relaxation experiments.     

Enhanced temporal and spatial resolution in super-resolution covariance imaging algorithm with deconvolution optimization

Based on the numerical analysis that covariance exhibits superior statistical precision than cumulant and variance, a new SOFI algorithm by calculating the n orders covariance for each pixel is presented with an almost -fold resolution improvement, which can be enhanced to 2ⁿ via deconvolution. An optimized deconvolution is also proposed by calculating the (n + 1) order SD associated with each n order covariance pixel, and introducing the results into the deconvolution as a damping factor to suppress noise generation. Moreover, a re-deconvolution of the covariance image with the covariance-equivalent point spread function is used to further increase the final resolution by above 2-fold. Simulated and experimental results show that this algorithm can significantly increase the temporal–spatial resolution of SOFI, meanwhile, preserve the sample's structure. Thus, a resolution of 58 nm is achieved for 20 experimental images, and the corresponding acquisition time is 0.8 seconds.     

Picosecond laser-fluorometer with simultaneous time and wavelength resolution for monitoring decay spectra of photoinhibited Photosystem II particles at 277 K and 10 K

The present study describes a novel fluorometer system which permits the simultaneous monitoring of the time and wavelength dependence of chlorophyll fluorescence in the picosecond time domain. The key element of this equipment is a microchannel-plate photomultiplier with delay-line anode. The comparatively short acquisition times in combination with full spectral and temporal resolution of this device are of high advantage, especially for measurements on photosynthetic samples at cryogenic temperatures. For a convenient numerical data evaluation of complex overlapping spectra a decay-associated gaussian deconvolution technique was installed by developing a fitting program with graphical user-interface. In order to illustrate the potential of the new set-up, surface plots and decay spectra gathered from measurements with anaerobically photoinhibited Photosystem II particles at 277 K and 10 K are presented.     

Comparison of Deconvolution Filters for Photoacoustic Tomography

In this work, we compare the merits of three temporal data deconvolution methods for use in the filtered backprojection algorithm for photoacoustic tomography (PAT). We evaluate the standard Fourier division technique, the Wiener deconvolution filter, and a Tikhonov L-2 norm regularized matrix inversion method. Our experiments were carried out on subjects of various appearances, namely a pencil lead, two man-made phantoms, an in vivo subcutaneous mouse tumor model, and a perfused and excised mouse brain. All subjects were scanned using an imaging system with a rotatable hemispherical bowl, into which 128 ultrasound transducer elements were embedded in a spiral pattern. We characterized the frequency response of each deconvolution method, compared the final image quality achieved by each deconvolution technique, and evaluated each method’s robustness to noise. The frequency response was quantified by measuring the accuracy with which each filter recovered the ideal flat frequency spectrum of an experimentally measured impulse response. Image quality under the various scenarios was quantified by computing noise versus resolution curves for a point source phantom, as well as the full width at half maximum (FWHM) and contrast-to-noise ratio (CNR) of selected image features such as dots and linear structures in additional imaging subjects. It was found that the Tikhonov filter yielded the most accurate balance of lower and higher frequency content (as measured by comparing the spectra of deconvolved impulse response signals to the ideal flat frequency spectrum), achieved a competitive image resolution and contrast-to-noise ratio, and yielded the greatest robustness to noise. While the Wiener filter achieved a similar image resolution, it tended to underrepresent the lower frequency content of the deconvolved signals, and hence of the reconstructed images after backprojection. In addition, its robustness to noise was poorer than that of the Tikhonov filter. The performance of the Fourier filter was found to be the poorest of all three methods, based on the reconstructed images’ lowest resolution (blurriest appearance), generally lowest contrast-to-noise ratio, and lowest robustness to noise. Overall, the Tikhonov filter was deemed to produce the most desirable image reconstructions.     

Estimation of the rate and extent of chiral inversion using linear systems analysis

An alternative method based on linear systems analysis is presented for the analysis of concentration-time data for the enantiomers of the 2-arylpropionic acids. This approach uses deconvolution to estimate the rate and extent of chiral inversion with respect to time, assuming linear pharmacokinetics and time invariance, without the need for complicated modelling procedures. Application to data for the chiral inversion of ibuprofen in the rat indicates that this approach provides a valid alternative to previous procedures for the analysis of chiral inversion data. © 1995 Wiley-Liss, Inc.     

Semi-Blind Ultrasound Image Deconvolution from Compressed Measurements

The recently proposed framework of ultrasound compressive deconvolution offers the possibility of decreasing the acquired data while improving the image spatial resolution. By combining compressive sampling and image deconvolution, the direct model of compressive deconvolution combines random projections and 2D convolution with a spatially invariant point spread function. Considering the point spread function known, existing algorithms have shown the ability of this framework to reconstruct enhanced ultrasound images from compressed measurements by inverting the forward linear model. In this paper, we propose an extension of the previous approach for compressive blind deconvolution, whose aim is to jointly estimate the ultrasound image and the system point spread function. The performance of the method is evaluated on both simulated and in vivo ultrasound data.     

Optimised generalized polarisation analysis of C-laurdan reveals clear order differences between T cell membrane compartments

Heterogenous packing of plasma membrane lipids is important for cellular processes like signalling, adhesion and sorting of membrane components. Solvatochromic membrane fluorophores that respond to changes from liquid-ordered (l_o) phase to liquid-disordered (l_d) by red shifts in their emission spectra are often used to assess lipid packing. Their response can be quantified using generalized polarisation (GP) using fluorescence microscopy images from two emission ranges, preferably from a region of interest (ROI) limited to a specific membrane compartment. However, image quality is limited by Poisson noise and convolution by the point spread function of the imaging system. Examining GP-analysis of C-laurdan labelled T cells using the image restoration procedure deconvolution, we demonstrate that deconvolution substantially improves the image resolution by making the plasma membrane clearly discernible and facilitating plasma membrane ROI selection. We conclude that automatic ROI selection has advantages over manual ROI selection when it comes to reproducibility and speed, but reliable GP-measurements can also be obtained by manually demarcated ROIs. We find that deconvolution enhances the difference in GP-values between the plasma and intracellular membranes and demonstrate that moving an intensity defined plasma membrane ROI outwards from the cell further improves this differentiation. By systematically changing the key deconvolution regularization parameter signal to noise, we establish a protocol for deconvolution optimisation applicable to any solvatochromic dye and imaging system. The image processing and ROI selection protocol presented improves both the resolution and precision of GP-measurement and will enable detection of smaller changes in membrane order than is currently achievable.     

基于频域反卷积滤波器的光声成像

采用探测器的脉冲响应在频域反卷积滤波光声信号以进一步提高光声成像的分辨率.由仿真和实验结果表明,频域反卷积滤波重建相对于时域反投影重建和滤波反投影重建具有更好的成像效果,明显地提高重建图像的分辨率,经仿真结果的计算,其重建图像的分辨率由2.58 mm提高到了0.16 mm.实验所用的光源为YAG激光器,波长为1064 ...     

Calibration of Wide-Field Deconvolution Microscopy for Quantitative Fluorescence Imaging

Deconvolution enhances contrast in fluorescence microscopy images, especially in low-contrast, high-background wide-field microscope images, improving characterization of features within the sample. Deconvolution can also be combined with other imaging modalities, such as confocal microscopy, and most software programs seek to improve resolution as well as contrast. Quantitative image analyses require instrument calibration and with deconvolution, necessitate that this process itself preserves the relative quantitative relationships between fluorescence intensities. To ensure that the quantitative nature of the data remains unaltered, deconvolution algorithms need to be tested thoroughly. This study investigated whether the deconvolution algorithms in AutoQuant X3 preserve relative quantitative intensity data. InSpeck Green calibration microspheres were prepared for imaging, z-stacks were collected using a wide-field microscope, and the images were deconvolved using the iterative deconvolution algorithms with default settings. Afterwards, the mean intensities and volumes of microspheres in the original and the deconvolved images were measured. Deconvolved data sets showed higher average microsphere intensities and smaller volumes than the original wide-field data sets. In original and deconvolved data sets, intensity means showed linear relationships with the relative microsphere intensities given by the manufacturer. Importantly, upon normalization, the trend lines were found to have similar slopes. In original and deconvolved images, the volumes of the microspheres were quite uniform for all relative microsphere intensities. We were able to show that AutoQuant X3 deconvolution software data are quantitative. In general, the protocol presented can be used to calibrate any fluorescence microscope or image processing and analysis procedure.     

Investigation of stretching vibrations of glycosidic linkages in disaccharides and polysaccharides with use of IR spectra deconvolution

The results are presented for the deconvolution of IR spectra of disaccharides and polysaccharides with alpha and beta configurations of the 1 --> 4 glycosidic linkage (maltose, cellobiose, amylose, and cellulose), as well as of their corresponding monosaccharides (alpha- and beta-D-glucose) in the 1200-920 cm(-1) frequency range. It is established that a characteristic of di- and polysaccharides with 1 --> 4 glycosidic linkage is the appearance of new absorption bands in the 1175-1140 cm(-1) spectral range, as opposed to the IR spectra of monosaccharides. This can be a spectroscopic manifestation of the glycosidic linkage formation. In the 1000-970 cm(-1) frequency range, absorption bands, which are not observed in the monomer spectrum, are separated as a result of the deconvolution of the IR spectra of cellobiose and cellulose. The number of bands in this range remains unchanged for maltose and amylose, as compared to the monomer spectra. It is shown that the application of the method of deconvolution leads to a considerable enhancement in the resolution of the absorption bands in the IR spectra of mono-, di-, and polysaccharides.     

A Deconvolution-Based Method with High Sensitivity and Temporal Resolution for Detection of Spontaneous Synaptic Currents In?Vitro and?In?Vivo

Spontaneous postsynaptic currents (PSCs) provide key information about the mechanisms of synaptic transmission and the activity modes of neuronal networks. However, detecting spontaneous PSCs in vitro and in vivo has been challenging, because of the small amplitude, the variable kinetics, and the undefined time of generation of these events. Here, we describe a, to our knowledge, new method for detecting spontaneous synaptic events by deconvolution, using a template that approximates the average time course of spontaneous PSCs. A recorded PSC trace is deconvolved from the template, resulting in a series of delta-like functions. The maxima of these delta-like events are reliably detected, revealing the precise onset times of the spontaneous PSCs. Among all detection methods, the deconvolution-based method has a unique temporal resolution, allowing the detection of individual events in high-frequency bursts. Furthermore, the deconvolution-based method has a high amplitude resolution, because deconvolution can substantially increase the signal/noise ratio. When tested against previously published methods using experimental data, the deconvolution-based method was superior for spontaneous PSCs recorded in vivo. Using the high-resolution deconvolution-based detection algorithm, we show that the frequency of spontaneous excitatory postsynaptic currents in dentate gyrus granule cells is 4.5 times higher in vivo than in vitro.     

Simultaneous analysis of multiple fluorescence decay curves by Laplace transforms. Deconvolution with reference or excitation profiles

The properties and potentials of the noniterative Laplace deconvolution (LAP2) (M. Ameloot and H. Hendrickx, Biophys. J. 44 (1983) 27) are further investigated. It is shown that LAP2 is exact and that no extrapolations have to be calculated or assumed for the data measured in the actual time window if the impulse response function of the investigated system can be described by a sum of exponentials. The formulas for the LAP2 deconvolution against the measured decay of a reference compound instead of the recorded excitation profile are derived. The procedure for the simultaneous analysis of multiple fluorescence decay curves by LAP2 is described in detail. This global analysis allows one to link any decay parameter, is fast and compares favorably with the nonlinear least-squares iterative reconvolution methods. Because of its short computation time the global analysis by LAP2 provides an efficient way to analyze the fluorescence decay surface in terms of decay associated spectra.     

Structure determination of lipid bilayers

A method of determining the phases of X-ray reflections from oriented model membrane systems at low resolution is described. The method involves deconvolution and requires that d less than or equal to 2v where v is the width of the head group region within the bilayer and d is the thickness of the bilayer. The method can be used with a single set of X-ray data and applies to lipid bilayers which have a relatively constant density in the hydrocarbon region. Phases for the first five or six orders of phosphatidylethanolamine and lecithin are derived. A refined analysis based upon deconvolution but using information inherent in the Fourier profile is also described.     

用去卷积法提高超声调制光学成像的空间分辨率

超声调制光学成像的空间分辨率取决于光在组织中的散射程度和扫描超声束的聚焦大小。由于组织是强散射介质,实际应用中的超声束都有一定的聚焦宽度(通常是毫米数量级),所以该技术成像空间分辨率一直无法提高。针对这个问题,首次将去卷积图像处理法运用在超声调制光学成像技术中,有效地解决了扫描超声束带来的信号展开,分辨率下降的影响。理论和防真结果表明,处理后的成像分辨率大大提高,图像质量明显改善。该方法无须对系统装置做任何改动,只利用适当的数据处理,就实现了成像超分辨,具有应用价值。     

Computational Methods for Estimation of Cell Cycle Phase Distributions of Yeast Cells

Two computational methods for estimating the cell cycle phase distribution of a budding yeast (Saccharomyces cerevisiae) cell population are presented. The first one is a nonparametric method that is based on the analysis of DNA content in the individual cells of the population. The DNA content is measured with a fluorescence-activated cell sorter (FACS). The second method is based on budding index analysis. An automated image analysis method is presented for the task of detecting the cells and buds. The proposed methods can be used to obtain quantitative information on the cell cycle phase distribution of a budding yeast S. cerevisiae population. They therefore provide a solid basis for obtaining the complementary information needed in deconvolution of gene expression data. As a case study, both methods are tested with data that were obtained in a time series experiment with S. cerevisiae. The details of the time series experiment as well as the image and FACS data obtained in the experiment can be found in the online additional material at http://www.cs.tut.fi/sgn/csb/yeastdistrib/.     

人脑源性神经营养因子cDNA在COS7细胞中的表达及活性研究

本文从质粒Ｍ１３ｍｐ１８－ｈＢＤＮＦ中酶切回收人脑源性神经营养因子（ｈＢＤＮＦ）全长基因,构建真核表达载体ｐＣＭＶ４－ｈＢＤＮＦ。利用脂质体的方法转染ＣＯＳ７细胞,对转染后的ＣＯＳ７细胞提取ＲＮＡ进行狭缝杂交分析和免疫细胞化学反应,分别从转录及翻译水平上检测ＢＤＮＦ基因在ＣＯＳ７细胞中的表达。实验还证实在ＣＯＳ７细胞中表达的ｈＢＤＮＦ蛋白可分泌至胞外并可促进中脑黑质细胞的发育和生长,具有良好的生物学活性。     

An improved method for the direct chromatographic resolution of abscisic acid enantiomers

An improved method for the direct chromatographic resolution of abscisic acid enantiomers using the commercially available Whelk-O 1 chiral stationary phase is presented. Previously reported strategies utilized in the chromatographic separation of abscisic acid enantiomers are summarized, and conditions for analytical and semipreparative separations using the Whelk-O 1 chiral stationary phase are described. This method offers the advantages of rapid analysis time and greatly increased capacity, allowing the resolution of more than 6 mg in 25 min using an analytical column (4.6 mm i.d. × 25 cm length). © 1993 Wiley-Liss, Inc.     

Precise Time Superresolution by Event Correlation Microscopy

Fluorescence imaging is often used to monitor dynamic cellular functions under conditions of very low light intensities to avoid photodamage to the cell and rapid photobleaching. Determination of the time of a fluorescence change relative to a rapid high time-resolution event, such as an action potential or pulse stimulation, is challenged by the low photon rate and the need to use imaging frame durations that limit the time resolution. To overcome these limitations, we developed a time superresolution method named event correlation microscopy that aligns repetitive events with respect to the high time-resolution events. We describe the algorithm of the method, its step response function, and a theoretical, computational, and experimental analysis of its precision, providing guidelines for camera exposure time settings depending on imaging signal properties and camera parameters for optimal time resolution. We also demonstrate the utility of the method to recover rapid nonstepwise kinetics by deconvolution fits. The event correlation microscopy method provides time superresolution beyond the photon rate limit and imaging frame duration with well-defined precision.     

High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy

Morphological features such as size, shape and density of dendritic spines have been shown to reflect important synaptic functional attributes and potential for plasticity. Here we describe in detail a protocol for obtaining detailed morphometric analysis of spines using microinjection of fluorescent dyes, high-resolution confocal microscopy, deconvolution and image analysis with NeuronStudio. Recent technical advancements include better preservation of tissue, resulting in prolonged ability to microinject, and algorithmic improvements that compensate for the residual z-smear inherent in all optical imaging. Confocal imaging parameters were probed systematically to identify both optimal resolution and the highest efficiency. When combined, our methods yield size and density measurements comparable to serial section transmission electron microscopy in a fraction of the time. An experiment containing three experimental groups with eight subjects each can take as little as 1 month if optimized for speed, or approximately 4-5 months if the highest resolution and morphometric detail is sought.     

F/F deconvolution of fluorescence decay data

An approach for the deconvolution of multiexponential fluorescence decay data in which a single exponential decay is used in place of the usual excitation profile is described. For analysis by the method of moments, the resulting decay lifetimes are identical to those in the multiexponential decay, while the pre-exponential factors are a simple function of the true values and the parameters of the single exponential decay. This approach, which we call the F/F deconvolution method, is capable of eliminating the errors in decay analyses which arise from the wavelength dependence of the instrument response function.