Dynamic Speckle Research Articles

The use of laser dynamical speckle interferometry in the study of cellular processes

A speckle interferometry setup is mounted, tested and approved that allows the analysis of intracellular processes with large optical magnification. For different part of the cell, the dependences of speckle image correlation coefficients upon time are found. It is shown that the method allows the detection of differences in the process that occur in the nutrient solution outside the cell and inside the cell, as well as those occurring in different parts of the cell.

Molecular Characterization of SRSF2 Mutation Identifies a Clinically Relevant Lncrna (MALAT1) in Chronic Myelomonocytic Leukemia (CMML)

SRSF2 is mutated at proline 95 (P95) in approximately 45% of patients with CMML. The consequence of SRSF2 P95H mutations on splicing has been associated with change in target RNA motif preference compared to wild type that favors CCNG over GGNG resulting in aberrant splicing abnormalities. Although this has led to several downstream mis-spliced candidates that may contribute to SRSF2 leukemic pathogenesis, the biologic consequences of SRSF2 mutation have not been fully elucidated and its effect on non-splicing pathways has yet to be explored.SRSF2 has two major functions within the nucleus: (1) bind to cis elements on pre-mRNA transcripts that functionally redefines putative exon-intron boundaries and (2) interact with other members of the spliceosome complex at nuclear suborganelles known as speckles. To explore the effects of SRSF2 P95 mutation on nuclear speckle dynamics, we transfected HeLa cells with GFP-SRSF2 wildtype (WT) and mutant (MT) constructs and performed Fluorescent Recovery After Photo-bleaching (FRAP) to determine the mutant specific kinetics of SRSF2 molecules localized to the nuclear speckle. Using this approach, we confirmed that SRSF2 WTs are characterized by complete photo-recovery with a half-life of approximately 35 (30-42) seconds as previously reported. However, when we performed FRAP of GFP-SRSF2 MTs a statistically significant difference in photo-recovery was observed compared to WT cells (Fig. 1a). The difference between baseline fluorescence and maximal recovered fluorescence in SRSF2 MT cells, or the immobile fraction, suggests that trapped bleached SRSF2 molecules are sterically inhibiting the diffusion of unbleached SRSF2 molecules.We reasoned that the observed differences in RNA binding capacity and nuclear speckle dynamics could be related. This rationale identified MALAT1 as a link connecting both increased RNA binding and nuclear speckle trafficking abnormalities because it is a Long Non-Coding (lnc) RNA that localizes to and has a critical role in SR protein recruitment and retention to the nuclear speckle. MALAT1, which is highly enriched for CCNG motifs, has also been demonstrated to bind to SRSF2 directly and has been implicated in a wide spectrum of carcinomas making it an attractive intermediary to study in our system.To determine whether SRSF2 MTs have increased MALAT1 binding capacity compared to SRSF2 WT, we performed RNA IP of myc-his-SRSF2 WT and MT transfected HeLa cells. Using this approach, we demonstrated a 60% enrichment of MALAT1 in SRSF2 WT transfected cells and a 120-150% enrichment of MALAT1 in SRSF2 MT cells compared to the empty vector control (Fig. 1b-c). We next performed the above FRAP experiment in the context of MALAT1 depletion and demonstrated that observed immobile fraction seen in SRSF2 MT cells is rescued (Fig. 1d).To determine the clinical relevance of MALAT1 in CMML we profiled a cohort of 54 CMML cases for MALAT1 expression by PCR. As shown in Figure 2a-b and d-e, MALAT1 expression is statistically elevated in CMML BMNCs and is among the highest differentially expressed transcripts in CMML monocytes (CD14+). We additionally characterized SRSF2 expression in our CMML BMNCs and CD14+ cohort and identified a linear correlation (Pearson R=0.7 p<0.05) between SRSF2 expression and MALAT1 expression (Figure 2c). Further, CMML patients with high MALAT1 expression in the BMNC compartment had inferior overall and leukemia-free survival (Fig. 2f-g).Given its relevance in solid tumors and its over-expression in CMML, we explored the consequence of MALAT1 depletion on human monocytic leukemic cells. We first performed ultra-deep RNA sequencing of the THP-1 cell line treated with two MALAT-1 depleting ASOs or matched chemistry specific controls. IPA analysis of differentially expressed transcripts identified a c-Myc gene signature that was validated by western blot analysis demonstrating reduction of c-Myc in THP-1 MALAT1-depleted cells. Colony formation assays using the THP-1 cell line demonstrated that MALAT-1 depletion resulted in decreased colony-forming capacity (CFC). MALAT-1 ASOs were also capable of depleting MALAT-1 in primary CMML BMNCs and were associated with a decreased CFC suggesting that MALAT-1 is a potential therapeutic target. Taken together, our data identifies SRSF2 P95 mutation nuclear trafficking abnormalities and identifies a novel clinically relevant lncRNA in CMML. [Display omitted] [Display omitted] DisclosuresKomrokji:Celgene: Consultancy, Research Funding; Incite: Consultancy; Novartis: Speakers Bureau; GSK: Research Funding. List:Celgene Corporation: Honoraria, Research Funding.

Coordinated Dynamics of RNA Splicing Speckles in the Nucleus.

Despite being densely packed with chromatin, nuclear bodies and a nucleoskeletal network, the nucleus is a remarkably dynamic organelle. Chromatin loops form and relax, RNA transcripts and transcription factors move diffusively, and nuclear bodies move. We show here that RNA splicing speckled domains (splicing speckles) fluctuate in constrained nuclear volumes and remodel their shapes. Small speckles move in a directed way toward larger speckles with which they fuse. This directed movement is reduced upon decreasing cellular ATP levels or inhibiting RNA polymerase II activity. The random movement of speckles is reduced upon decreasing cellular ATP levels, moderately reduced after inhibition of SWI/SNF chromatin remodeling and modestly increased upon inhibiting RNA polymerase II activity. To define the paths through which speckles can translocate in the nucleus, we generated a pressure gradient to create flows in the nucleus. In response to the pressure gradient, speckles moved along curvilinear paths in the nucleus. Collectively, our results demonstrate a new type of ATP-dependent motion in the nucleus. We present a model where recycling splicing factors return as part of small sub-speckles from distal sites of RNA processing to larger splicing speckles by a directed ATP-driven mechanism through interchromatin spaces.

Following the drying process of Fevicol (adhesive) by dynamic speckle measurement

This work presents the study of drying of Fevicol (adhesive) using dynamic laser speckle technique. Dynamic laser speckle analysis is a non-invasive method that does not disturb the drying process. A low power laser light illuminates the sample and corresponding speckle images of the sample resulting from the phenomenon of interference are registered. If the sample shows activity, the speckle pattern changes in time. Following this evolution, we can analyze the drying process by means of image processing algorithms. In this work, the application of dynamic speckle technique to follow the drying process of Fevicol with different drying times is presented. Results obtained using numerical methods of Inertia Moment and cross-correlation show that quantitative evaluation of spatial temporal speckle activities can be used to monitor Fevicol drying process. The approaches such as auto-correlation and speckle contrast using temporal correlation allowed differentiating different stages of drying. In addition, the techniques allowed their applicability to follow the slow streaming of Fevicol from the coin surface.

Monitoring capillary blood flow using laser speckle contrast analysis with spatial and temporal statistics

Laser Speckle Contrast Imaging (LSCI) is a simple and powerful technique that is used for full-field imaging of blood flow. The technique analyzes fluctuations in a dynamic speckle pattern to detect the movement of particles. In this article Laser speckle contrast analysis based on spatial and temporal correlations (sLASCA and tLASCA) were used to provide a velocity map of the area of interest in real time without the need for scanning. Spatial and temporal laser speckle contrast analysis have been shown to be a viable real-time method for detecting variations in blood flow. The results of some initial experiments on volunteers are presented. Both the techniques namely sLASCA and tLASCA are able to pick up changes in blood flow, which may arise due to various physiological conditions. Further, more blood flow reflected due to less skin thickness was sensed by the tLASCA technique which can be put to good use in the follow-up of the treatment of certain diseases which may cause decreased blood flow. Thus, both the techniques can be used to compare differences in blood flow values at region in normal subjects and in diseased subjects.

Acousto-optical detection of hidden objects via speckle based imaging.

Optical detection of objects hidden behind opaque screening layers is a challenging problem. We demonstrate an optically detected echographic-like method that combines collimated acoustic and laser beams. The acoustic waves cross the screening layers, and their back-reflection from the hidden objects is detected through the analysis of a dynamic laser speckle pattern created at the outer surface of the screening layer. Real-time remote detection of moving targets 15 meters away, with a few mm resolution is demonstrated using a very sensitive camera detection scheme.

Motility Contrast Imaging for Drug Screening Applications

Motility contrast imaging is a coherence-domain imaging technique that uses cellular motility as a fully endogenous imaging contrast agent. Motility is measured inside tissue using a digital holographic coherence gate that extracts dynamic speckle from fixed depths. The dynamic speckle arises from the normal organelle motion inside cells, and from the movement of the cellular membranes driven by the cytoskeleton. It measures cellular activity and the effects of temperature and osmolarity. Motion is sensitive to cytoskeletal drugs, such as the antimitotic drugs used for cancer chemotherapy, and the effects of drug combinations also can be monitored. Motility contrast imaging is a potential tissue-based assay platform for highthroughput screening of pharmaceuticals.

Selectivity and balance of spatial filtering velocimetry of objective speckles for measuring out-of-plane motion

We probe the dynamics of objective laser speckles as the axial distance between the object and the observation plane changes. With the purpose of measuring out-of-plane motion in real time, we apply optical spatial filtering velocimetry to the speckle dynamics. To achieve this, a rotationally symmetric spatial filter is designed. The spatial filter converts the speckle dynamics into a photocurrent with a quasi-sinusoidal response to the out-of-plane motion. Our contribution presents the technology, discusses the selectivity of the spatial filter and proposes two solutions to balance phase-stepped photocurrents. Specifically, we discuss how the selectivity of the spatial filter with regard to radial speckle motion is influenced by a concurrent in-plane speckle motion. The spatial filter is emulated with a CCD camera and is tested on speckle acquisitions obtained from a controlled setup. Experiments with the emulated filters illustrate the performance and potential applications of the technology.

Dynamic speckle analysis using multivariate techniques

In this work we use principal components analysis to characterize dynamic speckle patterns. This analysis quantitatively identifies different dynamics that could be associated to physical phenomena occurring in the sample. We also found the contribution explained by each principal component, or by a group of them. The method analyzes the paint drying process over a hidden topography. It can be used for fast screening and identification of different dynamics in biological or industrial samples by means of dynamic speckle interferometry.

Dynamics of hnRNPs and omega speckles in normal and heat shocked live cell nuclei of Drosophila melanogaster.

The nucleus limited long-noncoding hsrω-n transcripts, hnRNPs, and some other RNA processing proteins organize nucleoplasmic omega speckles in Drosophila. Unlike other nuclear speckles, omega speckles rapidly disappear following cell stress, while hnRNPs and other associated proteins move away from chromosome sites, nucleoplasm, and the disappearing speckles to get uniquely sequestered at hsrω locus. Omega speckles reappear and hnRNPs get redistributed to normal locations during recovery from stress. With a view to understand the dynamics of omega speckles and their associated proteins, we used live imaging of GFP tagged hnRNPs (Hrb87F, Hrb98DE, or Squid) in unstressed and stressed Drosophila cells. Omega speckles display size-dependent mobility in nucleoplasmic domains with significant colocalization with nuclear matrix Tpr/Megator and SAFB proteins, which also accumulate at hsrω gene site after stress. Instead of moving towards the nuclear periphery located hsrω locus following heat shock or colchicine treatment, omega speckles rapidly disappear within nucleoplasm while chromosomal and nucleoplasmic hnRNPs move, stochastically or, more likely, by nuclear matrix-mediated transport to hsrω locus in non-particulate form. Continuing transcription of hsrω during cell stress is essential for sequestering incoming hnRNPs at the site. While recovering from stress, the sequestered hnRNPs are released as omega speckles in ISWI-dependent manner. Photobleaching studies reveal hnRNPs to freely move between nucleoplasm, omega speckles, chromosome regions, and hsrω gene site although their residence periods at chromosomes and hsrω locus are longer. A model for regulation of exchange of hnRNPs between nuclear compartments by hsrω-n transcripts is presented.

The spectral maps of dynamic speckle patterns: A new perspective to look at the motion-induced spatiotemporal cross-correlation

We introduce the spectral map method as a means to visualize the spatiotemporal correlation in dynamic speckles. Dynamic speckle patterns of moving diffuse objects are Fourier transformed to construct spectral maps in spatial domain (r-space) and spatial-frequency domain (k-space). The periodic patterns in r-space spectral maps demonstrate the spatiotemporal cross-correlation induced by the directional motions. To resolve the spectral bands in k-space spectral maps, and to observe the concomitant appearance of the periodic pattern in r-space spectral maps, the frequency must exceed the minimum values given by the speckle size and the speed. A bigger speckle size helps to reveal fast dynamics, and to ease the requirement on the minimum frequency. The transition from ordered translational speckles to chaotic boiling speckles at different speeds and speckle sizes is elucidated in connection with the change observed in spectral maps.

System of acquisition and processing of images of dynamic speckle

In this paper we show the design and implementation of a system to capture and analysis of dynamic speckle. The device consists of a USB camera, an isolated system lights for imaging, a laser pointer 633 nm 10 mw as coherent light source, a diffuser and a laptop for processing video. The equipment enables the acquisition and storage of video, also calculated of different descriptors of statistical analysis (vector global accumulation of activity, activity matrix accumulation, cross-correlation vector, autocorrelation coefficient, matrix Fujji etc.). The equipment is designed so that it can be taken directly to the site where the sample for biological study and is currently being used in research projects within the group.

Assessment of Fevicol (adhesive) Drying Process through Dynamic Speckle Techniques

Dynamic laser speckle (or biospeckle) analysis is a useful measurement tool to analyze micro-motion on a sample surface via temporal statistics based on a sequence of speckle images. The aim of this work was to evaluate the use of dynamic speckles as an alternative tool to monitoring Fevicol drying process. Experimental demonstration of intensity-based algorithm to monitor Fevicol drying process is reported. The experiment was explored with the technique called Inertia Moment of co-occurrence matrix. The results allowed verifying the drying process and it was possible to observe different activity stages during the drying process. Statistical Tukey test at 5% significance level allowed differentiating different stages of drying. In conclusion, speckle activity, measured by the Inertia Moment, can be used to monitor drying processes of the Fevicol.

Signal Feature Extraction Using Granular Computing. Comparative Analysis with Frequency and Time Descriptors Applied to Dynamic Laser Speckle Patterns

AbstractThe laser dynamic speckle is a phenomenon caused by the fluctuant interference of the laser light reflected from an illuminated surface where some kind of activity is taking place. Signals generated by the intensity changes in each pixel through the sequence are processed with the finality of identifying underlying activity in each point. In this work we compare the performance of a Rough Fuzzy Granular Descriptor (previously published) against a set of dynamic speckle descriptors based in time and frequency processing. To perform this evaluation a numerical simulation is proposed to explore their linearity, robustness, sensitivity related to the samples quantity, as well as also by their computing time. Also the robustness to inhomogeneous spatial intensity was evaluated in an experiment performed with the illuminated surface of an actual biological object.

纳米磁流体动态散斑干涉场的奇异场分布

纳米磁流体动态散斑干涉场的奇异场分布

Dynamic Speckle-Interferometry of Microscopic Processes in Thin Biological Objects

Speckle dynamics in a thin biological object illuminated through a diffusor box is studied both theoretically and experimentally. A model that allows for random time variations in the phase difference Δφ of pairs of waves transmitted through the object is used in the theoretical part of this study. Formulas for the time-averaged intensity of the radiation at a certain point of observation and a temporal autocorrelation function of the radiation intensity are deduced. The relationship between the characteristics of the random variable Δφ and the parameters of the speckle dynamics is discussed. The case of occurrence of a quasiperiodic variation in the radiation intensity is separately considered. The theoretical results are used for interpretation of the data obtained in the experiments on studying the metabolic activity of cells cultivated on a glass substrate.

Using Speckle Dynamics for Comparison of the Metabolic Activity of Different Cell Cultures

The dynamics of speckles in the image plane of a monolayer of cells cultivated on a glass substrate has been recorded. Cell cultures HEL-3, L-41, and Vero were selected as the objects of research. The digitized value of the radiation intensity I in one pixel and the parameter η characterizing the change in the intensity distribution on an 10 × 10 pixel area was recorded for 24 hours. The multiple determinacy coefficient of three cell cultures, which was obtained from the time dependences of η, was equal to 0.94.

毛玻璃转速对热光鬼成像质量的影响

In this paper,we experimentally studied how the rotational speed of ground-glass influences the quality of thermal ghost imaging.As the rotational speed of ground-glass increased,the dynamic speckles fluctuate faster,which reduced the coherence time of the speckle field,and the speckle pattern obtained by a CCD detector blurs.Yet,the contrast to noise ratio of the ghost image was virtually irrelevant to the rotational speed of ground-glass.Our experimental result showed that the quality of the ghost image did not degrade even when ground-glass rotates so fast that the blurred speckle pattern appears in CCD detector.

Dynamic speckle illumination wide-field reflection phase microscopy.

We demonstrate a quantitative reflection-phase microscope based on time-varying speckle-field illumination. Due to the short spatial coherence length of the speckle field, the proposed imaging system features superior lateral resolution, 520 nm, as well as high-depth selectivity, 1.03 μm. Off-axis interferometric detection enables wide-field and single-shot imaging appropriate for high-speed measurements. In addition, the measured phase sensitivity of this method, which is the smallest measurable axial motion, is more than 40 times higher than that available using a transmission system. We demonstrate the utility of our method by successfully distinguishing the motion of the top surface from that of the bottom in red blood cells. The proposed method will be useful for studying membrane dynamics in complex eukaryotic cells.

Pointwise implementation of dynamic laser speckle technique

The paper proposes pointwise implementation of correlation-based algorithms for processing time sequences of 2D dynamic speckle patterns of a diffuse object illuminated with a laser light in order to obtain a spatial map of physical or biological activity across its surface. Both temporal correlation and structure functions were studied with and without the pointwise normalization by the variance of intensity fluctuations in time. The performance of the algorithms was checked by simulation of dynamic speckle patterns at a given spatial distribution of the temporal correlation radius of activity and by dynamic speckle measurement of a test object. The obtained set of high contrast and high spatial resolution activity maps proves the efficiency of the normalized correlation-based algorithms to indicate regions of varying activity even at non-uniform illumination and reflectivity across the object.