Tagged Region Research Articles

STAR-FDTD: space-time modulated acousto-optic guidestar in disordered media

Abstract We developed a 2D Finite-difference time-domain (FDTD) method for modeling a space-time modulated guidestar targeting wavefront shaping applications in disordered media. Space-time modulation in general (a particular example being the acousto-optic effect) is used here as a guidestar for the transverse confinement of light around the tagged region surrounded by disorder. Together with the guidestar, the iterative optical phase conjugation (IOPC) method is used to overcome the diffusion of light due to multiple scattering. A phase sensitive lock-in detection technique is utilized to estimate the steady-state amplitude and phase of the modulated wavefronts emerging from the guidestar region continuously operating in the Raman-Nath regime. As the IOPC scheme naturally converges to the maximally transmitting eigenchannel profile, one could use the position of the guidestar within the disorder to channelize the maximal transmission through the tagged region. The associated code developed in MATLAB® is provided as an open source (The MIT License) package. The code package is referred by the acronym STAR-FDTD where STAR stands for Space-Time modulated Acousto-optic guidestaR.

Microwave re-excitation of femtosecond laser tagging for highly flexible velocimetry.

Molecular tagging velocimetry is typically species specific and limited by excited state/species lifetimes. We utilize laser-generated ionization, long-lived anions, and a time-delayed microwave pulse to monitor the tagged region up to several milliseconds. This non-resonant excitation and microwave interaction is demonstrated in a range of gas mixtures. Signal levels show up to 1000-fold improvement, and the flexibility in interrogation time allows for velocity measurements over a large dynamic range (1-100 m/s) with single-shot precision of <5%. This approach has the potential for wide application over a range of relevant gas compositions, temperatures, and pressures.

A simple approach to generate transgenic cell lines with high efficiency using CRISPR and flippase-mediated recombination

Clustered regularly interspaced short palindromic repeats (CRISPR) can be concocted to cleave any desired sequence in the genome. We combine its precision with the efficiency of flippase based homologous recombination (HR) to insert genes at a safe genomic locus like Adeno-associated virus integration site 1 (AAVS1) in seven different cell lines. Our approach involves the creation of an acceptor cell line tagged with flippase recognition target (FRT), which accelerates the generation of the desired transgenic cell line with a single-copy insertion at a specific locus. To create this acceptor cell line, we designed a series of guide RNAs (gRNAs) that recognize and cleave a specific sequence within the AAVS1 locus. These validated gRNAs were co-expressed with an FRT-tagging plasmid, which expresses reporter genes (fluorescence and/or antibiotic resistance) flanked by FRT-recombination and genomic sequences adjacent to the CRISPR site. This process resulted in the generation of an acceptor cell line tagged with FRT and reporter genes, using CRISPR-induced HR. Subsequently, a donor plasmid containing mCherry (our gene of interest/GOI) and reporter genes flanked by FRT was introduced into the acceptor cell line. This led to the insertion of the GOI and reporter genes in the tagged region at a neutral locus of the genome. Our strategy helps generate transgenic cell lines with precision, thereby offering a streamlined and efficient approach to genetic manipulation.

Subcutaneous injection of visible implant elastomer in sterlet (Acipenser ruthenusLinnaeus, 1758): a study on compatibility and retention

Summary The purpose of this study was to mark endangered sterlet (Acipenser ruthenus) with visible implant elastomer (VIE) in order to assess mortality, compatibility, retention, persistence and histological reactions resulting from this tagging technique. It was hypothesized there would be only minor effects on the fishes' health, and assumed that acute effects would be more pronounced than long-term effects. On 11 September 2013, 20 specimens were tagged ventrally with visible implant elastomer, 20 received a subcutaneous injection with 0.9% NaCl solution, and another 20 served as untreated control. Mean total length was 28.0 ± 1.8 cm and mean body mass 64.1 ± 12.0 g. The sterlets were kept in four 4,000-L tanks filled with 2,400-L water. Acute effects were monitored for 95 days, where fish were held at temperatures between 2.4°C and 15.2°C, reflecting outdoor conditions. Chronic effects were examined 282 days post-tagging through histological sections of the tagging region in five sterlets. During the first 95 days of observation, tag retention was 100%. No signs of incompatibility were detected. Body mass did not significantly differ between VIE-tagged fish and controls. At day 282 post-tagging, however, distinct tissue reactions were visible at the tagging sites of nine fish. Histological examination of five fish revealed a variable degree of infiltration with leukocytes in the areas around the elastomer, which did not necessarily correspond with the externally visible degree of inflammation. After medical treatment, the lesions healed without complications, whereas the retention rate of the VIE tags was 5%. According to the findings, the tag location rather than the tag itself was responsible for the externally visible irritations, indicating that the ventral subcutis of sterlet is not a suitable site, even for small VIE tags in long-term studies. The results of this study also suggest that VIE marking should in general be critically evaluated before application in field studies.

Signature-tagged mutagenesis of Vibrio vulnificus.

Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.

Measurement of liquid sheet using laser tagging method by photochromic dye

Liquid atomization system has been extensively applied as the most significant process in many industrial fields. In the internal combustion engine, the combustion phenomenon is strongly influenced by the spray characteristics of the fuel given by the atomization process. In order to completely understand the whole atomization process, a detail investigation of relations between the liquid jet characteristics and the breakup phenomenon is required. In this study, a non-intrusive method called as laser tagging method by photochromic dye has been developed with aim to study the breakup process of liquid sheet in detail, covering from the behavior in film until disintegrated into ligament and droplets. The laser tagging method by photochromic dye is based on a shift in the absorption spectrum of photochromic dye molecules tagged by ultraviolet laser. The shift results a color change at the tagged region of liquid containing the dye. In this study, the motions of the dye traces were analyzed as the liquid surface velocity. As a result, liquid sheet was found to keep its velocity constantly in film before suddenly increase around broken point. However, it then decreased after broken into droplets. By forming a set of four points of dye traces on the liquid sheet, the change of relative position of the set enabled the measurement of deformation and rotational motion of the liquid sheet. As a result, the normal strain of the liquid sheet parallel to the flow direction depended on the flow behavior of ligament formation.

A Nearest Neighbor Finite Element Method (NNFEM) for Validating Left-Ventricular Regional Strain from Displacement Encoding with Stimulated Echoes (DENSE) MRI, Compared to Tagged MRI

Cardiovascular magnetic resonance (CMR) is a magnetic resonance imaging (MRI) technique that is considered the most viable noninvasive technology for quantifying and visualizing regional myocardial function. CMR is expensive but characterized by higher spatial resolution and functional observations. The attribute of high spatial resolution allows quantitative assessment of cardiac wall motion and computation of transmural strains, allowing phenotyping cardiovascular physiopathologies [1-9]. Currently two CMR techniques are accepted as standard research practice which are 1. MRI tissue tagging (TMRI) [1-3] and 2. Stimulated echoes [6-9]. The first of the two, TMRI, is a method for tracking myocardial motion which places noninvasive markers (tags) within the tissue by locally induced perturbations of the magnetization. The altered magnetization shows as dark lines in the tagged region in successive images and myocardial deformation during the cardiac cycle is tracked [2,3]. However the intrinsic problem with tag lines is their fading after several cardiac phases. Hence, in addition to improvements toward longer tag persistence, parallel advancements in non-TMRI quantitative gradient technologies have also been made. One such technique is displacement encoding with stimulated echoes (DENSE) which directly encodes displacements into MRI phase data in three orthogonal phase encoding directions and facilitates rapid quantification of myocardial displacement through the cardiac cycle [6-9]. It is noted that while DENSE uses high displacement encoding frequencies resulting in phase wrapping, accurate measurements of displacements can be obtained using quality-guided spatio-temporal phase unwrapping algorithms [9].

Identification of AtWRKY75 as a transcriptional regulator in the defense response to Pcc through the screening of Arabidopsis activation-tagged lines

The necrotrophic pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc) causes soft rot in a broad range of plant hosts. Approximately 60,000 independent seeds from Arabidopsis activation tagging lines were inoculated with Pcc and screened for resistant mutants. An Rpe1 (resistance protein to Pectobacterium 1) mutant, which had more resistance to Pcc than wild-type (WT) plants, was selected for further study. The T-DNA inserting locus in Rpe1 was located on the middle of chromosome V by flanking sequence analysis. Through expression analysis with several genes adjacent to the T-DNA tagging region, AtWRKY75 gene was highly up-regulated in the Rpe1 mutant compared to the WT plant. The up-regulation of AtWRKY75 gene was shown to be correlated on the induction of the PDF1.2, VSP1 and PR1 genes compared to the WT plant. AtWRKY75 over-expression lines exhibited reduced Pcc bacterial growth compared to WT. Taken together, our data suggest that AtWRKY75 should be a positive regulator in the JA- or SA-mediated defense signaling responses to Pcc.

Regional effects of magnetization dispersion on quantitative perfusion imaging for pulsed and continuous arterial spin labeling

Most experiments assume a global transit delay time with blood flowing from the tagging region to the imaging slice in plug flow without any dispersion of the magnetization. However, because of cardiac pulsation, nonuniform cross-sectional flow profile, and complex vessel networks, the transit delay time is not a single value but follows a distribution. In this study, we explored the regional effects of magnetization dispersion on quantitative perfusion imaging for varying transit times within a very large interval from the direct comparison of pulsed, pseudo-continuous, and dual-coil continuous arterial spin labeling encoding schemes. Longer distances between tagging and imaging region typically used for continuous tagging schemes enhance the regional bias on the quantitative cerebral blood flow measurement causing an underestimation up to 37% when plug flow is assumed as in the standard model.

Near-Wall Motion of Caged Fluorescent Dye in Microchannel Flows Obtained from Evanescent Wave Molecular Tagging

A molecular tagging technique utilizing evanescent wave illumination was developed to investigate the motion of a caged fluorescent dye in the vicinity of the microchannel wall surface in electroosmotic and pressure-driven flows. A line pattern in a buffer solution was written by a pulsed UV laser and the uncaged dye was excited by the evanescent wave with total internal reflection inside the glass wall using an objective lens. The velocities calculated by the measured displacement of the near-wall tagged region were compared with the results of molecular tagging using volume illumination, which represents the bulk flow information. Concerning electroosmotic flow, the micro-PIV technique using a confocal microscope system was applied to the microchannel rinsed by the caged fluorescein beforehand in comparison with a pure glass-PDMS microchannel to examine the effect of dye adsorption to the wall on the electroosmotic mobility. The electroosmotic mobility obtained by evanescent wave molecular tagging (EWMT) showed close to the micro-PIV measurement result near the glass wall for the rinsed case and the uncaged dye at the almost constant velocity remained in the depthwise illumination region. On the other hand, the dye velocity in pressure-driven flow by EWMT increased rapidly with respect to time. The uncaged dye convected to the streamwise direction dispersed toward the wall due to the concentration gradient of the dye, which was confirmed by the numerical simulations.

The Effects of Flow Dispersion and Cardiac Pulsation in Arterial Spin Labeling

The blood in the carotid arteries exhibits time-varying flow velocity as a function of cardiac phases. Despite this flow velocity variation, most current methods set forth for the analysis of arterial spin labeling (ASL) data have assumed that the tagged blood is delivered from the tagging region to the imaging region via simple plug flow, i.e., a single transit delay (deltat). In this study, we used a pulse oximeter to synchronize image acquisition at systole and diastole separately. The deltat dispersion was modeled with a Gaussian distribution and the effect of cardiac pulsation upon the ASL signal was evaluated on five healthy volunteers. ASL signals were collected at a series of inflow times (TI) using PICORE QUIPSS II: TR/TE/TI1 = 2400/3.2/700 ms, TI = {300, 500, 700, 900, 1100, 1300, 1500} ms, matrix size = 64 x 64, repetition = 100. Transit delay was found significantly shorter in systolic tag than diastolic tag (paired student's t-test, p < 0.001; mean difference across subjects = 54 ms). When the tag was applied in late systole, the ASL signal arrived in the target brain slice earlier, and was higher by 16% with TI = 700 ms. Intervoxel dispersion (-350 ms) dominated over intravoxel dispersion (< 200 ms). The disparity of ASL signals found between systolic and diastolic tags indicated that ASL imaging was sensitive to cardiac pulsations. We conclude that both flow dispersion and fluctuations in the ASL signal due to cardiac pulsations are significant.

Comparison of multislice and single-slice acquisitions for pulsed arterial spin labeling measurements of cerebral perfusion

Multislice Q2TIPS is a widely used pulsed arterial spin labeling (PASL) technique for efficient and accurate quantification of cerebral blood flow (CBF). Slices are typically acquired inferior to superior from a tagging plane. Superior slices show signal loss greater than the loss expected from blood T1 decay. In order to assess the reasons for this additional signal loss, three single-slice acquisition studies were compared to multislice acquisition (six slices) in healthy volunteers. In Study 1 (n=8), the tagging plane was fixed in location, and the inversion time (TI2) was 1500 ms for each slice. For Study 2 (n=12), the tagging plane was fixed as in Study 1; however, TI2 increased as slices were acquired further from the tagging plane. In Study 3 (n=9), the tagging plane was kept adjacent to the imaging slice, and TI2 was 1500 ms for every slice. Gray matter (GM) and white matter (WM) signal-to-noise ratio (SNR) and CBF were measured per slice. GM SNR from single-slice acquisitions was significantly higher at slices 4-6 in Study 2 and at slices 2-6 in Study 3 compared to multislice acquisitions. Signal loss in distal slices of multislice acquisitions can be attributed to the destruction of tagged bolus in addition to blood T1 decay. If limited brain coverage is acceptable, perfusion images with greater SNR are achievable with limited slices and placement of the tagging region immediately adjacent to the site of interest.

Quantification of regional pulmonary blood flow using ASL‐FAIRER

Pulsed arterial spin labeling (ASL) techniques have been theoretically and experimentally validated for cerebral blood flow (CBF) quantification. In this study ASL-FAIRER was used to measure regional pulmonary blood flow (rPBF) in seven healthy subjects. Two general ASL strategies were investigated: 1) a single-subtraction approach using one tag-control pair acquisition at an inversion time (TI) matched to the RR-interval, and 2) a multiple-subtraction approach using tag-control pairs acquired at various TIs. The mean rPBF averaged 1.70 +/- 0.38 ml/min/ml when measured with the multiple-subtraction approach, and was approximately 2% less when measured with the single-subtraction method (1.66 +/- 0.24 ml/min/ml). Assuming an average lung density of 0.33 g/ml, this translates into a regional perfusion of approximately 5.5 ml/g/min, which is comparable to other measures of pulmonary perfusion. As with other ASL applications, a key problem with quantitative interpretation of the results is the physical gap between the tagging region and imaged slice. Because of the high pulsatility of PBF, ASL acquisition and data analysis differ significantly between the lung and the brain. The advantages and drawbacks of the single- vs. multiple-subtraction approaches are considered within a theoretical framework tailored to PBF.

Quantifying CBF with arterial spin labeling

The basic principles of measuring cerebral blood flow (CBF) using arterial spin labeling (ASL) are reviewed. The measurement is modeled by treating the ASL method as a magnetic resonance imaging (MRI) version of a microsphere study, rather than a diffusible tracer study. This approach, particularly when applied to pulsed ASL (PASL) experiments, clarifies that absolute calibration of CBF primarily depends on global properties of blood, rather than local tissue properties such as the water partition coefficient or relaxation time. However, transit delays from the tagging region to the image voxel are a potential problem in all standard ASL methods. The key to quantitative CBF measurements that compensate for this systematic error is to create a well-defined bolus of tagged blood and to ensure that all of the bolus has been delivered to an imaging voxel at the time of measurement. Two practical technical factors considered here are 1) producing a tagged bolus with a well-defined temporal width and 2) accounting for reduction in magnitude of the tagged magnetization due to relaxation. The ASL approach has the potential to provide a robust estimation of CBF, although the timing of water exchange into tissue and the effects of pulsatile flow require further investigation.

Spatially‐confined arterial spin‐labeling with FAIR

To investigate the effectiveness of slab-selective inversion in pulsed arterial spin labeling with body coil excitation as a means to reduce large vessel contamination of the perfusion signal. Studies were conducted by varying the tagging width in multislice flow-sensitive alternating inversion recovery (FAIR) in conjunction with body coil excitation on a Siemens Sonata whole-body 1.5-T scanner. The results of spatially-confined tagging were then compared with conventional nonselective tagging in the presence and absence of a bipolar gradient crusher pair in order to determine the effectiveness of suppressing vascular signal and to estimate the bolus width that reaches the capillary bed. It is shown in five volunteers, ages 23-38 years, that depending on the average velocity of the arterial blood flow in the tagging region, a bolus of 6-8 cm in width reaches the capillary bed at a fixed inversion time TI of 1.4 seconds, while a bolus of 11.2-16.5 cm in width enters the imaging region. Further, noticeable velocity differences have been found among the participating subjects, with averages ranging from 10.1 to 13.9 cm/second. The data suggest that it is advantageous to replace nonselective global tagging in FAIR perfusion imaging with body coil excitation by spatially-confined tagging to reduce undesired residual tagged blood in large vessels.

Two analytical solutions for a model of pulsed arterial spin labeling with randomized blood arrival times

A fairly general theoretical model for pulsed arterial spin labeling perfusion methods has been available for some time but analytical solutions were derived for only a small number of arterial blood input functions. These mostly assumed a sudden and simultaneous arrival of the tagged blood into the imaged region. More general cases had to be handled numerically. We present analytical solutions for two more realistic arterial input functions. They both allow the arrival times of the molecules of tagged arterial blood to be statistically distributed. We consider cases of (1) a uniform distribution on a finite time interval and (2) a normal distribution characterized by its mean and standard deviation. These models are physiologically meaningful because the statistical nature of the arrival times reflects the distribution of velocities and path lengths that the blood water molecules undertake from the tagging region to the imaged region. The model parameters can be estimated from the measured dependency of the perfusion signal on the tag inversion time.

Flow tagging velocimetry in a superorbital expansion tube

A non-intrusive laser-based method for direct velocity measurements has been demonstrated in a superorbital flow facility. The method is based upon laser enhanced ionisation velocimetry in which a tagged region is created by two step excitation of sodium and subsequent collisional ionisation. The achieved depletion of neutral atoms is then interrogated by planar laser induced fluorescence. The velocities were measured in the freestream at a superorbital condition yielding 9.20 +/- 0.11 km/s. These results compare favourably with the measured shock speeds in the facility.

MSL1 plays a central role in assembly of the MSL complex, essential for dosage compensation in Drosophila.

In male Drosophila, histone H4 acetylated at Lys16 is enriched on the X chromosome, and most X-linked genes are transcribed at a higher rate than in females (thus achieving dosage compensation). Five proteins, collectively called the MSLs, are required for dosage compensation and male viability. Here we show that one of these proteins, MSL1, interacts with three others, MSL2, MSL3 and MOF. The latter is a putative histone acetyl transferase. Overexpression of either the N- or C-terminal domain of MSL1 has dominant-negative effects, i.e. causes male-specific lethality. The lethality due to expression of the N-terminal domain is reduced if msl2 is co-overexpressed. MSL2 co-purifies over a FLAG affinity column with the tagged region of MSL1, and both MSL3 and MOF co-purify with the FLAG-tagged MSL1 C-terminal domain. Furthermore, the MSL1 C-terminal domain binds specifically to a GST-MOF fusion protein and co-immunoprecipitates with HA-tagged MSL3. The MSL1 C-terminal domain shows similarity to a region of mouse CBP, a transcription co-activator. We conclude that a main role of MSL1 is to serve as the backbone for assembly of the MSL complex.

QUIPSS II with thin-slice TI1 periodic saturation: A method for improving accuracy of quantitative perfusion imaging using pulsed arterial spin labeling

Quantitative imaging of perfusion using a single subtraction, second version (QUIPSS II) is a pulsed arterial spin labeling (ASL) technique for improving the quantitation of perfusion imaging by minimizing two major systematic errors: the variable transit delay from the distal edge of the tagged region to the imaging slices, and the contamination by intravascular signal from tagged blood that flows through the imaging slices. However, residual errors remain due to incomplete saturation of spins over the slab-shaped tagged region by the QUIPSS II saturation pulse, and spatial mismatch of the distal edge of the saturation and inversion slice profiles. By replacing the original QUIPSS II saturation pulse with a train of thin-slice periodic saturation pulses applied at the distal end of the tagged region, the accuracy of perfusion quantitation is improved. Results of single and multislice studies are reported.

QUIPSS II with thin‐slice TI1 periodic saturation: A method for improving accuracy of quantitative perfusion imaging using pulsed arterial spin labeling

Quantitative imaging of perfusion using a single subtraction, second version (QUIPSS II) is a pulsed arterial spin labeling (ASL) technique for improving the quantitation of perfusion imaging by minimizing two major systematic errors: the variable transit delay from the distal edge of the tagged region to the imaging slices, and the contamination by intravascular signal from tagged blood that flows through the imaging slices. However, residual errors remain due to incomplete saturation of spins over the slab-shaped tagged region by the QUIPSS II saturation pulse, and spatial mismatch of the distal edge of the saturation and inversion slice profiles. By replacing the original QUIPSS II saturation pulse with a train of thin-slice periodic saturation pulses applied at the distal end of the tagged region, the accuracy of perfusion quantitation is improved. Results of single and multislice studies are reported. Magn Reson Med 41:1246–1254, 1999. © 1999 Wiley-Liss, Inc.