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OSA Publishing > Biomedical Optics Express > Volume 11 > Issue 12 > Page 6812
Christoph Hitzenberger, Editor-in-Chief
Three-dimensional label-free imaging and quantification of migrating cells during wound healing
Ariel J. Lee, Herve Hugonnet, WeiSun Park, and YongKeun Park
Ariel J. Lee,1,2 Herve Hugonnet,1,2 WeiSun Park,1,2 and YongKeun Park1,2,3,*
1Department of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea
2KAIST Institute for Health Science and Technology, KAIST, Daejeon, Republic of Korea
3Tomocube Inc., Daejeon, Republic of Korea
*Corresponding author: yk.park@kaist.ac.kr
YongKeun Park https://orcid.org/0000-0003-0528-6661
H Hugonnet
W Park
Y Park
•https://doi.org/10.1364/BOE.405087
Ariel J. Lee, Herve Hugonnet, WeiSun Park, and YongKeun Park, "Three-dimensional label-free imaging and quantification of migrating cells during wound healing," Biomed. Opt. Express 11, 6812-6824 (2020)
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Label-free characterization of white blood cells by measuring 3D refractive index maps (BOE)
Correlative three-dimensional fluorescence and refractive index tomography: bridging the gap between molecular specificity and quantitative bioimaging (BOE)
Three-dimensional label-free observation of individual bacteria upon antibiotic treatment using optical diffraction tomography (BOE)
Table of Contents Category
Optical Biophysics and Photobiology
Diode pumped lasers
Phase imaging
Three dimensional imaging
Original Manuscript: August 10, 2020
Revised Manuscript: October 12, 2020
Manuscript Accepted: October 12, 2020
The wound-healing assay is a simple but effective tool for studying collective cell migration (CCM) that is widely used in biophysical studies and high-throughput screening. However, conventional imaging and analysis methods only address two-dimensional (2D) properties in a wound healing assay, such as gap closure rate. This is unfortunate because biological cells are complex 3D structures, and their dynamics provide significant information about cell physiology. Here, we presented 3D label-free imaging for wound healing assays and investigated the 3D dynamics of CCM using optical diffraction tomography. High-resolution subcellular structures as well as their collective dynamics were imaged and analyzed quantitatively.
© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
Most biological systems are composed of multiple cells. These collective cell systems involve complex interactions between cells, resulting in a completely different picture compared to single-cell activity [1]. Collective cell migration (CCM) includes multiple important phenomena, such as cancer progression, wound healing, and morphogenesis [2]. CCM has been widely studied using the wound healing model owing to its simplicity of sample generation and ease of analysis [3]. Furthermore, it provides an effective tool for high-throughput screening by observing the effect of various chemical treatments on wound healing speed [4]. Numerous studies have observed novel mechanical and molecular interactions between cells during the healing process using this assay [5,6].
The most common and conventional format of the wound healing model is the two-dimensional (2D) cell monolayer, and the migration of cells is usually imaged using 2D bright-field, phase-contrast, or fluorescence microscopy [7–9]. These imaging techniques are utilized to measure the wound healing rate based on the size of the wound or the total number of cells inside the initial wound area. Multiple algorithms and software programs have been developed for the analysis of 2D wound healing assay images [10,11]. However, these assay analyses do not consider the three-dimensional (3D) characteristic structures of the cells. The 3D structure and dynamics of subcellular organelles have not been addressed in the context of CCM in a wound healing assay. This is mainly because conventional imaging methods for the study of CCM do not consider 3D subcellular imaging of individual cells.
Here, we presented 3D label-free imaging and quantitative analysis of CCM in a wound healing assay. Exploiting optical diffraction tomography (ODT), a 3D quantitative phase imaging (QPI) technique [12], we demonstrated that refractive index (RI) tomogram measurements reveal the 3D high-resolution structures of individual cells in CCM. By stitching multiple 3D RI tomograms measured at various positions and times, we illustrated long-term and large-scale CCM. We studied both the overall shape and subcellular structures of individual cells using 3D RI information during a time-lapse. Multiple 3D quantities, such as the thickness of cells or RI distribution inside the nuclei in groups of cells with different chemical treatments or wound boundary locations, were explored. This new biophysical approach would readily enable various investigations of the wound healing mechanism, and how the cells react to various chemical signals.
2.1 Wound healing assay
NIH3T3 cells (ATCC CRL-1658) were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Invitrogen) 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a 10% CO2 incubator. Approximately 450,000 cells were seeded in TomoDish (Tomocube Inc., Daejeon, Korea), a specially designed cell culture dish to maximize the quality of the tomogram during ODT data acquisition. The TomoDish was coated using 0.01% poly-D-lysine for 15 min and washed 3 times with distilled water. After washing, it was fully dried and stored until use in the experiment. The cells were left to adhere to the dish surface and grow into a confluent monolayer for 28-30 h. The wound was formed by a scratch assay using a pipette tip. After scratching, the healing process of the wound was monitored using an ODT microscope. NIH3T3 cells were treated with 1 µg/mL Cytochalasin D (Cyto D; Sigma) was applied at 4 µm for 30 min, which was added to the cell medium at the start of the migration period.
2.2 Optical diffraction tomography
To measure the 3D RI tomograms of cells, we utilized ODT, also known as holotomography (HT). As an optical analogy to X-ray computed tomography, ODT exploits the RI as an intrinsic imaging contrast and reconstructs the 3D RI distribution of unlabeled cells [13–15]. Furthermore, ODT does not require the use of exogenous labeling agents or dyes. It not only greatly simplifies the experimental steps but also eliminates obstacles, such as photobleaching or phototoxicity, that originate from the limitations of labeling. This gives ODT an incomparable advantage in monitoring the healing process in 3D for an extended period compared to other imaging techniques.
The ODT measurements of live cells were performed since the cells were maintained at 37 °C and 10% CO2 using a live-cell imaging chamber (Tomochamber; Tomocube Inc, Daejeon, South Korea). ODT of wound healing assay cells was performed using a commercial ODT microscope (HT-2H; Tomocube Inc., Daejeon, South Korea). The ODT system used is based on a Mach-Zehnder interferometric microscope equipped with a digital micromirror device (DMD) [16]. A coherent laser beam from a diode-pumped solid-state laser (λ=532 nm) was used as the illumination source. The beam from the laser is split by a 2 × 2 single-mode fiber coupler. One beam illuminates a sample as a plane wave, and its incident angle is controlled by projecting time-multiplexed holograms onto the DMD [17]. Following this, the diffracted beam for the sample is imaged onto a camera plane, where it generates spatial interference patterns or holograms with a reference laser beam split from the fiber coupler.
For each hologram, both the amplitude and phase images are retrieved using a phase retrieval algorithm [18]. Subsequently, from multiple 2D optical holograms of a sample acquired with various illumination angles, the 3D RI distribution of the sample is reconstructed by inversely solving the Helmholtz equation with the Rytov approximation of weak scattering [14]. The theoretically calculated lateral and axial spatial resolutions of the optical imaging system used were 110 nm and 360 nm, respectively [19,20]. The required time for reconstruction is highly dependent on the computing power and field-of-view (FoV), but typically takes less than 10 seconds.
Owing to the limited numerical aperture of the condenser and objective lenses, side scattering signals are not collected, which results in the degradation of image quality in reconstructed 3D RI tomograms [21]. To address this missing cone problem, an iterative regularization algorithm based on the non-negative constraint was used. Details on the reconstruction procedure and algorithm can be found elsewhere [15,22].
2.3 Large field-of-view (FoV) imaging
The FoV of each 3D RI tomogram was 82 µm × 82 µm. To perform large FoV measurements, multiple 3D RI tomograms were measured at various lateral positions and stitched into one large FoV tomogram. To accomplish this, a motorized sample stage was synchronized with the image acquisition, and the stitching algorithm was used to seamlessly connect multiple 3D RI tomograms [23].
Multiple field area stitching for wide-field imaging was performed using a stitching function in commercial software (Tomostudio; Tomocube Inc.). The stitching algorithm used in the software generates stitched RI tomogram from individual smaller RI tomograms at each tile position. The algorithm consists of two steps. The first part of the algorithm calculates the displacement information of each tile consisting of the entire field of view. This is done by calculating the Pearson correlation coefficient from the phase correlation of the overlapping region of the tomogram between the neighboring tiles. The second part of the algorithm blends the overlapping region to create smooth boundaries. The blending method, or feathering, is a widely-used technique in computer graphics software and the window size of feathering is determined to be identical to the size of the overlapping area. The overlap area between each tile was 30% of the area of a single FoV. A large area of a sample (up to 1 mm × 1 mm) was imaged with a high resolution (down to 110 nm and 360 nm for the lateral and axial spatial resolutions, respectively) at a high frame rate (up to 3 − 5 tomograms per s for a FoV of 82 µm × 82 µm). Tomograms were taken every few minutes to hours.
The resulting large-size tomogram after stitching contains 3D high resolution information on wide area, which requires large size of data storage. Typically, a stitched tomogram consisting of 45 tiles with each tile having the FoV of 82 µm × 82 µm requires 3.80 GB of data storage for the hologram acquisition and 8.6 GB for the reconstructed and stitched tomogram storage (uint16 TIFF file type). The data size can be further reduced to 766 MG when using uint8 compressed TIFF file type, which does not degrade the image quality.
2.4 Cell parameter analysis
3D tomogram imaging by ODT can provide information about the RI in the sample. The volume and thickness of the cells were calculated by thresholding the RI tomogram with respect to the background RI value using a custom-written MATLAB code. The thickness calculated using this method thus directly relates to the thickness of the cell monolayer.
Cell nuclei RI histogram analysis was performed using the ImageJ Analyze-Histogram tool (National Institutes of Health [NIH], Bethesda, MD, USA) after selecting the nuclei region using the freehand selection tool [24]. The regions were selected at different depths in the tomogram to obtain volumetric data. The analysis was conducted on 15 cells at the wound boundary (leading cells) and 17 cells that were located at least 8 − 10 rows of cells away from the boundary (lagging cells).
2.5 Particle image velocimetry (PIV) analysis
PIV analysis was performed using the PIV lab software [25] written in MATLAB. It calculates the velocity information in multiple grid positions from 2D cross-section image pairs. It is based on a discrete Fourier transform technique that calculates the correlation matrix using the Fourier transform of each grid tile and finds the relative position between tiles at different time points. The resulting velocity vectors indicate the change in the cell position as a function of both time and position.
2.6 Statistical analysis
Distributions obtained for RI inside the nuclei of cells in different categories were compared using the two-sample t-test. The shaded error bars in the thickness graph and nuclei RI histogram represent the standard deviation (SD). In the RI distribution graphs, the upper and lower box ranges indicate the 25th and 75th percentiles, respectively.
3.1 Optical diffraction tomography imaging of the collectively migrating cells
To demonstrate 3D label-free, large-scale FoV imaging of a wound healing assay, the CCM of NIH3T3 fibroblasts was measured (Fig. 1; see Methods). From the 2D multiple holograms measured at various illumination angles, the amplitude and phase images were retrieved using the phase retrieval algorithm (Fig. 1(A)). Following this, the 3D RI tomogram of a sample was reconstructed using the ODT algorithm. To cover a large FoV, multiple RI tomograms were measured at various lateral positions, from which the 3D RI tomogram with a large FoV was stitched.
Fig. 1. Wide-field imaging of a wound healing assay using optical diffraction tomography (ODT). (A) The reconstruction process of ODT. Multiple holograms are acquired and processed to retrieve the amplitude and phase delay information of the light passing through the sample. The tomogram reconstruction algorithm generates a three-dimensional refractive index (RI) map, which provides quantitative information from the sample. Then, the stitching algorithm based on the correlation method generates a single wide-field RI tomogram using multiple single-field RI tomograms. (B) Maximum intensity projection (MIP) image of a wide-field RI tomogram of a wound healing assay. NIH3T3 cells are collectively migrating into the wound area in the middle, which is an empty space generated by scratching with a pipette tip. (C) A lateral cross-section image of a RI tomogram shows NIH3T3 cells migrating towards the wounded region on the right. The lateral resolution of the imaging system is down to 110 nm, which allows for imaging of various subcellular organelles as shown in the region of interest (ROI) 1. The wound area has no cells, and the cells on the wound boundary form migrating structures as shown in ROI 2. The cell membrane, cytoskeletons, and subcellular organelles all have different morphologies and distributions for different axial positions. Image planes of two different heights are shown for ROI 2.
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The reconstructed large-scale RI tomogram of NIH3T3 cells presented large-scale, high-resolution volumetric imaging. Figure 1(B) is a representative image with the total FoV covering both sides of the wound (670 µm × 1,120 µm). Figure 1(C) shows the left side of a wound where the cells collectively migrate towards the right. The maximum intensity projection (MIP) along the axial direction visualizes the overall distribution of cells (Fig. 1(B)).
Unlike conventional imaging methods, HT enables the multiscale tomographic imaging of multicell systems with a large FoV covering both sides of the wound and down to high-resolution subcellular features (Fig. 1(C)). The cell membrane, nucleus membrane, nucleoli, mitochondria, and vesicles with high RI values were distinctly visualized (regions of interest [ROIs] 1 and 2 in Fig. 1(C)) without introducing any exogenous labeling. The magnified image of ROI 1 showed the cells that were away from the wound boundary, or so-called lagging cells. Here, the cells were surrounded by other cells, which resulted in a completely different morphology from the leading cells in ROI 2. At z = 0 µm (the bottom of the cell), the complex distribution of cytoskeletons around and at the bottom of the nuclei were clearly visible. The cytoskeletons were also located around the nuclei at z = 1.5 µm, but at this height, they expanded only to the closer peripheral area of the nuclei. Formation of lamellipodia was observed in both cross-sections, showing wrinkled cellular membranes where the cells extended out to the empty wound area. A high RI (bright pixels) was usually observed around and inside the nuclei. These results demonstrate the validity and applicability of HT in the study of CCM.
3.2 Monitoring the thickness of the cell layer
Optical diffraction tomography imaging allows for direct and exact examination of the z-directional thickness of the sample based on 3D quantitative information from the RI. 2D QPI techniques, such as digital holography, have been used for wound healing assays. However, 2D QPI techniques only measure the optical thickness, a coupled parameter between the physical thickness and RI [26,27], whereas ODT decouples physical thickness from a direct tomographic measurement and provides precise and quantitative thickness information about cells.
The thickness of the migrating cell monolayer was retrieved from a single tomogram as shown in Fig. 2(A). The thickness of the region surrounded by a dotted box at multiple time points is shown in Fig. 2(B). The lateral position describes the distance from the left end, and each thickness value is calculated from the YZ MIP image. The wound boundary created by scratching the confluent cell layer has regions with some cells that are jammed together. As time passes, these closely packed cells return to the ground. This overall process is shown in the time-lapse thickness data. There initially exists a peak on the frontmost borderline, and then the height gradually decreases with time. In addition, the tendency of leading cells to reach out to the empty wound area and start migration is illustrated by the consistently decreasing slope of the thickness graph line of the frontmost cell.
Fig. 2. Thickness of the cell monolayer changes over time. (A) Maximum intensity projection (MIP) images from a three-dimensional refractive index tomogram of a wound healing assay. The wound region without any cells is created in the center. The dotted box specifies the boundary region of the wound. Cells inside the dotted box are leading cells, which actively migrate toward the wound area. (B) Thickness value of the cell monolayer is calculated from the YZ MIP image at various time points.
3.3 Quantification of wound healing progress using chemical treatments
To perform 3D tomographic imaging and quantification of migrating cells upon chemical treatment, the physical thickness and 2D migration of individual cells were measured and analyzed simultaneously.
Quantification of wound healing progress or the amount of cell migration can be accomplished using the thickness distribution information of the cell layer in addition to the widely used 2D migration distance information. Figure 3(A) shows multiple MIP images of tomograms taken over a time-lapse as the cells migrate towards the empty wound area, which in this case is the upper direction. The control and Cyto D-treated groups were wounded, and their recovery processes were observed. The ODTs are visualized using 2D MIP images, which are similar to the imaging results from other widely used imaging methods, such as bright-field or fluorescence imaging. In addition, ODT can provide 2D images from any point of view using 3D tomographic data. For example, the YZ MIP images on the rightmost side in Fig. 3(A) demonstrate how the cells are migrating to heal the wound from a unique viewpoint. Based on these data, Fig. 3(B) shows the migration distance of cells from each group. As expected, the Cyto D-treated group showed a much slower migration speed than the control group.
Fig. 3. Wound healing process analysis in terms of three-dimensional morphology. (A) Wound healing progress shown in a refractive index tomogram for each control and Cytochalasin D (Cyto D)-treated group of cells. (B) Migration distance is compared for the control and Cyto D-treated cells. (C) Thickness values are calculated and compared. The shaded area represents the standard deviation of the thickness values of the points in the same lateral position. Thickness values in each lateral position do not significantly change after Cyto D treatment. (D) Change in the total standard deviation of thickness values is much smaller for Cyto D-treated cells.
The thickness values were also calculated, and their distributions were studied over time. The average thickness values of the points at the same lateral positions are represented by the solid line and the standard deviation as the shaded area. Thus, the effect of some empty areas between the cells can be reduced and allows for examination of the overall trend of thickness change over time. The standard deviation of all thickness values of each pixel point reflects how much the cells migrate and fill the empty spaces between the cells as well as how much the cells change their shape and become attached to the ground. Over time, the value tends to decrease as shown in Fig. 3(D), and the slope is much steeper in the control group compared to the Cyto D-treated group.
3.4 Nuclei of leading and lagging cells exhibit different RI distributions
Several previous studies have been conducted regarding changes in nuclear structure during cell migration. A number of them have revealed that directed cell migration is closely related to the condensation of chromatin fibers and shape of the nucleus [28–30]. In addition, it has been determined that the ratio of euchromatin to heterochromatin is a factor in determining nuclear stiffness and affects cell migration, which requires further investigation [31–33]. Other studies identified a link between the nucleoli and the extracellular force applied to the cell [34] as well as the role of A-type lamin for sustaining directed cell migration [35].
To demonstrate the capability of the present method, the nuclei of leading cells actively migrating towards the wound boundary and lagging cells surrounded by other cells far behind the wound boundary were compared. In Fig. 4(A), representative images of lagging and leading cell nuclei are shown as yellow solid circles and red dotted circles, respectively. The RI of a cell and its nuclei are strongly inhomogeneous (Fig. 4(B)). Higher RIs exist mainly inside or near the nucleus of a cell. For further investigation, multimodal ODT and fluorescence imaging were conducted to confirm the range of the RI distributions, which correlated well with some of the well-known molecular contents inside the nucleus. Hoechst dye, a blue fluorescent dye widely used for labeling DNA [36], showed a good correlation with the RI values from 1.364 to 1.373.
Fig. 4. Refractive index (RI) distribution inside the nuclei of cells near and away from the wound boundary. (A) Yellow solid circles represent the nuclei of lagging cells, whereas red dotted circles represent the nuclei of leading cells. The nuclear membrane is visible as a bright, thin circular shape in each cell. (B) RI tomogram visualization using different-colored voxels within certain RI ranges. The regions with RI values from 1.364 to 1.373 and the Hoechst fluorescence signal distributions overlap. (C1) RI histogram inside the volume of each nucleus within lagging and leading cells. (C2) Lagging cells have a higher portion of RI values in the low RI region than leading cells. On the contrary, leading cells have a higher portion of RI values in the high RI region than lagging cells. (C3) Cellular dry mass and cellular volume were calculated and compared between the lagging and leading cells. There is no meaningful difference between the two groups.
For systematic comparison, the RI histograms inside the 3D volume of nuclei in lagging and leading cells were compared (Fig. 4(C1)). The lagging and leading cells had opposite dominance in the low and high RI regions inside their nuclei (Fig. 4(C2)). The results showed that a higher RI range was included in the RI range that showed a good correlation with the Hoechst signal (1.364-1.373). The data demonstrated that the leading cells had more regions in their nuclei with higher RI values, which correlated well with the fluorescence signal labeling DNA. The RI values provide quantitative information on how optically densely the molecules are packed together. The dry mass, which is the total amount of molecules that exist inside each cell, and the volume of each cell revealed a negligible difference in the groups as shown in Fig. 4(C3). This data implies that leading cells have more densely packed DNA in their nuclei than lagging cells.
Overall, the aforementioned results suggest that the leading cells in directed migration motion have more condensed contents, including DNA, which constitute the higher RI regions inside their nuclei. This is further evidence that environmental cues alter the morphology of the nuclei and are related to the way cells react to them. Although RI tomograms do not generally have molecular specificity when compared to fluorescence or other labeling methods, they provide quantitative information about the whole nuclei without missing other unlabeled molecules. In addition, the RI values directly relate to how densely molecules are packed compared to previous studies that were necessary to find a quantity that indirectly represents the level of condensation of certain molecules. Some studies have proposed indices that are calculated indirectly based on thresholding or edge detection in the fluorescence image, but they fundamentally depend on the intensity of the fluorescence signals in the image [37,38].
3.5 Multilayer particle image velocimetry
To further demonstrate the potential of the present approach, we analyzed the collective motions of cells at various axial positions using PIV (Fig. 5). The boundary where the cells protrude from their original position to the empty area was examined at various depths. Figure 5(A) shows the results in which the green vectors indicate the migration velocity at each grid point and the yellow lines are the streamlines. From 0.5-1 h after scratching, most of the cells remained, except for the leading cells. The leading cells clearly showed the largest migration velocity, and their movement was larger near the bottom coverslip surface. The migration velocity and its directionality toward the wound area become larger as time passed, making the streamline perpendicular to the wound edge. Figure 5(B) represents the average map of all velocity vector map results at different time steps on the plane with a height of z = 2.34 µm. The point where all of the average streamlines start was where the highest number of cells per volume was located. This illustrated the pushing forces in the compact region between adjacent cells in addition to the different dynamics of leading and lagging cells. The migration velocity vectors (Fig. 5(C)) demonstrated an asymmetric distribution concerning the origin pointing toward the migration direction.
Fig. 5. Particle image velocimetry (PIV) analysis on cell migration using optical diffraction tomography. (A) PIV analysis of different planes of tomograms at various timepoints. Each green arrow shows the migration velocity at each grid point, and the yellow lines are the streamlines. They differ both temporally and spatially. Velocity magnitudes are generally larger in the lower part of the cells that are nearer to the wound boundary. The streamlines are initially aligned orthogonal to the migration direction but gradually become parallel over time in all planes. (B) Average velocity map and average streamline for all timepoints at z = 2.34 µm. The leader cell is characterized as the cell with the largest velocity magnitudes on the map. (C) The velocity vectors are plotted in a single graph. The vertical velocity distribution is asymmetric with respect to zero, showing that the cells are migrating toward the wound area in total.
To perform 3D label-free imaging and quantification of a wound healing assay, ODT imaging, synchronization of a motorized sample stage, and a stitching algorithm were combined. With the present approach, we investigated the wound healing mechanism in confluent monolayer NIH3T3 cells. This method enables the measurement of various biophysical parameters in a wound healing assay, which are inaccessible or challenging to access with conventional methods. We visualized and quantified individual migrating cells and their subcellular organelles. The physical thicknesses were directly retrieved from the tomographic measurements. Migration of cells at multiple depth positions was proven, which clearly demonstrates that cellular migration and associated subcellular movements in a wound healing model exhibit 3D characteristics, even for monolayered cells. Furthermore, the time-lapse measurement of the thickness of a confluent monolayer from the 3D tomogram data showed the effects of Cyto D treatment. Finally, the RIs inside the nuclei were distributed differently in leading cells compared to lagging cells.
Thus far, 2D QPI techniques, such as digital holographic microscopy, have been applied to the study of cell migration or wound healing assays [39–42]. Although the use of 2D QPI techniques has included label-free and quantitative imaging capabilities, only limited 2D optical thickness information can be measured, and the 3D morphology of individual cells and subcellular structures is not accessible.
In this study, we presented label-free tomographic imaging and quantification of a wound healing assay and illustrated the migration of NIH3T3 cells. We also demonstrated the proof of principle and expect that the current approach can be further exploited in various biophysical studies to stimulate new research approaches. For example, tomographic subcellular information in a migration assay will also promote the development of the theoretical modeling of wound healing [43]. There have been numerous types of research on understanding the fundamentals of collective cell migration using complex models and hypothesis. These models frequently utilize several biophysical parameters such as cellular polarity, center of mass, cell shape, and density [44–46]. These types of parameters are difficult to measure using conventional imaging methods. Thus, it was necessary to find alternative variables that are measurable through experiments to verify their theoretical predictions. However, when utilizing the proposed measurement method, these biophysical parameters can be directly derived from the refractive index and dry mass tomogram. Giving access to direct measurement of parameters that are widely used in various collective cell migration models through the presented method will allow more precise verification of these models and help develop more accurate theories that explain collective cell migration. In addition, as scientists' interest in the cell nucleus rotation or its morphology during cell migration increases, further examination of the RI inside the nuclei and other analyses become possible using the ODT wound healing data accompanied by quantitative analysis using the dry mass calculation from the raw data. Furthermore, our work was limited to 2D wound healing assays, but 2.5D or 3D wound healing models are readily accessible for examination using ODT. The proposed measurement method could be used on visualizing and studying the 3D collective cell migration and filopodia motion in hydrogel 3D wound healing assay or 3D cell culture. Previously, there were various studies observing the 3D cell migration process in 3D environment via fluorescence labeling [47–49]. Similar approach can be done for this method, but instead using the RI as the imaging contrast. This allows direct imaging of how cells migrate in a 3D extracellular matrix without labeling, and thus enables to circumvent problems related to the photobleaching effects of fluorescence molecules. Also, the observation of formations and motions of filopodia in 3D environments can be done by imaging with a shorter time interval, and several biophysical parameters such as RI or center of mass could be compared with the cells migrating in 2D environments. Finally, 3D PIV analysis combined with a 3D phase correlation algorithm would allow direct visualization of how leading cells protrude their pseudopods and move towards the empty area.
In sum, the present method provides a unique opportunity to study the behavior of migrating cells during the wound healing process. Our method would also be of particular interest to the pharmaceutical industry for drug discovery. It has a unique ability to examine 3D dynamics over a wide FoV for a long period of time and without any labeling. This provides an opportunity to study and analyze wound healing mechanisms using a new viewpoint. Studying wound healing models in a 3D environment by applying this technology will allow for a better and more systematic understanding of CCM as well as the wound healing mechanism.
National Research Foundation of Korea (2015R1A3A2066550, 2017M3C1A3013923, 2018K000396); BK21+ program; Tomocube; KAIST.
F (Financial Support): This work is partially supported by the financial support received from Tomocube Inc. I (Personal Financial Interest): Y.P. declare the employment by Tomocube Inc. E (Employment): Y.P. declare the employment by Tomocube Inc.
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17. S. Shin, K. Kim, J. Yoon, and Y. Park, "Active illumination using a digital micromirror device for quantitative phase imaging," Opt. Lett. 40(22), 5407–5410 (2015). [CrossRef]
18. S. K. Debnath and Y. Park, "Real-time quantitative phase imaging with a spatial phase-shifting algorithm," Opt. Lett. 36(23), 4677–4679 (2011). [CrossRef]
19. V. Lauer, "New approach to optical diffraction tomography yielding a vector equation of diffraction tomography and a novel tomographic microscope," J. Microsc. 205(2), 165–176 (2002). [CrossRef]
20. C. Park, S. Shin, and Y. Park, "Generalized quantification of three-dimensional resolution in optical diffraction tomography using the projection of maximal spatial bandwidths," J. Opt. Soc. Am. A 35(11), 1891–1898 (2018). [CrossRef]
21. J. Lim, K. Lee, K. H. Jin, S. Shin, S. Lee, Y. Park, and J. C. Ye, "Comparative study of iterative reconstruction algorithms for missing cone problems in optical diffraction tomography," Opt. Express 23(13), 16933–16948 (2015). [CrossRef]
22. K. Kim, H. Yoon, M. Diez-Silva, M. Dao, R. R. Dasari, and Y. Park, "High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography," J. Biomed. Opt. 19, 011005 (2013).
23. H. Hugonnet, Y. W. Kim, M. Lee, S. Shin, R. H. Hruband, S. Hong, and Y. Park, "Multiscale label-free volumetric holographic histopathology of thick-tissue slides with subcellular resolution," BioRxiv, 2020.2007.2015.205633 (2020).
24. J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, "Fiji: an open-source platform for biological-image analysis," Nat. Methods 9(7), 676–682 (2012). [CrossRef]
25. W. Thielicke and E. Stamhuis, "PIVlab – Towards User-friendly, Affordable and Accurate Digital Particle Image Velocimetry in MATLAB," J. Open Res. Software 2(1), e30 (2014). [CrossRef]
26. D. Bettenworth, P. Lenz, P. Krausewitz, M. Brückner, S. Ketelhut, D. Domagk, and B. Kemper, "Quantitative stain-free and continuous multimodal monitoring of wound healing in vitro with digital holographic microscopy," PLoS One 9(9), e107317 (2014). [CrossRef]
27. D. Bettenworth, A. Bokemeyer, C. Poremba, N. S. Ding, S. Ketelhut, P. Lenz, and B. Kemper, "Quantitative phase microscopy for evaluation of intestinal inflammation and wound healing utilizing label-free biophysical markers," Histol. Histopathol 33, 417–432 (2018).
28. G. Gerlitz, I. Livnat, C. Ziv, O. Yarden, M. Bustin, and O. Reiner, "Migration cues induce chromatin alterations," Traffic 8(11), 1521–1529 (2007). [CrossRef]
29. G. Gerlitz and M. Bustin, "The role of chromatin structure in cell migration," Trends Cell Biol. 21(1), 6–11 (2011). [CrossRef]
30. G. Gerlitz and M. Bustin, "Efficient cell migration requires global chromatin condensation," J. Cell Sci. 123(13), 2207–2217 (2010). [CrossRef]
31. F. J. Calero-Cuenca, C. S. Janota, and E. R. Gomes, "Dealing with the nucleus during cell migration," Curr. Opin. Cell Biol. 50, 35–41 (2018). [CrossRef]
32. J. D. Pajerowski, K. N. Dahl, F. L. Zhong, P. J. Sammak, and D. E. Discher, "Physical plasticity of the nucleus in stem cell differentiation," Proc. Natl. Acad. Sci. 104(40), 15619–15624 (2007). [CrossRef]
33. F. Erdel, M. Baum, and K. Rippe, "The viscoelastic properties of chromatin and the nucleoplasm revealed by scale-dependent protein mobility," J. Phys.: Condens. Matter 27(6), 064115 (2015). [CrossRef]
34. A. J. Maniotis, C. S. Chen, and D. E. Ingber, "Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure," Proc. Natl. Acad. Sci. 94(3), 849–854 (1997). [CrossRef]
35. F. Houben, C. H. M. P. Willems, I. L. J. Declercq, K. Hochstenbach, M. A. Kamps, L. H. E. H. Snoeckx, F. C. S. Ramaekers, and J. L. V. Broers, "Disturbed nuclear orientation and cellular migration in A-type lamin deficient cells," Biochimica et Biophysica Acta (BBA) - Molecular Cell Res. 1793(2), 312–324 (2009). [CrossRef]
36. J. Bucevicius, G. Lukinavicius, and R. Gerasimaite, "The Use of Hoechst Dyes for DNA Staining and beyond," Chemosensors 6(2), 18 (2018). [CrossRef]
37. J. Sosnik, W. A. Vieira, K. A. Webster, K. R. Siegfried, and C. D. McCusker, "A new and improved algorithm for the quantification of chromatin condensation from microscopic data shows decreased chromatin condensation in regenerating axolotl limb cells," PLoS One 12(10), e0185292 (2017). [CrossRef]
38. J. Irianto, D. A. Lee, and M. M. Knight, "Quantification of chromatin condensation level by image processing," Med. Eng. & Phys. 36(3), 412–417 (2014). [CrossRef]
39. O. Tolde, A. Gandalovičová, A. Křížová, P. Veselý, R. Chmelík, D. Rosel, and J. Brábek, "Quantitative phase imaging unravels new insight into dynamics of mesenchymal and amoeboid cancer cell invasion," Sci. Rep. 8(1), 12020–13 (2018). [CrossRef]
40. P. Guo, J. Huang, and M. A. Moses, "Quantitative phase imaging characterization of tumor-associated blood vessel formation on a chip," in Quantitative Phase Imaging IV (International Society for Optics and Photonics, 2018), p. 105031O.
41. P. Langehanenberg, L. Ivanova, I. Bernhardt, S. Ketelhut, A. Vollmer, D. Dirksen, G. K. Georgiev, G. von Bally, and B. Kemper, "Automated three-dimensional tracking of living cells by digital holographic microscopy," J. Biomed. Opt. 14(1), 014018 (2009). [CrossRef]
42. M. Rezaei, J. Cao, K. Friedrich, B. Kemper, O. Brendel, M. Grosser, M. Adrian, G. Baretton, G. Breier, and H.-J. Schnittler, "The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor–endothelial cell interactions," Histochem. Cell Biol. 149(1), 15–30 (2018). [CrossRef]
43. J. C. Arciero, Q. Mi, M. F. Branca, D. J. Hackam, and D. Swigon, "Continuum model of collective cell migration in wound healing and colony expansion," Biophys. J. 100(3), 535–543 (2011). [CrossRef]
44. R. Alert and X. Trepat, "Physical Models of Collective Cell Migration," Annu. Rev. Condens. Matter Phys. 11(1), 77–101 (2020). [CrossRef]
45. T. Ohta, M. Tarama, and M. Sano, "Simple model of cell crawling," Phys. D 318-319, 3–11 (2016). [CrossRef]
46. B. A. Camley and W.-J. Rappel, "Physical models of collective cell motility: from cell to tissue," J. Phys. D: Appl. Phys. 50(11), 113002 (2017). [CrossRef]
47. L. K. Fritz-Laylin, M. Riel-Mehan, B.-C. Chen, S. J. Lord, T. D. Goddard, T. E. Ferrin, S. M. Nicholson-Dykstra, H. Higgs, G. T. Johnson, E. Betzig, and R. D. Mullins, "Actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes," eLife 6, e26990 (2017). [CrossRef]
48. Y. Xiao, R. Riahi, P. Torab, D. D. Zhang, and P. K. Wong, "Collective Cell Migration in 3D Epithelial Wound Healing," ACS Nano 13, 1204–1212 (2019).
49. M. Anguiano, C. Castilla, M. Maška, C. Ederra, R. Peláez, X. Morales, G. Muñoz-Arrieta, M. Mujika, M. Kozubek, A. Muñoz-Barrutia, A. Rouzaut, S. Arana, J. M. Garcia-Aznar, and C. Ortiz-de-Solorzano, "Characterization of three-dimensional cancer cell migration in mixed collagen-Matrigel scaffolds using microfluidics and image analysis," PLoS One 12(2), e0171417 (2017). [CrossRef]
Article Order
M. Sadati, N. Taheri Qazvini, R. Krishnan, C. Y. Park, and J. J. Fredberg, "Collective migration and cell jamming," Differentiation 86(3), 121–125 (2013).
[Crossref]
P. Friedl and D. Gilmour, "Collective cell migration in morphogenesis, regeneration and cancer," Nat. Rev. Mol. Cell Biol. 10(7), 445–457 (2009).
A. Grada, M. Otero-Vinas, F. Prieto-Castrillo, Z. Obagi, and V. Falanga, "Research Techniques Made Simple: Analysis of Collective Cell Migration Using the Wound Healing Assay," J. Invest. Dermatol. 137(2), e11–e16 (2017).
K. Muniandy, S. Gothai, W. S. Tan, S. S. Kumar, N. Mohd Esa, G. Chandramohan, K. S. Al-Numair, and P. Arulselvan, "In Vitro Wound Healing Potential of Stem Extract of Alternanthera sessilis," Evid. Based Complement Alternat. Med. 2018, 1–13 (2018).
R. Riahi, J. Sun, S. Wang, M. Long, D. D. Zhang, and P. K. Wong, "Notch1–Dll4 signalling and mechanical force regulate leader cell formation during collective cell migration," Nat. Commun. 6(1), 6556 (2015).
C. Plutoni, E. Bazellieres, M. Le Borgne-Rochet, F. Comunale, A. Brugues, M. Séveno, D. Planchon, S. Thuault, N. Morin, S. Bodin, X. Trepat, and C. Gauthier-Rouvière, "P-cadherin promotes collective cell migration via a Cdc42-mediated increase in mechanical forces," The Journal of Cell Biology 212(2), 199–217 (2016).
A. Stamm, K. Reimers, S. Strauß, P. Vogt, T. Scheper, and I. Pepelanova, "In vitro wound healing assays - State of the art," BioNanoMaterials 17(1-2), 79–87 (2016).
C.-C. Liang, A. Y. Park, and J.-L. Guan, "In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro," Nat. Protoc. 2(2), 329–333 (2007).
P. Y. K. Yue, E. P. Y. Leung, N. K. Mak, and R. N. S. Wong, "A Simplified Method for Quantifying Cell Migration/Wound Healing in 96-Well Plates," J. Biomol. Screening 15(4), 427–433 (2010).
A. Zaritsky, S. Natan, J. Horev, I. Hecht, L. Wolf, E. Ben-Jacob, and I. Tsarfaty, "Cell motility dynamics: a novel segmentation algorithm to quantify multi-cellular bright field microscopy images," PLoS One 6(11), e27593 (2011).
T. Gebäck, M. M. P. Schulz, P. Koumoutsakos, and M. Detmar, "TScratch: a novel and simple software tool for automated analysis of monolayer wound healing assays," BioTechniques 46(4), 265–274 (2009).
Y. Park, C. Depeursinge, and G. Popescu, "Quantitative phase imaging in biomedicine," Nat. Photonics 12(10), 578–589 (2018).
D. Kim, S. Lee, M. Lee, J. Oh, S.-A. Yang, and Y. Park, "Refractive index as an intrinsic imaging contrast for 3-D label-free live cell imaging," BioRxiv, 106328 (2017).
E. Wolf, "Three-dimensional structure determination of semi-transparent objects from holographic data," Opt. Commun. 1(4), 153–156 (1969).
K. Kim, J. Yoon, S. Shin, S. Lee, and S.-A. Yang, "Optical diffraction tomography techniques for the study of cell pathophysiology," Journal of Biomedical Photonics Engineering 2, 2 (2016).
S. Shin, K. Kim, T. Kim, J. Yoon, K. Hong, J. Park, and Y. Park, "Optical diffraction tomography using a digital micromirror device for stable measurements of 4D refractive index tomography of cells," in Quantitative Phase Imaging II (International Society for Optics and Photonics, 2016), p. 971814.
S. Shin, K. Kim, J. Yoon, and Y. Park, "Active illumination using a digital micromirror device for quantitative phase imaging," Opt. Lett. 40(22), 5407–5410 (2015).
S. K. Debnath and Y. Park, "Real-time quantitative phase imaging with a spatial phase-shifting algorithm," Opt. Lett. 36(23), 4677–4679 (2011).
V. Lauer, "New approach to optical diffraction tomography yielding a vector equation of diffraction tomography and a novel tomographic microscope," J. Microsc. 205(2), 165–176 (2002).
C. Park, S. Shin, and Y. Park, "Generalized quantification of three-dimensional resolution in optical diffraction tomography using the projection of maximal spatial bandwidths," J. Opt. Soc. Am. A 35(11), 1891–1898 (2018).
J. Lim, K. Lee, K. H. Jin, S. Shin, S. Lee, Y. Park, and J. C. Ye, "Comparative study of iterative reconstruction algorithms for missing cone problems in optical diffraction tomography," Opt. Express 23(13), 16933–16948 (2015).
K. Kim, H. Yoon, M. Diez-Silva, M. Dao, R. R. Dasari, and Y. Park, "High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography," J. Biomed. Opt. 19, 011005 (2013).
H. Hugonnet, Y. W. Kim, M. Lee, S. Shin, R. H. Hruband, S. Hong, and Y. Park, "Multiscale label-free volumetric holographic histopathology of thick-tissue slides with subcellular resolution," BioRxiv, 2020.2007.2015.205633 (2020).
J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, "Fiji: an open-source platform for biological-image analysis," Nat. Methods 9(7), 676–682 (2012).
W. Thielicke and E. Stamhuis, "PIVlab – Towards User-friendly, Affordable and Accurate Digital Particle Image Velocimetry in MATLAB," J. Open Res. Software 2(1), e30 (2014).
D. Bettenworth, P. Lenz, P. Krausewitz, M. Brückner, S. Ketelhut, D. Domagk, and B. Kemper, "Quantitative stain-free and continuous multimodal monitoring of wound healing in vitro with digital holographic microscopy," PLoS One 9(9), e107317 (2014).
D. Bettenworth, A. Bokemeyer, C. Poremba, N. S. Ding, S. Ketelhut, P. Lenz, and B. Kemper, "Quantitative phase microscopy for evaluation of intestinal inflammation and wound healing utilizing label-free biophysical markers," Histol. Histopathol 33, 417–432 (2018).
G. Gerlitz, I. Livnat, C. Ziv, O. Yarden, M. Bustin, and O. Reiner, "Migration cues induce chromatin alterations," Traffic 8(11), 1521–1529 (2007).
G. Gerlitz and M. Bustin, "The role of chromatin structure in cell migration," Trends Cell Biol. 21(1), 6–11 (2011).
G. Gerlitz and M. Bustin, "Efficient cell migration requires global chromatin condensation," J. Cell Sci. 123(13), 2207–2217 (2010).
F. J. Calero-Cuenca, C. S. Janota, and E. R. Gomes, "Dealing with the nucleus during cell migration," Curr. Opin. Cell Biol. 50, 35–41 (2018).
J. D. Pajerowski, K. N. Dahl, F. L. Zhong, P. J. Sammak, and D. E. Discher, "Physical plasticity of the nucleus in stem cell differentiation," Proc. Natl. Acad. Sci. 104(40), 15619–15624 (2007).
F. Erdel, M. Baum, and K. Rippe, "The viscoelastic properties of chromatin and the nucleoplasm revealed by scale-dependent protein mobility," J. Phys.: Condens. Matter 27(6), 064115 (2015).
A. J. Maniotis, C. S. Chen, and D. E. Ingber, "Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure," Proc. Natl. Acad. Sci. 94(3), 849–854 (1997).
F. Houben, C. H. M. P. Willems, I. L. J. Declercq, K. Hochstenbach, M. A. Kamps, L. H. E. H. Snoeckx, F. C. S. Ramaekers, and J. L. V. Broers, "Disturbed nuclear orientation and cellular migration in A-type lamin deficient cells," Biochimica et Biophysica Acta (BBA) - Molecular Cell Res. 1793(2), 312–324 (2009).
J. Bucevicius, G. Lukinavicius, and R. Gerasimaite, "The Use of Hoechst Dyes for DNA Staining and beyond," Chemosensors 6(2), 18 (2018).
J. Sosnik, W. A. Vieira, K. A. Webster, K. R. Siegfried, and C. D. McCusker, "A new and improved algorithm for the quantification of chromatin condensation from microscopic data shows decreased chromatin condensation in regenerating axolotl limb cells," PLoS One 12(10), e0185292 (2017).
J. Irianto, D. A. Lee, and M. M. Knight, "Quantification of chromatin condensation level by image processing," Med. Eng. & Phys. 36(3), 412–417 (2014).
O. Tolde, A. Gandalovičová, A. Křížová, P. Veselý, R. Chmelík, D. Rosel, and J. Brábek, "Quantitative phase imaging unravels new insight into dynamics of mesenchymal and amoeboid cancer cell invasion," Sci. Rep. 8(1), 12020–13 (2018).
P. Guo, J. Huang, and M. A. Moses, "Quantitative phase imaging characterization of tumor-associated blood vessel formation on a chip," in Quantitative Phase Imaging IV (International Society for Optics and Photonics, 2018), p. 105031O.
P. Langehanenberg, L. Ivanova, I. Bernhardt, S. Ketelhut, A. Vollmer, D. Dirksen, G. K. Georgiev, G. von Bally, and B. Kemper, "Automated three-dimensional tracking of living cells by digital holographic microscopy," J. Biomed. Opt. 14(1), 014018 (2009).
M. Rezaei, J. Cao, K. Friedrich, B. Kemper, O. Brendel, M. Grosser, M. Adrian, G. Baretton, G. Breier, and H.-J. Schnittler, "The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor–endothelial cell interactions," Histochem. Cell Biol. 149(1), 15–30 (2018).
J. C. Arciero, Q. Mi, M. F. Branca, D. J. Hackam, and D. Swigon, "Continuum model of collective cell migration in wound healing and colony expansion," Biophys. J. 100(3), 535–543 (2011).
R. Alert and X. Trepat, "Physical Models of Collective Cell Migration," Annu. Rev. Condens. Matter Phys. 11(1), 77–101 (2020).
T. Ohta, M. Tarama, and M. Sano, "Simple model of cell crawling," Phys. D 318-319, 3–11 (2016).
B. A. Camley and W.-J. Rappel, "Physical models of collective cell motility: from cell to tissue," J. Phys. D: Appl. Phys. 50(11), 113002 (2017).
L. K. Fritz-Laylin, M. Riel-Mehan, B.-C. Chen, S. J. Lord, T. D. Goddard, T. E. Ferrin, S. M. Nicholson-Dykstra, H. Higgs, G. T. Johnson, E. Betzig, and R. D. Mullins, "Actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes," eLife 6, e26990 (2017).
Y. Xiao, R. Riahi, P. Torab, D. D. Zhang, and P. K. Wong, "Collective Cell Migration in 3D Epithelial Wound Healing," ACS Nano 13, 1204–1212 (2019).
M. Anguiano, C. Castilla, M. Maška, C. Ederra, R. Peláez, X. Morales, G. Muñoz-Arrieta, M. Mujika, M. Kozubek, A. Muñoz-Barrutia, A. Rouzaut, S. Arana, J. M. Garcia-Aznar, and C. Ortiz-de-Solorzano, "Characterization of three-dimensional cancer cell migration in mixed collagen-Matrigel scaffolds using microfluidics and image analysis," PLoS One 12(2), e0171417 (2017).
Adrian, M.
Alert, R.
Al-Numair, K. S.
Anguiano, M.
Arana, S.
Arciero, J. C.
Arganda-Carreras, I.
Arulselvan, P.
Baretton, G.
Baum, M.
Bazellieres, E.
Ben-Jacob, E.
Bernhardt, I.
Bettenworth, D.
Betzig, E.
Bodin, S.
Bokemeyer, A.
Brábek, J.
Branca, M. F.
Breier, G.
Brendel, O.
Broers, J. L. V.
Brückner, M.
Brugues, A.
Bucevicius, J.
Bustin, M.
Calero-Cuenca, F. J.
Camley, B. A.
Cao, J.
Cardona, A.
Castilla, C.
Chandramohan, G.
Chen, B.-C.
Chen, C. S.
Chmelík, R.
Comunale, F.
Dahl, K. N.
Dao, M.
Dasari, R. R.
Debnath, S. K.
Declercq, I. L. J.
Depeursinge, C.
Detmar, M.
Diez-Silva, M.
Ding, N. S.
Dirksen, D.
Discher, D. E.
Domagk, D.
Ederra, C.
Eliceiri, K.
Erdel, F.
Falanga, V.
Ferrin, T. E.
Fredberg, J. J.
Friedl, P.
Friedrich, K.
Frise, E.
Fritz-Laylin, L. K.
Gandalovicová, A.
Garcia-Aznar, J. M.
Gauthier-Rouvière, C.
Gebäck, T.
Georgiev, G. K.
Gerasimaite, R.
Gerlitz, G.
Gilmour, D.
Goddard, T. D.
Gomes, E. R.
Gothai, S.
Grada, A.
Grosser, M.
Guan, J.-L.
Guo, P.
Hackam, D. J.
Hartenstein, V.
Hecht, I.
Higgs, H.
Hochstenbach, K.
Hong, K.
Hong, S.
Horev, J.
Houben, F.
Hruband, R. H.
Huang, J.
Hugonnet, H.
Ingber, D. E.
Irianto, J.
Ivanova, L.
Janota, C. S.
Jin, K. H.
Johnson, G. T.
Kamps, M. A.
Kaynig, V.
Kemper, B.
Ketelhut, S.
Kim, K.
Kim, Y. W.
Knight, M. M.
Koumoutsakos, P.
Kozubek, M.
Krausewitz, P.
Krishnan, R.
Krížová, A.
Kumar, S. S.
Langehanenberg, P.
Lauer, V.
Le Borgne-Rochet, M.
Lee, D. A.
Lee, K.
Lee, M.
Lenz, P.
Leung, E. P. Y.
Liang, C.-C.
Lim, J.
Livnat, I.
Long, M.
Longair, M.
Lord, S. J.
Lukinavicius, G.
Mak, N. K.
Maniotis, A. J.
Maška, M.
McCusker, C. D.
Mi, Q.
Mohd Esa, N.
Morales, X.
Morin, N.
Moses, M. A.
Mujika, M.
Mullins, R. D.
Muniandy, K.
Muñoz-Arrieta, G.
Muñoz-Barrutia, A.
Natan, S.
Nicholson-Dykstra, S. M.
Obagi, Z.
Oh, J.
Ohta, T.
Ortiz-de-Solorzano, C.
Otero-Vinas, M.
Pajerowski, J. D.
Park, A. Y.
Park, C.
Park, C. Y.
Park, J.
Park, Y.
Peláez, R.
Pepelanova, I.
Pietzsch, T.
Planchon, D.
Plutoni, C.
Popescu, G.
Poremba, C.
Preibisch, S.
Prieto-Castrillo, F.
Ramaekers, F. C. S.
Rappel, W.-J.
Reimers, K.
Reiner, O.
Rezaei, M.
Riahi, R.
Riel-Mehan, M.
Rippe, K.
Rosel, D.
Rouzaut, A.
Rueden, C.
Saalfeld, S.
Sadati, M.
Sammak, P. J.
Scheper, T.
Schindelin, J.
Schmid, B.
Schnittler, H.-J.
Schulz, M. M. P.
Séveno, M.
Shin, S.
Siegfried, K. R.
Snoeckx, L. H. E. H.
Sosnik, J.
Stamhuis, E.
Stamm, A.
Strauß, S.
Swigon, D.
Taheri Qazvini, N.
Tan, W. S.
Tarama, M.
Thielicke, W.
Thuault, S.
Tinevez, J.-Y.
Tolde, O.
Tomancak, P.
Torab, P.
Trepat, X.
Tsarfaty, I.
Veselý, P.
Vieira, W. A.
Vogt, P.
Vollmer, A.
von Bally, G.
Wang, S.
Webster, K. A.
White, D. J.
Willems, C. H. M. P.
Wolf, E.
Wolf, L.
Wong, P. K.
Wong, R. N. S.
Xiao, Y.
Yang, S.-A.
Yarden, O.
Ye, J. C.
Yoon, H.
Yoon, J.
Yue, P. Y. K.
Zaritsky, A.
Zhang, D. D.
Zhong, F. L.
Ziv, C.
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Link to Trigger Publishing and Ebook
Published by: Upside Down Books, an imprint of Trigger Publishing.
Superheroes don't get scared...oh yes they do!
Last night, my daughter sat on the bed with a wobbly tooth that has been troubling her for days.
"Why don't you let me pull it out?" I asked gently. "It's only hanging by a thread!"
Her eyes lit up only to cloud over seconds later.
"But I'm scared!" she said.
"Never fear, let's read this!" I jumped at the chance to share this new, brilliantly crafted and superbly illustrated picture book with her. "It's okay to be scared. Even Superheroes get scared!"
Dazzled by the front cover and eager to find answers within that she could relate to, she dived in.
Superheroes Don't Get Scared, written in simple but catchy rhyme, has a clear message told in a fun and modern way. Forget Spiderman, Superman and Wonder Woman! These Superheroes are far more up to date and they are about to show Maisie Brown that it's okay to have fears- she just needs to find the strength to face them.
Bursting with phobias that children (and adults) can easily relate to, this book will lead readers on an epic journey through the ocean, the jungle and deep, dingy caves with loveable, larger -than-life characters and unique and vibrant villains.
As Maisie watches her idols overcome the creatures they are most afraid of, she comes to a simple conclusion... if they can face their fears, she can. And an act of bravery is an act of bravery, no matter how big or small.
This picture book is an empowering treat! The characters and settings are vividly brought to life with outstanding illustrations from Clare Elsom and, although the topic is a serious and super-important one, debut author Kate Thompson tells it in a highly engaging and unthreatening way. Both girls and boys alike will be so busy being wowed by the Superheroes' antics that they are very likely to be distracted from the things troubling them. And, when they remember, they will hopefully have absorbed enough power to face it.
Safe to say, we got that tooth out with a nervous squeeze, bags of bravery and a big smile!
So if your little ones love superheroes, are feeling anxious or are struggling to find their feet this is the perfect, uplifting read. It even gave me the boost and positivity to face another day in isolation head-on!
SUPERHEROES DON'T GET SCARED is available now as an ebook and available to pre-order from online retailers for release in September. Link to ebook and pre-order
Labels: Picture books, UPCOMING RELEASES
Funny Investigations with Kes Grey and Jim Field
As our house descends into pretty much 'anything goes' (my daughter is at this very minute covering my husband in paint), books and the weather continue to be my salvation. This week my son went rooting through the bookshelves and rediscovered some brilliantly funny picture books by Kes Gray. Not only have they given us a much needed laugh but they gave me some ideas for silly investigations in Science, English and Maths.
If you're not familiar with Kes Gray, he is a well established children's author, well-known for his Daisy chapter books and Oi! picture book series; Oi Frog! Oi Cat! Oi Dog! Oi Platypus!, Oi Puppies! and soon to be released, Oi Aardvark! These, brilliantly illustrated by Jim Field, can in themselves spark off some fun with rhyme. But there are a few, maybe lesser known, stories that he's written which are fantastic for inducing both giggles and creative learning.
You're called What?!
Ever heard of a Blue-footed Booby, a Fried Egg Jellyfish or a Aha ha wasp? Well, not only do they make exceptionally humorous book characters but they also actually exist in real life.
Yes! This book features a collection of the strangest-named animals on the planet and on the back pages you will find actual pictures of them. Which leads me to the question:
Can you find or name any more hilarious creatures?
This story is great for sparking up a naming game. Can kids re-name well known animals with a more humorous title? Can they make up their own weird and wonderful creature? Can they research more about these weird and wonderful creatures? And can they find more that actually exist? For those really wanting to go all out - can you work out why the creatures evolved this way and how it helps them to survive?
Whether you do all or none of the activities, this is a really funny read, which always brings me to my own curious question: What is it with the name, Dave?
Quick Quack Quentin
Quentin has a problem! He's lost the 'A' from his Quack and a Quck will never do. In order to sound like a duck again, Quentin must visit the farm and the zoo and find an animal who can lend him an 'A'. Easier said than done when everyone needs to hang on to them...or do they?
This book is genius. Simple but funny and brilliant for a phonics investigation. Who has an 'A' in their name? What would happen if you lost it? Who could you borrow one from?
Of course, it doesn't have to be an 'A' you lose? What about another letter? What if some animals lost the first letter of their name? What would their new name be?
This is also great for word building and lateral thinking. What is 'crow' without the 'c'? What is 'cow' without the 'c'? What is 'hat' with a 'w' in front of it?
Let's get some silly word play going on!!!
How Many Legs?
Moving on to Maths, this rhyming picture book asks readers to add up the number of legs of the party guests. Problem is, they just keep coming...
Once you've worked out the total number on the page, how about working out the number of legs in your house? Or fingers, toes, feet, eyes...? Can you include your pets? Maybe even the table (as the book suggests?) Is the answer going to be even or odd?
Whilst reading Planet Stan by Elaine Wickson this week, I also stumbled on another maths idea which the Gran in the story uses to entertain her very lively grandson. On your daily walk, why not work out how many hands high the gate is or how many feet wide the stream is? How many bums wide is the bench or the bridge or anything else that might keep them occupied for a few minutes. This one I trialled immediately and it worked! It didn't work the following day but, hey, I was winning for a little bit. And, right now, every tiny victory counts right?
The good thing is, many books can trigger curiosity and investigations whether they are fiction or non-fiction. There is a currently a huge demand for children's non-fiction and narrative non-fiction books (this is non-fiction told in a story format, rather like 'You're Called What?). We particularly like these ones because they are so much fun to read but look out for How to be Extraordinary by Rashmi Sirdeshpande or Nosy Crow's HerStory by Katherine Halligan and Wildlives by Ben Lerwill for more serious but interesting research projects. And if you come across a good one, please let us know. Anything that gets us through the morning in a relatively fun and enjoyable way is in serious demand over here ;)
Labels: HOMESCHOOLING WITH STORIES
The Unadoptables by Hana Tooke
Link to Goodreads
Published by: Puffin, July 2020
Oh my heart! 💔 I'm not sure I have the words for this beautiful, gut-wrenching but gripping tale, exquisitely told by Hana Took. It is a future classic - a tale of hope, sorrow and joy that will evoke flowing tears, a fierce attachment to characters that you just want to bring home and a roiling hatred for villains so vile, you'll want to finish them off yourself.
Welcome to 'Little Tulips' - the bleakest orphanage in the city of Amsterdam- where in 1880, five babies were abandoned in the most unusual manner. Here starts the journey of Milou, Sem, Egg, Lotta and Fenna - the 'unadoptables' unable to find a home.
When cruel matron, Gassbeck, decides to sell the orphans off to sinister sailor, Rotman, the five inseparables have no choice but to run for their lives, to the outskirts of the city where Milou believes her real family awaits her. Following the clues from her past, she leads them to an abandoned windmill where a puppet-maker once lived. Convinced that the windmill holds the key to her identity, Milou convinces the others to stay and forge a home. But curious eyes are watching, Rotman is lurking and the Kinderbureau are smelling a rat. Will Milou ever uncover who her true family are and find her happy end?
Although predominantly Milou's story, this book would be nothing without the unity of the five adorables who you will want to adopt by the end of chapter one. Bursting with heart and soul, this book weaves a tale of gothic intrigue - puppets, nightmares and clock-makers- with the cruel reality of life as destitute orphans. Milou's flawed but desperate tenacity will gouge claw-marks into your heart and the orphans' unbreakable bond is beyond endearing. Gassbeck and Rotman are villains to rival Cruella de Vil and Fagin and the role of the puppets is truly captivating.
Not all the adults are rotters, however. Edda the curious clock-maker, although somewhat unfathomable, brings a sense of stability and safety to the story and plays a key role in the ending. As far as endings go, this one will not disappoint. It keeps you guessing until the end and is one of the most satisfying and moving conclusions that I have read.
For me, this novel, although fresh and original, had echoes of 'The Sound of Music', 'Oliver Twist' and 'A Little Princess.' The storytelling felt traditional, the setting was gritty and the plot intoxicating. It is a love story, a gothic nightmare and a tale of friendship, the tendrils of which will entwine around every heartstring you own and stay there.
'The Unadoptables' is available to pre-order via online retailers.
Labels: Middle Grade, UPCOMING RELEASES
Hotels are closed, right? Nope, not these ones!
Just because hotels are closed for business, doesn't mean you have to forego your holiday completely. Why not escape but stay safe? Go on a thrilling journey without moving a muscle? Chase adventure whilst sat on the sofa?
Sound good? Great! Because these two fantastic books offer the perfect place to visit.
Hotel Flamingo by Alex Milway
Published: 2019, Piccadilly Press
Hotel Flamingo is a charming, feel-good chapter book about a hotel full of colourful and loveable animals. From flamboyant flamingos to otter lifeguards to cockroach guests, this heartwarming tale will make you feel cosy right down to your toes.
When Anna inherits the Hotel Flamingo, things seem hopeless. After being overshadowed by the glamour of The Glitz, the rundown wreck doesn't stand a chance of getting back on it's feet. Yet, with Anna's endless optimism and some loyal members of staff, a wind of change is in the air. Before long, Hotel Flamingo is bursting with a new lease life - a cacophony of chaos and joy! But challenges are waiting in the shadows. Can Anna and her team overcome a fierce competitor and a ruthless critic in order to stay in business?
The story is truely enchanting and full of vibrant and cuddly characters you'll want to meet again and again. Anna is a strong-willed, determined lead who champions teamwork and inclusivity and has a clear idea of what she stands for. The cockroach family are a genius touch, the flamingos add a fabulous flair and even the trickier characters remain unthreatening. This is a must read for any animal lover and perfect for more sensitive readers who relish a safe and comforting read. Put your slippers on and curl up with a cup of cocoa or put on some overalls and get stuck in!
The Nothing to see Here Hotel by Steven Butler (Illustrated by Steven Lenton
Published: February 22nd 2018 by Simon Schuster Children's UK
WOW! This has to be the most high octane chapter book I have ever read. Packed with magical creatures and non-stop action, I read this book in a single sitting and came away breathless.
Frankie Banister and his family are from troll descent. Now they run a hotel for all kinds of magical creatures. With a terrifying grandmother in the attic, a scatty spider cook, a gardener married to a Venus Fly Trap and guests ranging from mermaids to tooth fairies, Frankie is kept incredibly busy. But when Prince Grobah from the 'dark and dooky deep' announces his arrival, the hotel descends into total chaos. Overrun by greedy goblins, Frankie and his parents have no chance of maintaining control. That is, until the goblin pirates arrive and battle commences...
The imagination and fast-pace of this story blew me away. Whereas Hotel Flamingo provides a rather more chilled getaway, this is a hurricane of a holiday. There is no down time or lull in the plot. Instead, expect page after page of high-action adventure bursting with characters so zany, they'll leave your head spinning. The story itself is funny, well-plotted and original. There were times when I felt I needed to catch my breath and that the main character, Frankie, felt more of a spectator than a participant in the story. The goblins of the 'dark and dooky deep' were rather condemned as a race and there possibly could have been more redemption here. However, the ending was spectacular, the ghost pirate was phenomenal and the moving lawn was genius. There is no doubt whatsoever that we will be reading the whole series in our house.
More in the series: You ain't seen nothing, Yeti!
Sea-ing is believing!
The Fiend of the Seven Sewers - released on September 3rd 2020
Labels: Chapter books, Young Middle Grade: 7-9
When children self-initiate...go with it!
With the Easter holidays ending, the blurry prospect of home-schooling is back on the horizon. After long days of playing in the garden and allowing the kids to lead the action, the thought of re-introducing 'learning', however play or outdoor based it is, is making me nervous. Yet, we don't always have to initiate an activity to feel we are fulfilling our role.
Today, on our annual Easter egg hunt around the garden the children unearthed three eggs hidden in a small Peter Rabbit suitcase which had arrived by Easter Bunny post (thanks Nan!). After the chocolate had been eaten and the excitement had passed, I found my little boy playing happily with the suitcase.
"Look Mum!" he said, marching past me with it clutched in his hand. "I'm like the BFG!"
Okay, didn't see that one coming but I jumped on it.
"Yeah, you do! Why don't you go and catch some dreams?" I picked up an empty chocolate wrapper. "Could this be a chocolate dream?"
Enough said! With a big smile on his face he put the wrapper in the case and went off on a search. Within five minutes he had a petal for flower dreams and his toy soldiers for 'bad dreams' and so on and so on. He kept the case by his side all evening.
So tomorrow, if he's still keen, then we can take the suitcase on our daily walk to collect more dreams. We can combine two or three items together to create a more complex dream. We can storyboard that dream, make potions of dreams and label the jars. We can Skype family members and ask them to tell us stories about their dreams. What is the science of dreams? What causes dreams? What will we dream about this week? Can we use our fitbit to track our sleep? How many hours have we slept? What time did we go to bed? Can we walk like a giant? Suddenly we have covered literacy, maths, science, PE, art and bags of creativity. All from one story.
This is the fundamental power of stories. Although we haven't read the BFG from beginning to end yet, the kids know it is one of my favourites, they have seen the film, we've read parts of it and listening to it on audio in the car. Stories throw children into new worlds, trigger the imagination and help them to relate to our own world. They explore new perspectives, viewpoints and empathy. And they stick in the memory.
I would never have made that connection from the suitcase to the BFG but my son did all on his own. With a little enthusiasm and encouragement we suddenly have a whole array of activities to get stuck into this week, including reading some of the book again. Yet, he could have easily made a similar connection to Paddington or Narnia (evacuees) or any other stories that involve a suitcase. (For older children I really recommend the new 'Strangeworlds Travel Agency' by L.D. Lapinski).
Stories are usually a great way for an adult to initiate and kickstart activities and get a child onboard. Yet, if it feels forced then don't push it. For every success, ten will probably flop and end in frustration. Just enjoying books and stories together and leaving it there is fine. Children will sit on it, process it and then, possibly, act on it, just like my little one did today. If they do then support it but don't take over. Allow them to lead and…who knows where you'll end up.
Super Happy Magic Forest and the Humongous Fungus by Matty Long
Published: April 2020, Oxford University Press
A victory pose is definitely in order. We have finished the first, long anticipated, Super Happy Magic Forest chapter book and it is a fizzing frolic of fun!
For those that are not familiar with Super Magic Happy Forest, it is a picture book bursting to the brim with super settings, hapless (or not so hapless) heroes and an epic quest to save the 'Magic Crystals of Life'. Told in a comic-style format, this 'Lord of the Ring' spoof is imaginative, hilarious and sparkles like sunbeams on the sea. Thank goodness the incredibly talented Matty Long added to the collection with Salty Dogs, Super Happy Frozen Forest and Super Happy Magic Forest: Slug of Doom. My son has requested them all.
The chapter book format didn't disappoint and is the perfect next step for those wanting more. The plot is deliciously fiendish yet drenched with a happy light-heartedness. The five heroes are loveable yet totally madcap. Trevor the Mushroom is a wonderful champion for disability, Blossom blasts apart gender stereotype and Herbert is a whizz at finding sprite jam. Who'd have thought?
To save the Rainbow Dragon and the whole of Super Happy Magic Forest from the festering, filth of the Humongous Fungus and his spores, the five heroes are entrusted by Gnomedalf and Shroomsworth to find five key ingredients for an antidote. But when the medicine fails to work, will they ever be able to stop the toxic spread of Fungellus' evil?
The story offers entertainment for the whole family. Aged from 4-40, we giggled, frolicked and quested with the heroes until we could quest no more and we found that the book does offer an antidote that works! It is a much needed dose of silliness to counteract these serious times.
We thought there were a few too many characters - some a bit similar to others - and weren't sure whether the 'Happiness Committee' were all needed. However, they offered some fun parodies to real life which added an extra layer for adult readers. The ice-cream tree, candyfloss cave and hugberry island were massive hits with my little ones and got them imagining other delicious places which they could add to the map. My four year old enjoyed the story so much, he is still delightfully carrying the book around with him whilst my autistic daughter, aged 9, is still giggling over the unicorn poop.
So to round things up: This is a slapstick sizzler of a story full of smiles, spores and 'fun guys'! We can't wait for the release of Happy Magic Forest and the Portals of Panic - coming October 1st 2020.
Pre-order available
Crater Lake by Jennifer Killick
Published: March 2020, Firefly Press
Don't.Ever.Fall.Asleep...
No chance reading this book!
It's been a long time since I read a seriously spooky story and I'd forgotten how much I enjoy them. Taking me right back to the days of Goosebumps and Point Horror, this fantastic read by Jennifer Killick kept me wide awake and reading late into the night.
Staying awake may have been easy for me but it's not so easy for the group of Year Six's taking a residential trip to the newly opened Crater Lake Activity Centre. Yet, Lance and his tightknit gang of friends soon realise that if they have any chance of surviving the creepy camp then that's exactly what they have to do.
With Lance's group being the only ones left standing, they must do everything in their power to protect themselves from a sinister alien fate. But waspish Miss. Hoche is on their trail, the exits are guarded and all phone signals are unavailable. Do they stand any chance of saving their classmates and getting out alive?
The first brilliant thing about this book is that you won't have to wait long to be gripped. The plot kicks off with an unnerving incident which immediately draws you into a fast-paced and twisty journey of zombies, alien spores and the urgent need to escape. Whilst the book has a classic feel of both horror and sci-fi about it, the story offers an original and spine-tingling take on familiar themes.
The tale is perfect for middle-grade readers looking for something daring, exhilarating and very, very spooky. The heart-thumping narrative will leave them gasping for breath but isn't too frightening to be age inappropriate. Counter-balancing the horror is a spot-on and very heart-warming account of group friendship which I found to be the highlight of the book (and tantamount to the group in The Goonies). Lance's group are a loveable mix of characters - savvy, contemporary, smart and everything that is wonderful about the youth of today. I loved their style, their language, their warmth and their loyalty and was left feeling full of hope and promise at the end of the novel (plus desperate to meet them again).
Jennifer Killick's writing is outstanding. She interweaves a web of horror and intrigue with the comforting safety blanket of kinship. I don't think there will be any stopping this novel...film? cinema? TV? I certainly hope so!
Labels: Middle Grade
UPCOMING RELEASE: THE STRANGEWORLDS TRAVEL AGENGY by L.D Lapinski
Published: April 30th by Orion Children's Books
Escaping into one fantasy world is tempting enough but when there are a whole rainbow of magical worlds to explore in one book, it sounds almost too good to be true...
Rest assured, it is definitely not too good to be true! The Strangeworlds Travel Agency sweeps you up and whisks you away to places that crackle with imagination and magical delight.
From a bouncy world full of sweets to a forest that has echoes of Peter Pan's famous 'Lost Boys', from a sinister, abandoned lighthouse to the bustling 'City of Five Lights', this story whisks you on an epic adventure through worlds that will make you gasp in wonder.
Twelve year old Flick is the epitome of 'normal'. Her family are normal and her life is normal. That is until she discovers The Strangeworld's Travel Agency, run by eccentric Jonathan Mercator.
When Jonathan realises that Flick can see magic, he welcomes her into an exclusive society - one that allows members to travel across the multiverse through suitcases. But, whilst Flick is dazzled by her new adventures, it is clear that Jonathan is keeping dark secrets. Desperate to find his missing father, he embarks on a journey that plunges both himself and Flick into deadly danger. For the worlds are changing - threatened by a leaking magic - and some people will do anything to escape them...
This book, filled to the brim with L.D Lapinski's phenomenal creativity, is the ultimate fantasy read. It begins with the joyful feel of a kid in a sweet shop before twisting into a dark, breath-taking adventure. It charts not only the discovery of worlds that we have all dreamed of ourselves, but also Flick's discovery of who she is and where she fits within the magic.
The character of Jonathan is unusual. Here we have an older - almost adult- mentor showing Flick the ropes of the magical society that he is Head Custodian of. Whilst his scathing sarcasm is fun to read, his guarded attitude and wariness is a stark contrast to Flick's jubilant innocence and his story just as compelling. It is Jonathan's turmoil that drives the first part of the novel, with Flick very much in the passenger seat. However, when she discovers the extent of Jonathan's lies, she is forced to come into her own with a power and strength she could never have dreamed of.
As this is the first in a series, the story leaves the readers with a whole suitcase full of unanswered questions and an insatiable thirst for more. With parallels to well-loved magical lands such as 'Peter Pan' 'Oz' and Phillip Pullman's trilogy 'Northern Lights' (in particular the world-jumping that occurs in 'The Subtle Knife), it promises to become a memorable and epic quest steeped in a depth of magic that I haven't experienced for a long time.
An absolutely cracking read!

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: Updating metro-config.js React Native I've installed react-native-svg and updated metro-config.js according to the docs:
metro-config.js
...
const { getDefaultConfig } = require('metro-config');

module.exports = (async () => {
  const {
    resolver: { sourceExts, assetExts },
  } = await getDefaultConfig();
  return {
    transformer: {
      babelTransformerPath: require.resolve('react-native-svg-transformer'),
    },
    resolver: {
      assetExts: assetExts.filter(ext => ext !== 'svg'),
      sourceExts: [...sourceExts, 'svg'],
    },
  };
})();

Now I need to install react-native-tcp-socket and add the following lines to the metro-config.js:
const {getDefaultConfig} = require('metro-config');
const defaultConfig = getDefaultConfig.getDefaultValues(__dirname);

module.exports = {
  resolver: {
    assetExts: [...defaultConfig.resolver.assetExts, 'pem'],
  },
  // ...
};

But I don't know how to combine it together. I've tried the following:
resolver: {
  assetExts: assetExts.filter(ext => ext !== "svg") + [...defaultConfig.resolver.assetExts, 'pem'],
  sourceExts: [...sourceExts, "svg"],
},

But it doesn't work as expected. Any ideas?

A: Replace resolver part like this:
resolver: {
  assetExts: [...assetExts.filter(ext => ext !== "svg"), 'pem'],
  sourceExts: [...sourceExts, "svg"],
}

Why? You don't need defaultConfig.resolver.assetExts as assetsExts is coming from the default config. You are only taking the svg out of it (read more about filter function) and so you will only add 'pem' to final config array.

redpajama_set_name: RedPajamaStackExchange


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text:
Biserica "Nașterea Sfintei Fecioare Maria" din Vama este o biserică romano-catolică construită în anul 1886 în satul Vama din comuna Vama (județul Suceava). Ea se află de-a lungul drumului care face legătura între orașele Suceava și Câmpulung Moldovenesc, la o distanță de 16 km sud-est de orașul Câmpulung Moldovenesc.

Comunitatea germană din Vama
Așezat între cele două râuri, Moldova și Moldovița, satul Vama se află la o distanță de 16 km de orașul Câmpulung Moldovenesc. Aflat la intrarea în Carpații Orientali și străjuit de Obcina Feredeului, acest sat a îndeplinit rolul de localitate de vamă între Principatul Moldovei și Principatul Transilvaniei. Aici erau vămuite mărfurile care veneau dinspre Ardeal sau plecau într-acolo.

Inițial, localitatea se numea Vama Moldoviței și apare într-un privilegiu comercial din 6 octombrie 1408 în care se stabilește ca  toți negustorii care vor duce mărfuri din Țara Ungurească în Moldova să plătească taxe vamale de "câte un groș și jumătate din grivnă" de povară - la dus și "câte doi groși" de povară la întoarcerea din Ardeal.  Domnitorul Alexandru cel Bun (1400-1432), într-un document din 18 noiembrie 1409, a scos satul Vama din Ocolul Câmpulung și l-a dat Mănăstirii Moldovița, împreună cu veniturile vămii.

După anexarea Bucovinei de către Imperiul Habsburgic (1775), autoritățile austriece au adus aici coloniști de origine germană, care proveneau din diverse regiuni ale imperiului și din landurile germane. Primii coloniști germani au sosit la Vama (în ) în anul 1807, ei lucrând la exploatările miniere din zonă. De asemenea, în capătul dinspre Câmpulung Moldovenesc al satului Vama, în lunca împădurită de sub culmea Hurghiș, s-au așezat în perioada 1807-1808 mineri germani din Grundler (Slovacia) care au format o colonie germană cu numele de Eisenau (în ).

Comunitatea germană a continuat să crească până în 1918. Satul avea învățători care predau copiilor în limba germană.

După Unirea Bucovinei cu România (1918), numărul germanilor din Vama s-a menținut constant. În perioada interbelică, Vama a avut rangul de comună urbană (oraș), făcând parte din Plasa Moldovei a județul Câmpulung. În anul 1930, populația orașului Vama era de 5.315 locuitori, dintre care 3.237 români (60,90%), 1.579 germani (29,70%), 392 evrei (7,37%), 44 polonezi, 25 ruteni, 12 ruși, 12 țigani, 3 cehi și slovaci, 2 unguri și 8 de alte naționalități.  După religie, locuitorii satului erau grupați astfel: 3.250 ortodocși (61,14%), 971 evanghelici (luterani) (18,26%), 635 romano-catolici (11,94%), 392 mozaici (7,37%), 50 greco-catolici, 1 armeano-gregorian, 1 lipovean, 1 adventist și 14 fără religie (liber-cugetători).

Etnicii germani au fost mutați în anul 1940 în zone ocupate de Germania Nazistă. România și Germania au încheiat o convenție prin care statul român se obliga să achite despăgubiri pentru fiecare clădire în parte, inclusiv pentru terenurile și pădurile germanilor care au emigrat.

Biserica romano-catolică din Vama
Încă de la început, comunitatea romano-catolică din Vama a fost păstorită de preoții din Câmpulung Moldovenesc. Decretul nr. 4445 din 7 februarie 1854 al Guvernului Bucovinei, prin care s-au stabilit jurisdicțiile parohiilor romano-catolice din ducat, a stabilit în jurisdicția Parohiei Câmpulung comunitățile romano-catolice din localitățile: Argel, Ciumârna, Freudenthal, Frumosu, Gemenea, Moldovița, Ostra, Rus pe Boul (Paltin), Sadova, Slătioara, Vama și Vatra Moldoviței.

Evidențele parohiale din 1857 consemnează existența unui număr de 281 credincioși romano-catolici la Vama.  Ca urmare, s-a propus Arhiepiscopiei de Lemberg să se înființeze o parohie la Vama, dar acest lucru nu s-a realizat.  După cum atestă o însemnare din 23 decembrie 1875 în Cronica Bisericii parohiale din Câmpulung, slujbele religioase se oficiau la Vama într-o capelă.

Ca urmare a creșterii numărului de credincioși, comunitatea germanilor de religie catolică din satul Vama, aflat pe atunci în Ducatul Bucovinei, a construit în anul 1886 o biserică din cărămidă. La momentul acela, locuiau mulți etnici germani în sat. Lăcașul de cult a fost sfințit la 8 septembrie 1886. Cu acest prilej, deasupra intrării în biserică s-a încastrat în zid o placă de marmură cu următoarea inscripție în limba germană: "Am 8ten September g:h: die K: weihe 1886." (Am 8. September 1886 g[escha]h die K[irchen]weihe). Traducerea aproximativă a acesteia în limba română este următoarea: "La 8 septembrie 1886 s-a înfăptuit sfințirea bisericii.". Biserica a fost construită de-a lungul drumului care leagă Suceava de Câmpulung Moldovenesc. Ea are hramul Nașterea Sfintei Fecioare Maria (sărbătorit la 8 septembrie).

Evidențele parohiale din 1925 consemnează existența a unui număr de 425 credincioși romano-catolici la Vama, care erau păstoriți de preotul profesor Leonhard Haschler. În acel an, preotul ajutător Iosef Ettmayer locuia la Vama.  Liturghia la biserica romano-catolică din sat era oficiată o dată pe lună. După datele Recensământului general al populației României din 29 decembrie 1930, în orașul Vama (care includea și satul Prisaca Dornei) locuiau 635 credincioși romano-catolici (reprezentând o pondere de 11,94% din populația localității).

Prin decret episcopal, la 1 august 1939 se înființează Parohia Vama prin desprindere de Parohia Câmpulung, ca paroh fiind numit preotul Ernst Haik, născut în Iacobeni. Noii parohii îi sunt arondate comunitățile catolice din Argel, Deia, Frumosu, Moldovița, Molid, Rus pe Boul (Paltin), Vatra Moldoviței, Eisenau (Prisaca) și Freudenthal.  Parohia Vama s-a desființat în timpului celui de-al doilea război mondial, ca urmare a emigrării în Germania a majorității credincioșilor romano-catolici (care erau de etnie germană), iar biserica din Vama a revenit în administrarea Parohiei Câmpulung. În timpul războiului, liturghiile din Vama au fost oficiate de preotul Eugen Baltheiser, fără să primească salariu.

În perioada regimului comunist, numărul credincioșilor romano-catolici din Vama s-a micșorat în mod continuu, majoritatea lor emigrând în Germania. De comunitatea catolică din Vama s-au îngrijit preoții din Câmpulung Moldovenesc, printre care Mihai Serafim Bejan (1950-1952), Franz Astalosch (1966-1985), Silvester Bejan (1985-1988) și Francisc Simon (1988-2006).

Aflat la 21 martie 2004 într-o vizită pastorală în Parohia Câmpulung Moldovenesc, episcopul Petru Gherghel a celebrat o liturghie în biserica filială din Vama.

În ianuarie 2010, conform evidențelor Parohiei Câmpulung, comunitatea romano-catolică din Vama era formată din 8 familii cu 12 credincioși.

În prezent, Biserica romano-catolică din Vama este filială a Parohiei "Înălțarea Domnului" din Câmpulung Moldovenesc. În această biserică sunt celebrate liturghii o dată la două săptămâni, precum și de sărbători.

Preoți
La această biserică au slujit următorii preoți-parohi:
 pr. Ernst Haik (n. 7 iulie 1908, Iacobeni, județul Suceava - d. 11 martie 1980, Oberstaufen, Bavaria, RFG) - A absolvit Institutul Teologic Romano-Catolic "Sf. Iosif" din Iași. A fost sfințit preot la Trier (Germania), la 17 martie 1934. A activat ca vicar la Rădăuți (octombrie 1934 - 1 august 1939), apoi paroh la Vama (1 august 1939 - 1940). A emigrat în Germania în 1940, păstorind ca paroh la Bärnwald (azi satul Neratov din Cehia) (1943-1945). A decedat la Oberstaufen.

Imagini

Note

Bibliografie

 Vasile Sfarghiu, Otilia Sfarghiu - "Câmpulungul Moldovenesc: confesiuni, lăcașuri de cult și slujitori ai acestora" (Ed. Axa, Botoșani, 2007), p. 208-221

Biserici din județul Suceava
Biserici romano-catolice din România

redpajama_set_name: RedPajamaWikipedia


04/16/2019 - The Senate Committee on Agriculture, Nutrition and Forestry held a hearing with witnesses emphasizing the importance of improving the delivery and structure of child nutrition programs.
04/16/2019 - Last week, multiple agencies signed a memorandum that highlights the agencies' commitment to increasing the collaboration and coordination in existing federal programs to reduce food loss and waste.
04/09/2019 - Experts from several organizations recently convened to conduct a reassessment of U.S. policies related to breastfeeding support.


I'm using IntelliJ 4.0. I'd like to install the plugin ReferenceScanner.
When updating my available plugins via the Settings | Plugins, I do not get ReferenceScanner in the list. The list contains 133 items. The web site says there are more than 300 plugins. The ReferenceScanner plugin indicates that it is valid for 4.0.
How do I get the plugin installed?
What is the plugin going to do for me?
If I can get it installed, the next question will be how do I get it to work. My run/debug configuration is set up to run my application, and I can't figure out how to run my application AND the ReferenceScanner.
I've already downloaded the Reference Scanner utility and added its .jar file to my lib path. When I open the browser and use localhost:4040, I get "The page cannot be displayed". I simply need the steps to get these things to work together.


text:
Q: Docker initialization on OSX: is a dial error normal? I ran boot2docker for the first time. At the end of the /usr/local/bin/boot2docker up process, I got the following:
Get http:///var/run/docker.sock/v1.19/version: dial unix /var/run/docker.sock: no such file or directory. Are you trying to connect to a TLS-enabled daemon without TLS?
bash-4.3$

Do I worry?

A: That indicates that something is wrong.
The "dial unix..." error is saying that the docker executable is failing to connect. You'll mostly get this if the VM isn't running or isn't configured correctly. In this case, it looks like the docker service on the VM had some problem and the error is coming from inside the VM (that's why it's /var/run/docker.sock instead of an https connection to the VM).
It's not possible to diagnose without more logs, but I recommend docker-machine as the new replacement for boot2docker. (It still runs the boot2doocker distro, but it's a little more sophisticated at setting up the VM.)


A band with a lot of potential.
You have to applaud Corey's song writing skills and the ability to produce uplifting indie pop rock so well. 'About to Be' would fit well into any soundtrack romantic comedy and the blend of Corey's and bass player Julie Holmes' vocals work wonderfully. If Smashing Pumpkins are your cup of tea then 'Out There' is a song that you will most certainly enjoy. 'Plans' has a potent lyrical content and the dual vocals add a much needed sensitivity to the mix.
A particular favourite song for me is 'Dusk' a song with a sense of mystery which again benefits tremendously from Julie Holmes accompanying vocals. The music treads closer to U2s better moments from the 80s and the chorus sounds uncannily like some of Lacuna Coil's earlier work. 'Surrounded' continues in the Smashing Pumpkins style before 'The Impossible' jangles along on a potent rhythm piece and the chorus jumps out into the sunshine. The two part finish of 'Rain Prt 1' and 'Rain Part 2' are ambitious moments that show a side to the band not explored on previous offerings. The moody intro of the first part is not unlike something New Order might have offered but is over a little too soon before the second part awakens in a roll of percussion and guitar.
I cannot help but imagine if these guys got a slot at the Reading festival they could really hit the big time, especially as there are less talented artists doing the rounds that have found acclaim. Surreal is a band with a lot of potential.


Wouldn't this be a nice entrance to one's home…or maybe it's a bit too pretentious for a country cottage! This is the old entrance to Kilronan Castle just outside the village of Keadue in County Roscommon. It was here we met today for the second Bards in the Woods of the year. The first was last week at Hazelwood near Sligo but I didn't get to that one.
Just beyond the entrance is this impressive folly which is known locally as the Fairy House. It is believed that it was used by the occupants of the castle as a place where they could play at being peasants. The roof is long gone as are the toffs in the castle. Very pretty isn't it? The castle has a new lease of life now as a popular hotel.
As I waited for the others to arrive I walked a little by the shore of Lough Meelagh. Something so very soothing about the sound of lapping water.
About a dozen people turned up and we set off happily on our walk. The weather was ideal…sunny but not too warm.
Spotted this Oak tree with what looks like a deer head growing out of it.
At the place called The End of the World we rested a while and had a cup of tea.
John recited a couple of his own compositions here.
What more could a body want on a beautiful sunny Sunday? Beside a beautiful lake, listening to poetry, the company of good friends and a picnic when we returned to the folly. Perfection!
A lovely Sunday in the woods!