dapi staining nucleus

Tap the tube to loosen the pellet and add 5 mL of PBS at room temperature. What is the purpose of DAPI staining in confocal microscopy? Analyze by flow cytometry in the presence of the dye. National Library of Medicine sharing sensitive information, make sure youre on a federal (a) Represents pixel value in 1st Square ring (S) at j th position. IEEE Trans. The proposed approach includes third plane information using hyperplanes into the design of the Sorted Random Projections (SRP) texture feature and is evaluated on publicly available 3D image datasets of human fibroblast and human prostate cancer cell lines obtained from the Statistics Online Computational Resource. This site needs JavaScript to work properly. For the PC3 data set, on the other hand, there is a decrease in the intensity and HC aggregation, which signifies a decondensation or open chromatin state on the transition to the EMT state (see Supplementary Figs. As further application of our method, it will be interesting to see the results of the proposed HC/EC computation on the 2D fluorescence microscopy images of cells such as white blood cells obtained from a liquid biopsy. and JavaScript. Chromosoma 121, 527538 (2012). \end{aligned}$$, $$\begin{aligned} \textit{Null hypothesis }{H_0}= \textit{Pseudo 3D outperforms 3D.} Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Be sure to protect from direct light. Before The HC/EC ratio was also computed following the same method with the original pixel values without random projections, and it was observed that the obtained p-value was higher. In the meantime, to ensure continued support, we are displaying the site without styles in 2015 IEEE 12th International Symposium on Biomedical Imaging (ISBI), 58 (IEEE, 2015). If you're seeing similar staining, like the. Nat. 16, 021001 (2019). DAPI staining is normally performed after all other staining. \end{array}\right. } Protocols for fixation and DAPI staining of uncultivable microbial eukaryotes. Following segmentation, cell objects are cropped from the image slices and stacked to form a 3D cell object for classification. Dahl, K. N. & Luxton, G. G. A special topic on nuclear mechanobiology. The increase or decrease in image gradient leads to the corresponding rise or drop in the slope of the curve of HC aggregate, which in turn indicates condensation or decondensation of chromatin, respectively. Kalinin, A. Evolut. Oncol. government site. SRP is based on the dimensionality-reduction technique known as Random Projections (RP), which compresses high dimensional data, captures salient information without information loss and preserves inter-distance of data values while projecting to a lower dimensional space. As described in the SRP section, the radial and angular descriptor function for the i th patch of an image with n cubic patches is computed as [\(f_{R}]_{CP_{i}}\) and [\(f_{A}]_{CP_{i}}\), respectively. in 2011 International Conference on Computer Vision, 391398 (IEEE, 2011). S4 and S5. DAPI Nucleic Acid Stain | 4 2.3 Tap the tube to resuspend the pellet in the residual liquid and add 1 mL of PBS at room temperature. CAS Their study discarded connected cell objects; however, this study employed a semi-automated approach where results from CellProfiler are visually inspected, and the watershed algorithm is applied to segment connected cell objects, leading to a higher number of SS, EMT and EPI cell objects. Song, Y. et al. The radial and angular feature descriptor vectors obtained from n cubic patches of the nuclear image ([\(f_{R,A}]_{Nucleus}\)) are traversed, and the values above the threshold are identified corresponding to HC ([\(f_{R,A}]_{HC}\)). DAPI staining (blue) depicts the nucleus with an intranuclear inclusion, which contains the autophagy-associated proteins ubiquitin and p62. doi: 10.1128/spectrum.01733-22. D1306,D3571,D21490,S33025,10010023,P36930, P36934,S36936,S36937, Copyright 2006-2023 Thermo Fisher Scientific Inc. All rights reserved, Spectroscopy, Elemental and Isotope Analysis, Adherent cells for fluorescence microscopy, Properties of classic nucleic acid stainsTable 8.4, Barcellona ML, Cardiel G, Gratton E (1990), Tanious FA, Veal JM, Buczak H, et al. FOIA Song, Y. etal. DAPI is a popular nuclear counterstain for use in multicolor fluorescent techniques. Changes in the chromatin pattern as implied by results in Table2 are in accordance with other relevant studies2,7,9,17. Med. All cell images have three channels showing different fluorophores: DAPI stain for nuclei, fibrillarin antibody (anti-fibrillarin) and ethidium bromide (EtBr) for staining nucleoli. Features are extracted from the segmented cell objects instead of whole image to avoid dilution of texture description by the image background and noise. A popular nuclear and chromosome counterstain, DAPI emits blue fluorescence upon binding to AT regions of DNA. Bischl, B., Mersmann, O., Trautmann, H. & Weihs, C. Resampling methods for meta-model validation with recommendations for evolutionary computation. Estimation of three-dimensional chromatin morphology for nuclear classification and characterisation, $$\begin{aligned} \textit{Null hypothesis }{H_0}= \textit{All classification models have the same mean rank.} Cells are segmented and cropped, followed by extraction of cellular texture features using proposed 3D SRP. Huisman, A. et al. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Incubate for 15 minutes at room temperature. Previously, the existence of two such EMT ICS has been found in mammary epithelium cells33. Pellet the cells by centrifugation and discard the supernatant. Nat. 3, different colours of the lines indicate different mean ranks and population. Cold Spring Harb Protoc. The length of the line represents the comparison interval, and the extent of overlap of lines implies the range of similarity of corresponding groups. Performance improvement is achieved over state-of-the-art classification, demonstrating the significance of texture features compared to shape features in the nuclear morphological study. In the current study, obtained metric values based on texture description are higher than the state-of-the-art (SOTA) works obtained by classification using shape-based features on the same dataset. Image Anal. 3b). 34, 574586 (2012). The test was conducted for all individual subsets, and the observations remained the same for all subsets except in one instance when the test was performed excluding one image subset (coded as 179 (\(EMT^{179}\))) comprising 25 volumetric images with 134 cells in the EMT class. Abbasi, S. & Tajeripour, F. Detection of brain tumor in 3d MRI images using local binary patterns and histogram orientation gradient. The area of each nucleus was . Note that 3D SRP, 3D LBP and FV-CNN curves are overlapping in (a). 12), to determine the chromatin pattern alterations in 3D. Both processes share a reciprocal relationship, where DNA methylation is enriched in transcriptionally inactive heterochromatin, while histone acetylation is associated with transcriptionally active (or potentially active) euchromatin27. In this method, k-means clustering is performed and the Sum of Squared Error (SSE) is obtained for different values of k (32, 64, 96, 128, 160). Estandarte, A. K., Botchway, S., Lynch, C., Yusuf, M. & Robinson, I. Subsequently, descriptors from the three planes are concatenated to obtain the final 3D SRP feature descriptor for a cubic patch with dimension 240 (80 bins \(\times\) 3 planes): The patch-based feature description results in a large set of local feature descriptors of cubic patches which are often redundant and could deteriorate the classification performance. In addition, the proposed metrics based on 3D SRP validate the change in intensity and aggregation of heterochromatin on transition to another state and characterise the intermediate and ultimate phenotypic states. Microbiol Spectr. In the current results, 22 more SS cell objects, 25 fewer PROLIF cell objects, 103 and 141 more EMT and EPI cell objects are detected, respectively. Cell Biol. Article Make DAPI staining solution fresh on the day by adding 1.5 l of 1 mg/ml DAPI stock solution to 1 ml of . Dilute the DAPI stock solution to 30 nM in PBS. (b) F1 Score. Its blue fluorescence stands out in vivid contrast to green, yellow, or red fluorescent probes of other structures. 3d shape modeling for cell nuclear morphological analysis and classification. The significance of including third plane information for low-resolution volumetric images is also investigated by comparing the performance of 3D texture descriptor with its respective pseudo 3D form that ignores the interslice intensity correlations. This is a reversible process; hence when active transcription is required, heterochromatin is converted to euchromatin which is lightly packed (histone acetylation)28. This intensifies the salient information of the data, providing global features, and therefore, statistical analysis demonstrates the distinction of ratios more significantly. Google Scholar. Abstract. Classification performance is evaluated using AUC (Area-Under-the-Curve) of the ROC (Receiver-Operating-Characteristic) curve and the F1 score. In Fig. Values from each ring are sorted, concatenated and projected to a lower dimensional space while preserving the original distances between data points14. Leave the cells in ethanol at 20C for 515 minutes. The proposed 3D approach extends SRP by extracting features from the third plane using hyperplanes built in the cubic patches of the volumetric image (explained in Methods). It is followed by computing the ratio of HC to EC corresponding to the respective pixel values and pixel differences obtained from SRP functions. Deep Active Learning for Automatic Mitotic Cell Detection on HEp-2 Specimen Medical Images. In this study, we developed protocols for DAPI (4',6-diamidino-2-phenylindole) staining of Arcellinida nuclei and adapted protocols for ciliates. Overall performance evaluation implies that 3D SRP and 3D LBP performed better than all other considered handcrafted feature descriptors, while 3D SRP achieved better results than 3D LBP for the PC3 data set. Comput. Nowacka-Zawisza, M. & Winik, E. Dna methylation and histone modifications as epigenetic regulation in prostate cancer. With VGG-16 (a CNN model trained on ImageNet), the input image goes through a series of convolutional layers before it finally produces a dense set of local feature descriptors of 512 dimensions at the last fully convolutional layer. Pattern Recognit. DAPI staining solution: dissolve 1 mg of 4,6-diamidino-2-phenylindole (DAPI, Sigma #D9542) in 1 ml of water, filter-sterilise and store 100 l aliquots at 20 C. The choice of LBP and SIFT is also motivated from a recent review of texture descriptors that mentions SIFT and LBP as milestone texture feature descriptors15. At present single-cell surface marker/RNA sequencing (scRNA-Seq) is the accepted method to identify the intermediate states that occur during EMT in metastasis. Following light microscopic analyses, the stained cells were processed for electron microscopy. https://doi.org/10.1038/s41598-021-82985-9, DOI: https://doi.org/10.1038/s41598-021-82985-9. 2023 Feb 14;11(1):e0173322. 29, 915928 (2007). J. The distribution of nuclei with exceptionally strong DAPI staining (red, ">2C" from 2A) relative to the profile of anti-NeuN immunostaining intensity are indicated. Chromatin domains on mutation undergo disarrangement of heterochromatin (HC) (condensed chromatin) organisation and observe coarsening or opening of HC, resulting in an increase or loss of HC aggregates all around the nuclear matrix region, respectively6,7,8. Sci. J. Comput. Average nuclear diameter was measured using ImageJ software. Sezgin, M. & Sankur, B. The CellProfiler modules (see Supplementary Fig. Bag of Visual Words (BOVW)15 is then applied to generate the final 3D feature descriptor. Rep. 8, 114 (2018). S2 and S6). As the onset of morphological changes transpires following genetic alterations in the chromatin configuration inside the nucleus, the nuclear texture as one of the low-level properties if detected and quantified accurately has the potential to provide insights on nuclear organisation and enable early diagnosis and prognosis. Hoechst and DAPI are popular blue fluorescent, nuclear-specific dyes that can be used to stain live or fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. \(EMT*\) refers to the image set without \(EMT^{179}\). For images with slices fewer than \([2(2n+1)]\), z can be the same as the number of slices. (A) Representative images of DAPI-stained nuclei showing successful isolation of a healthy single nucleus (top) and nuclei aggregation . This protocol describes the use of DAPI to label nuclear DNA of cells grown in culture. Since the nucleolus contains very few pixels, we study only the DAPI images. Unlike BOVW where the image is represented by the number of occurrences of the visual word, FV encodes the gradient of the log likelihood of features with respect to the GMM parameters (mean vector, standard deviation vector and mixing weights). Signal Process. J. Electron. This work proposes a new approach to extract 3D texture, where multiple hyperplanes are built within the cubic patch to extract 3D SRP features. ADS Furthermore, two different cellular states in EMT class are identified and characterised, which are considered to hold critical biological significance when studying cancer progression and drug resistance. It is believed that DAPI associates with the minor groove of double-stranded DNA, with a preference for the adenine-thymine clusters. DAPI can be excited with a xenon or mercury-arc lamp or with a UV laser. Medeiros, R., Scharcanski, J. 2014 Jul;255(1):7-19. doi: 10.1111/jmi.12133. Meyer, F. Topographic distance and watershed lines. doi: 10.1101/pdb.prot5557. 2.4 Transfer the full volume of resuspended cells to 4 mL of absolute ethanol at -20C by pipetting the cell suspension slowly into the ethanol while vortexing at top speed. BMC Bioinform. Heterochromatin to euchromatin ratio (HC/EC) of both cell states is computed as explained in Methods and statistically compared to inspect the nature of changes in chromatin pattern on transition to another phenotypic state. Mol. P.R. From bow to cnn: Two decades of texture representation for texture classification. It is excited by the violet (405 nm) laser line and is commonly used as a nuclear counterstain in fluorescence microscopy, flow cytometry, and chromosome staining. Therefore, image classification based on cellular feature descriptors is recommended to comprehend the variations. Mach. In order to evaluate the performance of cellular textural feature descriptors for volumetric image-based classification, 10-fold cross-validation is used. Store frozen and protected from light. 3D LBP and FV-CNN curves are overlapping in (b). Subsequently, the fraction of the HC intensity in the nucleus is estimated using the following equation: HC aggregation measure HC/EC ratio based on pixel differences estimates the fraction of the HC aggregates in the nucleus. Vital staining of nucleus is a useful method in plant cell culture and protoplast fusion studies. 5a, \(x^S_{i,j}\) is the pixel in the ith square ring (S) in jth position, \({x}^{Sqr}\) is the final vector built by concatenating sorted pixels of m concentric squares and \(p_{i}\) is the number of pixels in the ith square. e Fluorescence, phase and adipogenesis-related mRNA relative expression of undifferentiated and differentiated FAPs stained with Nile Red (red) and DAPI (blue). ISSN 2045-2322 (online). Mol. Unlike other texture descriptors utilised in this study, there is currently no 3D version of SRP. SRP. Rueden, C. T. et al. 20, 249275 (2012). This final rinse will help reduce nonspecific background staining on the glass. Federal government websites often end in .gov or .mil. Liu, L. et al. In addition, instances of connected nuclei are resolved by applying the watershed algorithm38 wherever required (see Supplementary Fig. Slider with three articles shown per slide. 11, e1004569 (2015). Google Scholar. MathSciNet FV for deep learning features is employed following the work of Song et al.45. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). (1992), DAPI dihydrochloride, Invitrogen FluoroPure grade (=98% pure), Optional: antifade reagent (ex. & Maddox, P. S. Cenp-a: The key player behind centromere identity, propagation, and kinetochore assembly. designed the methodology. As demonstrated in Fig. & Kim, D.-H. Deciphering nuclear mechanobiology in laminopathy. Careers. in Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition Workshops 22722280, (2018). The two-sided Wilcoxon rank-sum test at the significance level of 1% is utilised to measure the difference in \(HC/EC_{PixelValues}\) and \(HC/EC_{PixelDifferences}\) between two classes for both cell lines. P.R. Each volumetric image of both cell lines is in 3D TIFF format and has dimensions \(1024 \times 1024 \times Z\) voxels, where Z ranges from 30 to 40 slices in the fibroblast cell collection and 6580 slices in the PC3 cell collection. performed the experiments, analysed the results and wrote the manuscript. In the early years of 3D texture description, Gray-Level Co-Occurrence Matrix (GLCM) descriptors were widely used for fluorescence microscopy images19. The https:// ensures that you are connecting to the A number of fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells, including Hoechst, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, propidium iodide, and acridine orange. Images are preprocessed following quality protocols and segmented to obtain individual cells with minimal background. Feature learning with component selective encoding for histopathology image classification. Since EMT is a reversible transition from epithelial to mesenchymal state in which cells do not necessarily exist in pure epithelial or mesenchymal state and comprise of multiple intermediate cell states (ICS) with hybrid features of pure states32. A z-stack of the . RP matrices can be constructed in various ways. Alexandr A. Kalinin, Ari Allyn-Feuer, Ivo D. Dinov, Saradha Venkatachalapathy, Doorgesh S. Jokhun, G. V. Shivashankar, Diego Herrez-Aguilar, Elena Madrazo, Javier Redondo-Muoz, Jude M. Phillip, Kyu-Sang Han, Pei-Hsun Wu, Zeynab Mousavikhamene, Daniel J. Sykora, Neda Bagheri, Hemaxi Narotamo, Maria Sofia Fernandes, Joo Miguel Sanches, Timea Toth, Tamas Balassa, Peter Horvath, Scientific Reports ROC curves: 3D SRP vs Pseudo 3D SRP (a) Fibroblast dataset. Sci Rep 11, 3364 (2021). In this study 3D Cell Nuclear Morphology Microscopy Imaging Dataset is used; the largest available public 3D image set obtained from Statistics Online Computational Resource (SOCR)10,11. However, all other subsamples performed as well as 3D SRP and 3D LBP. Accessibility Mach. In cross disciplines such as mechanobiology, nuclear morphological quantification has emerged as a promising approach to study the effect of external signals on nuclear morphology and their further impact on enclosed protein organisation. Cell-death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques. Following a standard cross-validation setup46, in each iteration, one subset is held out for model testing (an independent test set), and the remaining nine subsets are used to train the model (outer training set). A graph of SSE for each value of k is plotted, which usually takes the shape of an arm, and the value of k corresponding to the elbow of the arm is chosen as an optimal value which represents the least value of k after which SSE scarcely varies. Estimation of three-dimensional chromatin morphology for nuclear classification and characterisation. According to Kalinin et al.10, their segmentation results do not represent ground truth, as they are not hand labelled by an expert.
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