PubMed · 9170189
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MOTIVATION: 10x Genomics VisiumHD enables spatial transcriptomics at 2 µm × 2 µm resolution but exhibits slide-specific, non-periodic striping artifacts due to lane-width variability. These multiplicative row/column effects distort bin total counts and can bias downstream analyses. The state-of-the-art destriping approach is the normalization procedure used as a preprocessing step in bin2cell; it applies sequential high-quantile row- then column-wise normalization, which is asymmetric and can introduce edge effects/macro-stripes and distortions of large-scale total-count structure. RESULTS: We propose a statistical destriping approach that leverages nuclei segmentation from the co-registered H&E image. Assuming transcript abundance is constant within each nucleus, we model bin counts with a negative binomial distribution whose mean is a product of a nucleus-specific concentration and row- and column-specific stripe-factors reflecting lane-width variation. We fit all parameters in a generalized linear modeling framework with cross-validated regularization on stripe-factors and iterative dispersion estimation, and use the fitted parameters to correct the observed counts into a destriped image. On synthetic data with known ground truth, our method improves stripe-factor estimation accuracy and reduces error in corrected counts relative to bin2cell and bin2cell-derived baselines. Across four public VisiumHD slides, it consistently lowers striping intensity while substantially better preserving biological signal present in the large-scale global count structure and avoiding the artifacts introduced by other methods. AVAILABILITY AND IMPLEMENTATION: All source code and links to publicly available data used for this study are available at https://github.com/paolamalsot/destriping-GLM.
Eduard Kellenberger understood that the conventional resin-embedding, he helped to develop (Ryter, A., Kellenberger, E., 1958. L'inclusion au polyester pour l'ultramicrotomie. J. Ultrastruct. Res. , 2, 200-214), was prone to aggregation artifacts (Kellenberger, E., 1987. The response to biological macromolecules and supramolecular structures to the physics of specimen cryo-preparation. In: Steinbrecht, R.A., Zierold, K. (Eds.), Cryo-techniques in Biological Electron Microscopy, Springer, Berlin, pp. 35-63). He was instrumental in developing various methods to overcome this limitation, for instance, by using low temperature-embedding and partially hydrophilic resins (Carlemalm, E., Garavito, R.M., Villiger, W., 1982. Resin development for electron microscopy and an analysis of embedding at low temperature. J. Microstruct., 126, 123-143; Villiger,W., 1993. Low temperature-embedding with Lowicryl resins. In: Robards, A.W., Wilson, A.J. (Eds.), Procedures in electron microscopy, Wiley, Chichester, UK, pp. 16:7.3-16:7.6). In principle, cryo-electron microscopy of vitreous sections is free of any aggregation artifact since the material remains fully hydrated and is free of chemical fixation or staining. The method is technically difficult still, but recent progress has made it amenable to routine practical applications. We compare here electron microscopical aspects of Zea mays meristem cells prepared by: (1) conventional resin-embedding and sectioning; (2) low temperature-embedding and sectioning of freeze substituted samples; and (3) cryo-sections of vitrified samples. The appearance of the extra-cellular space, the cytoplasm and the nucleoplasm are very different in conditions (1) and (3). They appear as compact, irregular and well delineated structures in conventional resin sections, whereas they are more diffuse and homogeneous in the vitreous sections. In the resin sections, the material seems to form a complex matrix, whereas it looks more like a thick soup in the vitreous sample. Low temperature-embedding (condition 2) shows an intermediate appearance. We suggest that regardless of the difference due to staining and different sectioning conditions, the other image differences are the consequence of aggregation artifacts in the resin-embedded specimens.
There is considerable current interest in the possible beneficial health effects of quercetin, catechins, epigallocatechins, epigallocatechin gallates, and related phenolic compounds found in teas, wines, and other plant products. As a result, many laboratories are studying the effects of these compounds on cells in culture. The present paper shows that addition of these compounds to commonly used cell culture media leads to generation of substantial amounts of hydrogen peroxide (H(2)O(2)). Dulbecco's modified Eagle medium gives the highest H(2)O(2) level for all the compounds tested, with levels reaching >400 microM within 2 h for addition of 1 mM concentrations of gallic acid, epigallocatechin gallate, and epigallocatechin. Catechin and quercetin produced lower, but still significant, levels of H(2)O(2). McCoy's 5A and RPMI 1640 media also promoted H(2)O(2) production from the above phenolic compounds. This rapid generation of H(2)O(2) could account for some or all of the reported effects of phenolic compounds on cells in culture.