Masks in imaging flow cytometry

Abstract

Data analysis in imaging flow cytometry incorporates elements of flow cytometry together with other aspects of morphological analysis of images. A crucial early step in this analysis is the creation of a mask to distinguish the portion of the image upon which further examination of specified features can be performed. Default masks are provided by the manufacturer of the imaging flow cytometer but additional custom masks can be created by the individual user for specific applications. Flawed or inaccurate masks can have a substantial negative impact on the overall analysis of a sample, thus great care must be taken to ensure the accuracy of masks. Here we discuss various types of masks and cite examples of their use. Furthermore we provide our insight for how to approach selecting and assessing the optimal mask for a specific analysis.

Imaging flow cytometry assays for quantifying pigment grade titanium dioxide particle internalization and interactions with immune cells in whole blood

“…Nonetheless, flow imaging based assays, similar to conventional flow cytometric assays, are able to incorporate additional read outs such as staining for markers of immune activation and function thereby allowing direct associations to be made between TiO 2 interactions with single cells (adhesion or internalization) and the functional consequences of such interactions on the same cell. Like others, we found imaging cytometry a powerful technique for the analysis of particle localization, this was dependant on tailoring custom masks in order to obtain an accurate analysis 9, 10, 11, 12, 13, 14, 15. There is always potential for some particles analyzed to be attached externally to the front or back of the cells, the occurrence of false positive internalization events is random and at the same frequency within all data files as it is dependent only on the orientation of the cells as they are imaged.…”

“…Boxplots display Q1-Q3 with whiskers set at 1.5 3 IQR (interquartile range) above the third quartile and 1.5 3 IQR below the first quartile, minimum or maximum values that have fallen outside this range are shown as outliers (small black dots). [Color figure can be viewed at wileyonlinelibrary.com] of particle localization, this was dependant on tailoring custom masks in order to obtain an accurate analysis (9)(10)(11)(12)(13)(14)(15). There is always potential for some particles analyzed to be attached externally to the front or back of the cells, the occurrence of false positive internalization events is random and at the same frequency within all data files as it is dependent only on the orientation of the cells as they are imaged.…”

“…Inevitably, the microscopy is not as high resolution or flexible as that of a high end dedicated microscope and neither is its flow cytometric capability as flexible or high throughput as a dedicated high end flow cytometer. However, when it comes to the study of cell-particle interactions in complex (mixed cell) suspensions, it is truly fit for purpose: in particular for the assessment of particle or bacteria internalization and for quantitation of particle-cell localization (9)(10)(11)(12)(13)(14)(15). TiO 2 is not fluorescent, but in pigment grade sizes, it is highly reflective with excellent light scattering properties.…”

“…Our approach was stepwise, first validating darkfield measures by imaging flow cytometry of TiO 2 -cell interactions in peripheral blood mononuclear cells (PBMCs) against conventional flow cytometry side scatter (SSC) measurements (18). We then determined internalization and outer membrane adherence of TiO 2 particles with cells residing within PBMC and whole blood by adapting conventional particle internalization methods for imaging flow cytometry (15). Finally, we demonstrated that the very low levels of TiO 2 that are anticipated to occur in whole blood in vivo following oral challenge are discernable by this technique and cell targets can be identified.…”

Detection and Quantification of Histone H4 Citrullination in Early NETosis With Image Flow Cytometry Version 4

“…For data analysis with the IDEAS software, custom masks should be employed because they represent more accurately the region of interest, rather than the default masks. Custom masks for total cell area and nucleus should be first validated for accuracy, by confirming that the masks are indeed masking the region(s) of interest (i.e., not missing parts of the target region or, on the opposite, masking "ghost" regions outside of what is relevant for the features) (17).…”