Using multispectral imaging flow cytometry to assess an in vitro intracellular Burkholderia thailandensis infection model

Abstract

The use of in vitro models to understand the interaction of bacteria with host cells is well established. In vitro bacterial infection models are often used to quantify intracellular bacterial load by lysing cell populations and subsequently enumerating the bacteria. Modern established techniques employ the use of fluorescence technologies such as flow cytometry, fluorescent microscopy, and/or confocal microscopy. However, these techniques often lack either the quantification of large data sets (microscopy) or use of gross fluorescence signal which lacks the visual confirmation that can provide additional confidence in data sets. Multispectral imaging flow cytometry (MIFC) is a novel emerging field of technology. This technology captures a bright field and fluorescence image of cells in a flow using a charged coupled device camera. It allows the analysis of tens of thousands of single cell images, making it an extremely powerful technology. Here MIFC was used as an alternative method of analyzing intracellular bacterial infection using Burkholderia thailandensis E555 as a model organism. It has been demonstrated that the data produced using traditional enumeration is comparable to data analyzed using MIFC. It has also been shown that by using MIFC it is possible to generate other data on the dynamics of the infection model rather than viable counts alone. It has been demonstrated that it is possible to inhibit the uptake of bacteria into mammalian cells and identify differences between treated and untreated cell populations. The authors believe this to be the first use of MIFC to analyze a Burkholderia bacterial species during intracellular infection.

Optimization of the culture condition of Bacillus mucilaginous using Agaricus bisporus industrial wastewater by Plackett–Burman combined with Box–Behnken response surface method

“…With an appropriate mixture of SYTO 9 and PI stains, bacteria with intact cell membranes stain fluorescent green, scored as viable ones; and bacteria with injured membranes stain fluorescent red, scored as non-viable ones (Leuko et al 2004; Hu et al 2017). And the fluorescent staining of bacterial cells was quantified with multispectral imaging flow cytometry by fluorescent signals and microscopic image (Berney et al 2007; Jenner et al 2016). Therefore, the B. mucilaginous viable and dead cells and particles in the fermented liquid were quickly and accurately identified and quantified by flow cytometry in a statistically sound manner.…”

Masks in imaging flow cytometry

“…A better version of Erode mask, called Adaptive Erode, erodes pixels adapting to the morphology of the object and can be applied for a variety of analysis including shape change. To analyze the bacterial burden in mammalian cells by IFC, Jenner and colleagues [2] made use of the Spot mask to identify the number of bacteria per cell, evaluating bright spots in the referenced channel of the bacteria stain in the cell. Because they were evaluating internalization of two different types of bacteria with distinct staining pattern, they decided to refine the Spot mask, using a Threshold mask with different cut-offs from the background to be able to identify each type.…”

“…The literature is replete with examples of Boolean logic applied to masks for IFC. For example, Jenner et al, [2] used a combination of an intracellular and spot counting masks to enumerate intracellular bacteria. In their study of radiation damage to lymphocytes, Durdik and coworkers [7] used custom masks which combined Spot, Peak, and Intensity features.…”

“…One way to do this is use the Raw Max Pixel feature to exclude those events close to the background and only analyze cells truly positive for the spot-signal to be counted. Jenner and colleagues [2] present excellent examples of good and poor masks used for spot counting in their study of intracellular Burkholderia thailandensis infection.…”

Veterinary Pathology – A Path Forward with New Directions and Opportunities

“…The introduction of powerful advanced techniques, such as imaging flow cytometry (19–21) and histology-directed imaging mass spectrophotometry (22), is yielding new insights into disease pathogenesis. As dedicated instruments become more commonplace and cost-effective, it will only be a matter of time before these technologies enter the diagnostic realm.…”