Abstract
There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.
Highlights
There has been an exponential increase in the release of anthropogenic pollutants, such as pesticides[1], toxins[2], pharmaceutical drugs[3], personal care products[4], microbeads[5], and nanoparticles[6], into our environment
We demonstrate for the first time simultaneous Atomic force microscopy (AFM)-quantitative imaging (QI)-Laser scanning confocal microscopy (LSCM) imaging of living bacteria, yeast and mammalian cells responding to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in situ, with the continuous monitoring of morphology, surface ultrastructure, mechanical properties, adhesion and tracking multiple fluorescently tagged molecules in real time
It was important to characterise changes to cell morphology, surface ultrastructure and physical properties under conditions favorable for E. coli proliferation to produce data relevant for assessing changes associated with exposure to stress
Summary
There has been an exponential increase in the release of anthropogenic pollutants, such as pesticides[1], toxins[2], pharmaceutical drugs[3], personal care products[4], microbeads[5], and nanoparticles[6], into our environment. Since conventional AFM and CM are temporally[17] and diffraction[18] limited, respectively, and both are capable of studying live cells under physiologically relevant conditions, they make an excellent pair for physical integration and routine simultaneous correlative imaging. Correlative AFM-LSCM has never successfully produced routine, high content data for live, actively growing cells, in particular bacteria[32], to assess their response to environmental contaminants in real-time (Fig. 1). We demonstrate for the first time simultaneous AFM-QI-LSCM imaging of living bacteria, yeast and mammalian cells responding to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in situ, with the continuous monitoring of morphology, surface ultrastructure, mechanical properties, adhesion and tracking multiple fluorescently tagged molecules in real time. The novel use of this method to probe Escherichia coli, Candida albicans and HEK 293 cells in response to a xenobiotic during active cell division highlights the versatility of the method, with future broad application for assessing the impact of virtually any type of anthropogenic contaminant on most cell types
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