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Seeing the Whole Elephant: Imaging Flow Cytometry Reveals Extensive Morphological Diversity within Blastocystis Isolates.

Yason JA, Tan KS - PLoS ONE (2015)

Bottom Line: The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans.Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate.We discuss the possible biological implications of these unusual forms.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT
Blastocystis is a common protist isolated in humans and many animals. The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans. There are biological and molecular differences between Blastocystis subtypes although microscopy alone is unable to distinguish between these subtypes. Blastocystis isolates also display various morphological forms. Several of these forms, however, have not been properly evaluated on whether or not these play significant functions in the organism's biology. In this study, we used imaging flow cytometry to analyze morphological features of Blastocystis isolates representing 3 subtypes (ST1, ST4 and ST7). We also employed fluorescence dyes to discover new cellular features. The profiles from each of the subtypes exhibit considerable differences with the others in terms of shape, size and granularity. We confirmed that the classical vacuolar form comprises the majority in all three subtypes. We have also evaluated other morphotypes on whether these represent distinct life stages in the parasite. Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate. Granular forms were present as a continuum in both viable and non-viable populations, with non-viable forms displaying higher granularity. By analyzing the images, rare morphotypes such as multinucleated cells could be easily observed and quantified. These cells had low granularity and lower DNA content. Small structures containing nucleic acid were also identified. We discuss the possible biological implications of these unusual forms.

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Initial gating strategy to analyze Blastocystis cells.Cells were gated for focused cells using brightfield channel, then selection of single cells using aspect ratio and area units, and finally to classify viable and non-viable cells using PI-staining characteristics. The above graphs shows the analysis for Blastocystis ST1-NUH9 isolate (A). Subsequent analyses made use of features arising from Hoechst and CFSE staining characteristics as well as features from brightfield and side-scatter channels (B).
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pone.0143974.g001: Initial gating strategy to analyze Blastocystis cells.Cells were gated for focused cells using brightfield channel, then selection of single cells using aspect ratio and area units, and finally to classify viable and non-viable cells using PI-staining characteristics. The above graphs shows the analysis for Blastocystis ST1-NUH9 isolate (A). Subsequent analyses made use of features arising from Hoechst and CFSE staining characteristics as well as features from brightfield and side-scatter channels (B).

Mentions: In order to analyze cultures at the same stage of growth, 2-day old cultures of Blastocystis isolates WR1 and B and 7-day old culture of isolate NUH9 were harvested. The cells were washed twice by centrifugation at 1,000 x g using warm PBS. 2 x 107 cells in 200 μL PBS were collected into 1.5 μL microtubes. The cell suspensions were then stained with 1 μg/mL propidium iodide (PI) (BioVision, Mountain View, CA), 5 μM carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies, Eugene, OR) and 1 μg/mL Hoechst 33342 (Life Technologies, Eugene, OR) for 15 mins. PI stain was used to select for viable and non-viable cells. Cells will only take up PI when there is membrane disruption. CFSE is used in proliferation studies and is found to stain vacuolar compartments of Blastocystis [8]. Hoechst stains the DNA and is useful for cell-cycle analyses. Actively dividing cells will have higher emission while dying cells undergoing DNA fragmentation will have lower fluorescence readings. The cells were then washed to remove excess stains and fixed in 2% formaldehyde. Single stained cells were also prepared and used to create a compensation matrix. Blastocystis cell suspension heated to 80°C for 15 mins was used as positive control for PI-staining. We used Amnis ImageStream MarkII (Merck Millipore, Seattle, WA) with 4-laser attachment (375, 488, 561 and 642) to acquire Blastocystis cell images. 2,000 events were obtained with low flow speed at 60x magnification. Images at extended depth of field (EDF) setting were also acquired. EDF involves deconvolution to obtain highly focused images. Gating strategy involved selecting for focused cells using RMS gradient values, then for single cells using brightfield aspect ratio (Fig 1). Viable Blastocystis cells were identified as those without PI-staining. Cell shapes were characterized using aspect ratios from brightfield and CFSE staining. Acquisition was done using 3 different batches of cultures. Analysis of images was performed using IDEAS software version 6.1.


Seeing the Whole Elephant: Imaging Flow Cytometry Reveals Extensive Morphological Diversity within Blastocystis Isolates.

Yason JA, Tan KS - PLoS ONE (2015)

Initial gating strategy to analyze Blastocystis cells.Cells were gated for focused cells using brightfield channel, then selection of single cells using aspect ratio and area units, and finally to classify viable and non-viable cells using PI-staining characteristics. The above graphs shows the analysis for Blastocystis ST1-NUH9 isolate (A). Subsequent analyses made use of features arising from Hoechst and CFSE staining characteristics as well as features from brightfield and side-scatter channels (B).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664278&req=5

pone.0143974.g001: Initial gating strategy to analyze Blastocystis cells.Cells were gated for focused cells using brightfield channel, then selection of single cells using aspect ratio and area units, and finally to classify viable and non-viable cells using PI-staining characteristics. The above graphs shows the analysis for Blastocystis ST1-NUH9 isolate (A). Subsequent analyses made use of features arising from Hoechst and CFSE staining characteristics as well as features from brightfield and side-scatter channels (B).
Mentions: In order to analyze cultures at the same stage of growth, 2-day old cultures of Blastocystis isolates WR1 and B and 7-day old culture of isolate NUH9 were harvested. The cells were washed twice by centrifugation at 1,000 x g using warm PBS. 2 x 107 cells in 200 μL PBS were collected into 1.5 μL microtubes. The cell suspensions were then stained with 1 μg/mL propidium iodide (PI) (BioVision, Mountain View, CA), 5 μM carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies, Eugene, OR) and 1 μg/mL Hoechst 33342 (Life Technologies, Eugene, OR) for 15 mins. PI stain was used to select for viable and non-viable cells. Cells will only take up PI when there is membrane disruption. CFSE is used in proliferation studies and is found to stain vacuolar compartments of Blastocystis [8]. Hoechst stains the DNA and is useful for cell-cycle analyses. Actively dividing cells will have higher emission while dying cells undergoing DNA fragmentation will have lower fluorescence readings. The cells were then washed to remove excess stains and fixed in 2% formaldehyde. Single stained cells were also prepared and used to create a compensation matrix. Blastocystis cell suspension heated to 80°C for 15 mins was used as positive control for PI-staining. We used Amnis ImageStream MarkII (Merck Millipore, Seattle, WA) with 4-laser attachment (375, 488, 561 and 642) to acquire Blastocystis cell images. 2,000 events were obtained with low flow speed at 60x magnification. Images at extended depth of field (EDF) setting were also acquired. EDF involves deconvolution to obtain highly focused images. Gating strategy involved selecting for focused cells using RMS gradient values, then for single cells using brightfield aspect ratio (Fig 1). Viable Blastocystis cells were identified as those without PI-staining. Cell shapes were characterized using aspect ratios from brightfield and CFSE staining. Acquisition was done using 3 different batches of cultures. Analysis of images was performed using IDEAS software version 6.1.

Bottom Line: The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans.Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate.We discuss the possible biological implications of these unusual forms.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT
Blastocystis is a common protist isolated in humans and many animals. The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans. There are biological and molecular differences between Blastocystis subtypes although microscopy alone is unable to distinguish between these subtypes. Blastocystis isolates also display various morphological forms. Several of these forms, however, have not been properly evaluated on whether or not these play significant functions in the organism's biology. In this study, we used imaging flow cytometry to analyze morphological features of Blastocystis isolates representing 3 subtypes (ST1, ST4 and ST7). We also employed fluorescence dyes to discover new cellular features. The profiles from each of the subtypes exhibit considerable differences with the others in terms of shape, size and granularity. We confirmed that the classical vacuolar form comprises the majority in all three subtypes. We have also evaluated other morphotypes on whether these represent distinct life stages in the parasite. Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate. Granular forms were present as a continuum in both viable and non-viable populations, with non-viable forms displaying higher granularity. By analyzing the images, rare morphotypes such as multinucleated cells could be easily observed and quantified. These cells had low granularity and lower DNA content. Small structures containing nucleic acid were also identified. We discuss the possible biological implications of these unusual forms.

Show MeSH