Fig 1.
The images in Fig 1 (all panels) show ARPE-19 cells incubated with 3 μg/ml AgNP citrate.
The cells were transfected with mitochondria-GFP (green), stained with CellMask Orange plasma membrane stain (C10045, red), fixed with 4% PF, and mounted with Prolong gold that contained DAPI to stain the nuclei (blue). This allowed for the visualization of cells, and nanoparticles (white) due to reflected light in the darkfield images. The images were observed using a 20x multi-immersion objective (NA 0.75). The images in each panel are a darkfield image and fluorescence images that were acquired sequentially and then combined into one composite image. Panel A. Image of four rings of silver nanoparticles on the cell culture slide. Cells are nearby or adjacent to the rings. Magnification 200x. Panel B. Image of five rings and adjacent cells. Magnification 200x. Panel C. Image of six rings and multiple cells. Magnification 200x. Panel D. Image of two circular structures consisting of nanoparticles, and a cell that was stained with CellMask Orange plasma membrane stain (C10045), transfected with mitochondria-GFP (green) and stained with DAPI (blue) to reveal the nucleus. The cell in the Panel D is about the same size as the nanoparticle circles in the image. Magnification 200x.
Fig 2.
Four different fields of cells treated with 3ug/ml AgNP citrate contained circles that was visualized obtained with darkfield optics using a 60-x plan Fluor objective with an iris diaphragm (NA 0.55–0.90).
Panel A. Two cells are located outside the nanoparticle circle that have an accumulation of intracellular nanoparticles that are brighter than the extracellular nanoparticles. The particles in the extracellular ring have similar brightness to other extracellular particles. Magnification 600x. Panel B. Two cells in mitosis is visible inside the nanoparticle circle. The cells were stained with CellMask Orange plasma stain (red) to reveal the cytoplasm and DAPI (blue) to reveal the nucleus. Since the mitotic cells are round, some of the colors of red cytoplasm have blended with the blue nuclei to yield a purple color. Cells were transfected with Mito-GFP (green). The two apparently mitotic cells are surrounded by a single nanoparticle ring structure. The circle diameter size is 54 μm, the center hole diameter is 30 μm and nuclear diameter is 18.7 μm. Magnification 600x. Panel C. Two post-mitotic daughter cells were observed near a large ring structure. The circle diameter is 56 μm, hole diameter is 23 μm, and circle wall thickness is 16 μm. Magnification 600x. Panel D. Two post-mitotic daughter cells were observed within a larger ring structure The circle diameter is 62 μm, the center diameter is 30 μm, and circle rim thickness is 15 μm. Magnification 600x.
Fig 3.
The four-panel figure shows the individual dark field and fluorescent images that were combined into a composite image.
A 60-x plan Fluor objective with an iris diaphragm (NA 0.55–0.90 was used to obtain the image. Panel A. A darkfield micrograph of a cell with an adjacent ring structure. The diameter of the circle is 53 μm and the hole is 32 μm. The longest direction is 90 μm and the nuclear diameter is 24 μm. The intensity of nanoparticles contained in the cell are brighter than the ones contained in the ring structure. Panel B. A composite image consisting of 3 fluorescent images and a darkfield image The composite image was deconvolved using Nikon Autoquant 2D “blind” program, which increased the resolution of the fluorescent probes, especially the Mito- GFP. Panel C. A fluorescent image showing mitochondrial Mito-GFP. (green). Nikon Autoquant 2D “blind” program was used which increased the resolution of the Mito-GFP probe. Panel D. A fluorescent image of the cytoplasm Cell Mask Orange membrane stain. Magnification 600x.
Fig 4.
A single ring structure was obtained with darkfield optics using a 60-x plan Fluor objective with an iris diaphragm (NA 0.55–0.90).
Panel A. A darkfield image of silver nanoparticles showing a concentrated ring structure. Panel B. A fluorescent image of the same field as Panel A after the slide was stained with Cell Mask Orange plasma membrane stain (red). A red ring of membrane proteins was observed corresponding to the circular formation of nanoparticles in Panel A. This suggests that cell membrane components remained on the slide after completion of mitosis. These residual materials were part of the cell membrane that preferentially attracted and/or bound silver nanoparticles that were deposited on the slide surface. The diameter of the circle was 53 μm, the inside circle was 27 μm, and the fluorescent ring was 37 μm. Magnification 600x., AgNP citrate 3ug/ml.
Fig 5.
Histogram consisting of 90 perpendicular diameter measurements from 45 nanoparticle circles.
Fig 6.
Four different fields of 3ug/ml AgNP citrate obtained with darkfield optics using a 60-x plan Fluor objective with an iris diaphragm (NA 0.55–0.90).
Panel A. Intracellular nanoparticles are brighter than the extracellular particles. Panel B. The cytoplasm was stained with CellMask Orange plasma membrane stain (red). Panel C. Cells transfected with Mito-GFP (green). Panel D. A composite image of fields A, B, and C were combined with an image of DAPI stained nuclei (blue). Magnification 600x.
Fig 7.
Four different fields of 3ug/ml AgNP citrate obtained with darkfield optics using a 60-x plan Fluor objective with an iris diaphragm (NA 0.55–0.90).
Panel A. Cells were stained with Lysosome- RFP (red) to reveal the lysosomes. Panel B. Intracellular nanoparticles (white) were condensed into the identical regions occupied by lysosomes in A. Panel C. Cells transfected with Mito-GFP (green). Panel D. A composite image of fields A, B, and C were combined with DAPI stained nuclei (blue). Magnification 600x.
Fig 8.
The four-panel figure of cell shown in Fig 2A was treated with 3ug/ml AgNP.
The individual dark field and fluorescent images were combined into a composite image (C) Panel A. Darkfield image of a cell with an adjacent ring structure. Panel B. Cell stained with CellMask Orange plasma membrane stain. Panel C. Composite image of fields A, B, and C combined with DAPI stained nuclei (blue). Panel D. Cells transfected with Mito-GFP (green). Magnification 600x.
Fig 9.
The four-panel figure of cell shown in Fig 2B was treated with 3ug/ml AgNP.
Panel A. Darkfield image of a ring structure with 2 newly divided cells laying on top of ring structure. Particles in cell are larger and brighter than particles in ring or extracellular. Panel B. The individual dark field (A) and fluorescent images (C, D) were combined into a composite image (B). DAPI stained nuclei (blue) image was added to the composite. Panel C. Cell stained with CellMask Orange plasma membrane stain. Panel D. Cells transfected with Mito-GFP (green). Magnification 600x.
Fig 10.
The four-panel figure of cell shown in Fig 2C was treated with 3ug/ml AgNP.
Panel A. The individual dark field (B) and fluorescent images (C, D) were combined into a composite image (A). DAPI stained nuclei (blue) image was added to the composite image. Panel B. Darkfield image of a ring structure with 2 newly divided cells adjacent to the top of ring structure. Panel C. Cells transfected with Mito-GFP (green). Panel D. Cell stained with CellMask Orange plasma membrane stain. Magnification 600x.
Fig 11.
The four-panel figure of cell shown in Fig 2D was treated with 3ug/ml AgNP.
Panel A. Darkfield image of a ring structure with 2 newly divided cells on top of ring structure. Panel B. Composite of individual dark field (A) and fluorescent images (C, D) were combined into a composite image (B). DAPI stained nuclei (blue) was added to the composite. Panel C. Cells transfected with Mito-GFP (green). Panel D. Cell stained with CellMask Orange plasma membrane stain. Magnification 600x.
Fig 12.
The four-panel figure of cell treated with 3ug/ml AgNP.
Panel A. Darkfield image of a ring structure with 2 newly divided daughter cells on top of ring structure. Panel B. The individual dark field (A) and fluorescent images (C, D) were combined into a composite image (B). DAPI stained nuclei (blue) image was added to the composite. Panel C. Cells transfected with Mito-GFP (green). Panel D. Cell stained with CellMask Orange plasma membrane stain. Magnification 600x.
Fig 13.
ARPE-19 cells incubated with 10 μg/ml AgNP citrate.
The cells were transfected with mitochondria-GFP (green), stained with CellMask Orange plasma membrane stain (C10045, red), fixed with 4% PF, and mounted with Prolong gold that contained DAPI to stain the nuclei (blue). Field shows a dense accumulation of nanoparticles, two ring structures and 5 cells. The nanoparticles inside the cell are brighter than the particles outside the cell. The image consists of a combination of one darkfield and three fluorescence images that were acquired sequentially and then combined into one composite. Magnification 200x.