Imaging Biologically Induced Mineralization
DNA stains SYTO-9 (green) and propidium iodide (red). According to Invitrogen, SYTO-9 can pene- trate intact cell walls and stain the DNA whereas propidium iodide cannot penetrate intact cell walls of live cells and thus indicates “dead” cellular material. Figure 6a reveals the formation
Figure 4: (a) Illustration of a 1 mm square glass capillary flow-cell system. Minerals and bacteria are imaged through the glass ceiling of the capillary. Figure adapted from Pitts and Stewart [6]. (b) Photograph of the reactor being imaged.
attached to the glass surface of the capillary flow cells are surrounded by bacterial cells and extracellular polymeric substances. A second study was conducted with the goal of visualizing
the calcite surface topography in addition to the bacteria. Tis was achieved by overlaying a series of images taken in confocal reflection contrast mode with a series of fluorescent CLSM images. In this experiment, Invitrogen Corporation’s LIVE/ DEAD® stain was used, which consists of the fluorescent
of small precipitates alongside attached bacteria, and Figure 6b reveals the surface topography of a precipitate attached to the glass surface. Very few cells were visible between the mineral and the glass surface, suggesting that cells were either absent from this region or that
Figure 5: CLSM images of cells and biofilm stained with SYTO-11 (green) and C2d (red) inside capillary. (a) Image of flow cell highlighting the intimate relationship between attached cells and calcite surfaces. (b) 3-D reconstructed image of the perspective from inside the capillary showing cells and cell matrix surrounding calcite crystals. (c) 2-D CLSM image slice highlighting a cross section at the interior of precipitates. (d) 3-D reconstructed image of cells and cell matrix from the perspective of outside of the capillary. Only the outside of these clusters is touching the glass, and the apparent void space in the center is occupied by calcite crystals, which were not visualized in this image (see Figure 6 for reflection image overlays illustrating the presence of calcium carbonate deposits). Scale bars are 150, 30, 20, and 20 μm for a, b, c, and d, respectively.
14
Figure 6: (a) CLSM images of cells and biofilm stained with SYTO-11 (green) and propidium iodide (red) inside capillary. A reflection contrast mode image is overlaid, showing small CaCO3 precipitates in close proximity to bacterial cells, as labeled. (b) CLSM image from the perspective of outside the capillary. The overlaid reflection image indicates the surface topography of a large precipitate attached to the glass, and the fluorescent stains show the attached cells and cell-matrix surrounding the precipitates. According to Invitrogen, SYTO-9 can penetrate intact cell walls and stain the DNA, whereas propidium iodide cannot penetrate intact cell walls of live cells and thus indicates “dead” cellular material that may exist in the biofilm surrounding the precipitate. Scale bars are 10 and 15 µm for a and b, respectively.
www.microscopy-today.com • 2011 September
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84