clsm-upright - nikon/ni-e  (Nikon)

Journal: PNAS Nexus

Article Title: Clear as mud redefined: Tunable transparent mineral scaffolds for visualizing microbial processes below ground

doi: 10.1093/pnasnexus/pgaf118

Figure Lengend Snippet: Pore size distributions of different freeze-cast synthetic cryolite scaffolds measured by mercury intrusion porosimetry (first column) and visualized in SEM images. Longitudinal (second column) and transverse (third column) cuts are shown for a) 12 vol.% dendritic, b) 24 vol.% dendritic, c) 12 vol.% columnar, and d) 12 vol.% isotropic scaffolds. The white arrow on the image of the 12 vol.% dendritic scaffold points to the pore wall of the primary pore channel, with darker secondary pores branching off from it. The fourth column shows the saturated pore space (in green) dyed with a fluorescein solution for the four different pore morphologies imaged by confocal microscopy at 500–550 nm. Images of additional fluorescence channels are provided in Fig. .

Article Snippet: All confocal images were acquired using an upright Zeiss 880 CLSM (Zeiss, Oberkochen) using 10× or 100× objectives (Plan-Apochromat 10×/0.45 M27 and alpha Plan-Apochromat 100×/1.46 Oil DIC M27 objectives).

Techniques: Pore Size, Confocal Microscopy, Fluorescence

Journal: PNAS Nexus

Article Title: Clear as mud redefined: Tunable transparent mineral scaffolds for visualizing microbial processes below ground

doi: 10.1093/pnasnexus/pgaf118

Figure Lengend Snippet: Comparison of cryolite transparency with other freeze-cast materials using micron-sized fluorescent beads. a) Confocal microscopy images of fluorescent beads at the surface (0 µm) and at 20 µm depth within scaffolds composed of SiOC, SiO 2 , and cryolite. Scale bar is 25 µm in each. b) The intensity of bead fluorescence (1 µm diameter), normalized to the maximum bead brightness and shown as a function of depth for the four cryolite scaffold morphologies. A comparison of bead brightness in scaffolds of SiOC and SiO 2 is shown. The intensities of 10 beads were averaged at each depth until the fluorescent beads were no longer visible (<20 µm in the case of SiOC). c) Lateral intensity profiles of 0.5, 1, and 2 µm diameter beads at the surface and at 100 µm depth in the cryolite scaffold with 95% CIs indicates cell-sized particles as small as 0.5 µm can still be confidently resolved to at least 100 µm depth.

Article Snippet: All confocal images were acquired using an upright Zeiss 880 CLSM (Zeiss, Oberkochen) using 10× or 100× objectives (Plan-Apochromat 10×/0.45 M27 and alpha Plan-Apochromat 100×/1.46 Oil DIC M27 objectives).

Techniques: Comparison, Confocal Microscopy, Fluorescence

Journal: PNAS Nexus

Article Title: Clear as mud redefined: Tunable transparent mineral scaffolds for visualizing microbial processes below ground

doi: 10.1093/pnasnexus/pgaf118

Figure Lengend Snippet: Tracking the movement of cell-sized fluorescent beads within different scaffold morphologies. Traces of the travel paths for 1 µm beads were visualized every 20 s over a 10-min period and overlaid on fluorescent (405 nm) confocal microscopy images for a) 12 vol.% dendritic, b) 24 vol.% dendritic, c) 12 vol.% isotropic, and d) 12 vol.% columnar porous cryolite scaffolds. In all images, the open-pore space is black, and the cryolite scaffold material appears blue due to some autofluorescence of cryolite in the UV range when excited by a 405-nm laser. e) The velocity of all measured 1 µm beads in each scaffold plotted against the intensity of blue pixels in the 25 × 25 pixel area surrounding each bead (with blue intensity serving as a proxy for the cryolite scaffold versus black open-pore space). f) Blue background intensities (a.u.) for representative 25 × 25 pixel areas with open-pore space showing low (blue background = 14 a.u.), medium (blue background = 61 a.u.), and high regions of cryolite (blue background = 229 a.u.) used in (e).

Article Snippet: All confocal images were acquired using an upright Zeiss 880 CLSM (Zeiss, Oberkochen) using 10× or 100× objectives (Plan-Apochromat 10×/0.45 M27 and alpha Plan-Apochromat 100×/1.46 Oil DIC M27 objectives).

Techniques: Confocal Microscopy

Journal: PNAS Nexus

Article Title: Clear as mud redefined: Tunable transparent mineral scaffolds for visualizing microbial processes below ground

doi: 10.1093/pnasnexus/pgaf118

Figure Lengend Snippet: Environmental deployment of a cryolite scaffold shows robust in situ microbial colonization deep into the cryolite pore network. A 12 vol.% dendritic cryolite scaffold was directly inserted into seagrass rhizosphere sediment for 4 weeks, fixed in paraformaldehyde, and colonizing microorganisms stained with the nucleic acid stain SYBR Gold. Confocal microscopy maximal depth projections at 10× (panels a–d) and 100× (panels e and f) magnification. a) The cryolite matrix (blue autofluorescence) and open-pore space (black) and b) a magnification of the inset box in (a), showing SYBR-stained environmental microorganisms (in green) within the cryolite matrix (blue). Cells were visible even at low magnification (10×) and deep within the scaffold (85 µm depth, still frame taken from ). c) A maximum intensity projection (MIP) of a 129-µm z -stack (associated with , representing an overlay of DAPI and FITC channels and d) the corresponding color-coded depth projection (CCP) of the same image showing environmental microorganisms distributed at multiple depths throughout the scaffold. The color scale of the CCP represents depth, with each color bar representing 8.08 µm increments. Filamentous cells marked with a blue arrow span 32 to 40 µm depth, while filamentous cells on the right with the red arrow were located deeper in the scaffold, between 80 and 100 µm. Single cells were resolved as deep as 125 µm (white arrow). Using a 100× objective allows higher resolution images of cells within the cryolite matrix, but at comparatively shallower depths due to the smaller depth of field of the objective. e) A higher magnification MIP (FITC channel only showing SYBR-stained cells) and corresponding (f) CCP of an 11-µm z -stack collected with a 100× objective show well-resolved cells of various morphologies, including fine filamentous cells, thicker chains of cells, single bacilli, and cocci. The color scale bar of CCP begins at 10 µm to indicate that imaging did not begin at the coverslip (0 µm) but deeper into the matrix, from 10 to 21 µm. Individual channel images are provided in Fig. .

Article Snippet: All confocal images were acquired using an upright Zeiss 880 CLSM (Zeiss, Oberkochen) using 10× or 100× objectives (Plan-Apochromat 10×/0.45 M27 and alpha Plan-Apochromat 100×/1.46 Oil DIC M27 objectives).

Techniques: In Situ, Staining, Confocal Microscopy, Imaging