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: Freeze-cast cryolite is compatible with FISH. Confocal image of a 12 vol.% dendritic cryolite scaffold colonized by a bacterial co-culture of P. carrageenovora and R. nubinhibens . Cells were fixed with paraformaldehyde and then hybridized with the general bacterial oligonucleotide probe, Eub338mix labeled with Alexa 488 and counterstained with the DNA stain DAPI (panels b and a). The passive dye AlexaFluor647 was additionally added to the aqueous solution visualize the pore structure (panel c). The overlay of the individual channels is displayed in (panel d). A negative control for FISH of the same co-culture without the addition of Eub338 is provided in Fig. .

Article Snippet: FISH-hybridized scaffolds were subsequently mounted on a coverslip with 500 μL of anti-fade Citifluor solution containing the general DNA stain DAPI (4.5 mg/mL final concentration).

Techniques: Co-Culture Assay, Labeling, Staining, Negative Control

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: FISH-hybridized scaffolds were subsequently mounted on a coverslip with 500 μL of anti-fade Citifluor solution containing the general DNA stain DAPI (4.5 mg/mL final concentration).

Techniques: In Situ, Staining, Confocal Microscopy, Imaging