Axioimager d1 fluorescence microscope

Small fragments of gypsum showing distinct signs of endolithic colonization with a green/orange layer beneath the rock surface and a green layer just above the bottom of the gypsum sample were scraped and suspended in double-distilled water. Suspensions were vortexed (2 min) and allowed to settle for 1 min before removing the supernatant, which was centrifuged for 10 min at 5000 g. The pellet was then stained with SYBR Green I (SBI) (Molecular Probes), a fluorocromes used for the specific staining of nucleic acids. Bright field images, SBI fluorescence and photosynthetic pigment autofluorescence were visualized in a Zeiss AxioImager D1 fluorescence microscope (Carl Zeiss, Jena, Germany). Detailed procedures are provided in Supplementary Section S4.

Small fragments of ignimbrite, showing distinct signs of endolithic colonization as a green-colored layer beneath the rock surface, were moistened with distilled water, and the autofluorescence of the cyanobacteria cell aggregates were visualized in situ using a Leica TCS-SP5 confocal laser scanning microscope (CLSM) (Leica Microsystems Heidelberg GmbH, Mannheim, Germany). Red autofluorescence was viewed in the red channel (640 to 785 nm emission) using a 561 nm laser diode.
Other fragments of rocks were cut perpendicularly to the rock surface with a diamond saw, and this plane was stained with SYBR Green (Molecular Probes), a fluorochrome used for specific staining of bacterial cell nucleic acids (NA). Next, the endolithic microbial colonies were observed in situ using a Zeiss AxioImager D1 fluorescence microscope (Carl Zeiss, Germany). Filter sets for eGFP (Zeiss Filter Set 38; Ex/Em: 450–490/500–550 nm) and rhodamine (Zeiss Filter Set 20; Ex/Em: 540–552/567–647 nm) were used for green and red (stained bacteria NA and cyanobacteria autofluorescence) signal visualization, respectively.