Filter set 20

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.

Electrophysiological recordings were essentially done as previously described (Kravic et al., 2016 (link), 2018 (link)). Isolated diaphragm-phrenic nerve preparations were maintained in Liley’s solution gassed with 95% O₂ and 5% CO₂ at room temperature (Liley, 1956 (link)). The recording chamber had a volume of ca. 1 ml and was perfused at a rate of 1 ml/min. The nerve was drawn up into a suction electrode for stimulation with pulses of 0.1 ms duration. The preparation was placed on the stage of a Zeiss Axio Examiner Z1 microscope fitted with incident light fluorescence illumination with filters for red (Zeiss filter set 20) fluorescing fluorophore (Carl Zeiss MicroImaging, Göttingen). At the beginning of the experiment, the compound muscle action potential (cMAP) was recorded using a micropipette with a tip diameter of ca. 10 μm, filled with a bathing solution. The electrode was positioned so that the latency of the major negative peak was minimized. The electrode was then positioned 100 μm above the surface of the muscle and cMAP was recorded.

Small pieces of gypsum, colonized by pigmented endoliths were scraped and suspended in double-distilled water. The suspension was stained with SYBR Green I (SBI) (Molecular Probes), which is a fluorochrome specifically used for the staining of nucleic acids. Observations were made first in differential interference contrast (DIC) using a Zeiss AXIO Imager M2 fluorescence microscope (Carl Zeiss, Jena, Germany) plus an Apochrome x60, n = 1.4 Zeiss oil-immersion objective. A CCD Axiocam HRc Rev. 2 camera and AXIOVISION 4.7 software (Carl Zeiss, Oberkochen, Germany) were used to capture and record the DIC images. Images were acquired using a Multichannel Image Acquisition system, employing an eGFP filter set (Zeiss Filter Set 38; Ex/Em: 450–490/500–550 nm), a DAPI filter set (Zeiss Filter Set 49; Ex/Em: 365/420–470 nm), and a Rhodamine filter set (Zeiss Filter Set 20; Ex/Em: 540–552/567–647 nm).