Filter set 09

Samples were imaged using an upright microscope (Axio Imager A.2; Carl Zeiss, Oberkochen, Germany), with a 40× /1.3 and 100× /1.3 oil-immersion objective lens (EC Plan-Neofluar; Carl Zeiss), with transmitted light and reflected fluorescence observation using cooled charge-coupled device cameras (Axiocam 503 color and Axiocam 503 monochrome; Carl Zeiss). Cameras were equipped with sensors having a physical pixel length of 4.54 µm, yielding image pixel sizes of 113.5 nm/pixel (40× objective) or 45.4 nm/pixel (100× objective). Fluorescence was excited using a broadband light-emitting diode source (X-Cite 120 LED; Excelitas Technologies Corp., Waltham, MA), and a mercury lamp for brightfield illumination. Green fluorescence labelled samples were imaged using a GFP filter (Filter Set 09; Carl Zeiss) and red fluorescence labelled samples were imaged using a TxRed filter (Filter Set 00; Carl Zeiss). Images were acquired using a Zeiss software (ZEN 2Blue, Carl Zeiss, Carl Zeiss, https://www.zeiss.com).

Light microscopical (LM) images of the filaments were taken on a Zeiss Axiovert 200 M light microscope (Carl Zeiss AG), equipped with an Axiocam HRc camera (Carl Zeiss AG) and a Zeiss Axiovision software. Cell measurements (n = 35) were then taken via ImageJ/ Fiji (Schindelin et al., 2012 (link)). Confocal laser scanning microscopy (CLSM) was performed with a Zeiss Pascal system under the control of Zen 2009 software, excitation was generated with an argon laser (488 nm), and emission was collected with a long pass filter (505 nm), and the chloroplast autofluorescence was false coloured red. Z‐stacks were generated from a series of 19 and 30 images for M. disjuncta and M. scalaris, respectively, at 1 μm distance and projected in the z‐axis. After the freezing experiments (described below), light, as well as fluorescence micrographs, were taken, with the chlorophyll (Chl) autofluorescence being visualized with a Zeiss Filter Set 09 (Excitation: band pass (BP) 450–490 nm and emission: long pass (LP) 515 nm). Images were processed using Adobe Photoshop Elements 11 (Adobe Inc., 2019 ).

Evaluation of GFP expression over time was achieved through the use of whole plant vacuum-agroinfiltration assays (as described by Chialva et al., 2018 (link)) along with gene transfer experiments in ‘Thompson Seedless’ somatic embryos. The GFP reporter gene expression was evaluated by epifluorescence microscopy in leaves at 1, 3, 5, 8, 13, 16, 19, and 23 days post-infiltration (dpi) (Figure 1A) and in embryogenic callus between 4 and 47 dpi (Figure 1B). Samples were observed using a Zeiss Axioscope Lab A.1 epifluorescence microscope equipped with Filter Set 09 (BP 450–490 nm) and Filter Set 38 (BP 470/540 nm; Zeiss, Oberkochen, Germany). The light source was a 470-nm LED lamp. Images were acquired with a Canon Rebel T3 camera using EOS Utility software (Canon Inc., Tokyo, Japan). The green channel was quantified via eight images per point using ImageJ software (National Institutes of Health, United States). Data was subjected to one-way ANOVA test with a 5% level of significance using Statgraphics Centurion XV (Manugistics Inc., Rockville, MD, United States).

The tracking of the rotation of the optically anisotropic particles
is based on a method reported by Niggel et al.^{37 (link)} First, the center of the particle is found in each frame
of a time series of standard wide-field epi-fluorescence microscopy
images (Axio Observer D1, 40× NA = 0.6 objective, Filter Set
09, Zeiss, Germany, 89 North Photofluor II, USA, Zyla 4.2, Andor,
UK, acquisition rate: 25 frames per second, 2048 × 2048 pixels).
Then, using image correlation, a reference image is compared with
sequentially rotated images of a subsequent frame to find the most
probable change in angular orientation. The instantaneous rotation
of the particle can be extracted and synchronized with the friction
force, as seen in Figure 1F–H. Alternatively, the cumulative rotation in one
scan can be determined and compared to the theoretical rolling without
slip of a particle of the same diameter over the same distance. The
rotation can be displayed as a percentage compared to pure rolling
without slip, as shown later in the article.

Light micrographs were taken with a Zeiss Axiovert 200 M light microscope (Carl Zeiss AG, Jena, Germany), equipped with an Axiocam HRc camera (Carl Zeiss AG, Jena, Germany) and Zeiss Axiovision software. Indian ink (Dr. Ph. Martin’s, Oceanside, United States) staining was employed to detect potential mucilage sheaths in live cells (according to Permann et al. [23 (link)]). Chlorophyll auto fluorescence was visualized with a Zeiss Filter Set 09 (Excitation: band pass (BP) 450–490 nm and emission: long pass (LP) 515 nm); the same filter set was used for fluorescent antibody detection in immunocytochemistry. Confocal laser scanning microscopy (CLSM) was performed with a Zeiss Pascal system control using Zen 2009 software, and excitation with an argon laser 488 nm, and emission was collected at 505–550 nm (false colored green).

Light microscopical investigations of the field sampled Mougeotia spp. filaments and zygospores were taken in the following three days after collecting, respectively. The maturation process was observed over a total period of four months, with images taken every 40 days. The analyses were performed using a Zeiss Axiovert 200 M light microscope (Carl Zeiss AG, Jena, Germany), equipped with an Axiocam HRc camera (Carl Zeiss AG, Jena, Germany) and Zeiss Axiovision software. Chlorophyll autofluorescence was visualized with a Zeiss Filter Set 09 (excitation: band pass (BP) 450–490 nm and emission: long pass (LP) 515 nm). A 1% calcolfluor white (Fluka Analytical, Cat# 18,909) staining following the protocol of Herburger and Holzinger (2016 ) was applied for cellulose and callose detection. The stained cells were than investigated using a Zeiss Filter Set 01 (excitation: BP 359–371 nm and emission: LP 397 nm). For mucilage sheath detection, stainings with Indian ink (Dr. Ph. Martin’s, Oceanside, USA) were performed.