Ec plan neofluar 20 0

For imaging, bones were held in 1% low melting agarose. Two-photon imaging was performed on a Zeiss LSM880 equipped with a EC Plan-Neofluar 20×/0.50 controlled by Zen Black 2 (Zeiss GmbH, Oberkochen, Germany). The excitation source was a Ti:Sapphire femtosecond laser cavity (Chameleon Vision II, Coherent), operating at 80 MHz and tuned to a wavelength of 750 nm for AlexaFluor 594 excitation and to a wavelength of 860 nm for second harmonic generation imaging of stromal collagen. To reconstruct large areas, 15% overlapping image tiles were acquired in Z-stack and processed using Zen Black 2 software, while 3D visualization was obtained using Zen Blue 2 (Zeiss GmbH).

For microscopy, around 1× 10⁴ spores were inoculated on thin LNA (1% agar 1.66 mM MgSO₄, 5.4 µM ZnSO₄, 2.6 µM MnSO₄, 18.5 µM FeCl₃, 13.4 mM KCl, 0.34 µM biotin, and 0.75 µM thiamin) and incubated for at least 2 h at 28°C. For conventional fluorescence images, a Zeiss AxioImager Z.1 microscope with either Plan-Apochromat 63×/1.4 Oil DIC, EC Plan-Neofluar 40×/0.75, EC Plan-Neofluar 20×/0.50, or EC Plan-Neofluar 10×/0.30 objective and an AxioCamMR was used. The images were taken using ZEN 2012 Blue Edition.
For confocal imaging, a Zeiss LSM900 microscope with Plan Apochromat 63×/1.4 Oil objective and Airyscan 2 detector in super resolution mode was used.

Fluorescent images were taken at room temperature with an AxioImager M2 microscope (Zeiss) equipped with an ApoTome.2 structured illumination unit, Orca-Flash 4.0 LT3 digital sCMOS camera (Hamamatsu Photonics), EC Plan-Neofluar 20×/0.50, Plan-Apochromat 40×/0.95 and Plan-Apochromat 63×/1.4 Oil objectives (Zeiss). Raw images were processed with ZEN2.3 lite Microscopy Software and Photoshop CS4 (Adobe Systems). To improve clarity in Figs. 2, 3, 4 and 6 consecutive optical slices spanning a depth of 3 µm were projected onto single images. Single focal planes were presented in other figures, including colocalization tests in Figs. 1 and 5.