Tcs sp8 inverted

Putative transformants were analysed as follows: Δpks4.2 transformants were sub-cultivated three times on MM agar medium and the spore colour formation compared to the wild-type strain. In case of questionable phenotypes, strains were further subjected to diagnostic PCR. Integration of Psnc1::gfp::snc1 was analysed using fluorescence microscopy. In brief, colonies cultivated on selective agar MM medium containing 15 mM acetamide for 24 h at 37 °C and fluorescence images were taken using an inverted TCS SP8 (Leica, Germany) as described earlier [30 (link)]. Most colonies with GFP-secretory vesicle signals had correct integration of the donor DNA at the snc1 locus as checked by diagnostic PCR (~ 99%). Consequently, transformants with GFP- secretory vesicle signals, were considered snc1 targeted. For Δpks4.1, Δalp1 and Δptf1 diagnostic PCR was done on the corresponding locus. For primer sequence information, see Additional file 5.

Different cultivation techniques were applied for microscopy. Cultivation for cLSM microscopy of A. niger was performed as described recently (Kwon et al., 2014 (link)). In brief, spores were spotted in MM plates, supplemented with different concentrations of doxycycline when needed and incubated at 22°C for 2 days, following cutting out of the colony and placing it upside down into a glass bottom petri dish (Kwon et al., 2014 (link)). Liquid MM medium (supplemented with the same concentration of doxycycline which was present in the MM plate) was added and cells were incubated at 22°C until they resumed growth. Fluorescence and DIC images were taken using an inverted TCS SP8 (Leica, Germany). For fluorescence microscopy, cultivation was performed as described earlier (Fiedler et al., 2014 (link)). In brief, cover slides were placed on the bottom of a Petri dish containing 5 ml MM medium (supplemented with 100 mM MES pH6.5 and different concentrations of doxycycline when needed), inoculated with 1 × 10⁵ spores/ml and cultivated at 28°C for 12 h. Images were acquired using a DMI6000 fluorescence microscope (Leica, Germany).

Tissues cleared by 3DISCO, PACT-sRIMS, CUBIC and SeeDB were imaged in their respective RI matching solutions in Ibidi μ-Dishes. PACT-RC-cleared tissues were mounted using iSpacer chambers in RC-CS Mounting Medium. Images were acquired using a Leica TCS SP8 inverted confocal microscope with 10×/0.4 or 20×/0.75 HC PL APO objective lenses. Laser power and gain were adjusted manually to give optimal fluorescence intensity for each fluorophore with minimal photobleaching. Step size and line averaging were kept constant for all main figures (line averaging, 16; step size 1–2), excluding CUBIC and SeeDB depth cueing examples. Imaging depths were recorded from the top of the epithelial structure being imaged (typically 350 μm through the native fat pad for the CUBIC and SeeDB protocols). Image reconstructions were generated using Imaris image management software (v8.0, Bitplane) or ImageJ (v1.50c, National Institutes of Health) [23 (link), 24 ]. Depth coding was performed using the Temporal Colour Code plugin with the spectrum LUT. De-noising of 3D image sequences was performed in MATLAB (R2014a, The Mathworks Inc.) [25 (link)].