Supplemental material of the Diploma Thesis "Holotomographic microscopy of subcellular structures and nuclear dynamics in filamentous fungi"

The supplemental online material of the Diploma Thesis "Holotomographic microscopy of subcellular structures and nuclear dynamics in filamentous fungi" includes two videos and an SOP (standard operating procedure) for data processing. Each file is briefly described below. For a more detailed description of the events depicted in the videos, please refer to the Diploma Thesis.

Video 1 shows the nuclei movement of the engineered Aspergillus niger strain A621_H2A:sGFP. The strain produces the histone H2A tagged with sGFP under the control of the inducible tet-on promoter. Images were recorded with the 3D Cell Explorer-fluo (Nanolive SA, Tolochenaz, Switzerland). The recording visualizes the movement of nuclei by superimposing the fluorescence signal onto the maximum intensity projection of the refractive index map. 

Observation of synchronous nuclei division is possible in the apical hyphal compartments located on the left and right sides of the focused hypha. In the middle, a single nucleus is located in a subapical compartment separated by two septa. The nucleus does not undergo any significant movement or division during the observed time frame and is therefore considered mitotically inactive.

Video 2 shows the behavior of the cyclin-dependent kinase nimX in the engineered Aspergillus niger strain A621_nimX:sGFP. The strain produces GFP-tagged nimX under the control of the inducible tet-on promoter. Images were recorded with the 3D Cell Explorer-fluo. The video displays the maximum intensity projection of the holotomographic slices on the left in black and white. On the right, the signal of the GFP-tagged nsimX is shown in green.

The video demonstrates the emergence of two germ tubes and the movement of accumulated nimX towards the growing hyphal tip. At this point, nimX is likely located in the nuclei, as previously reported for Aspergillus nidulans. In the last frame of the video, the GFP signal suddenly disappears, indicating that nimX is either exiting the nuclei or undergoing degradation.

The SOP aims to assist with processing data from the 3D Cell Explorer-fluo, mainly in Fiji/ImageJ. A short part describes data acquisition and export with the microscope software Steve. The fungi Aspergillus niger, Trichoderma reesei and Aureobasidium pullulans were used to create this SOP, so there may be differences in data processing when working with other organisms.