Automated Segmentation and 3D Reconstruction of Different Membranes from Confocal Z-Stacks.

Confocal laser scanning microscopy (CLSM) is an advanced microscopy technique based on fluorescence technology which produces sharp images of a specimen in a single focal plane. The optical sectioning by CLSM allows to have z-stacks which can be further processed into 3D reconstructions. These then provide the option of variable perspectives and additional precise data evaluation on structural and anatomical alterations. Here, we used CLSM to image the thylakoids of cyanobacteria and the endoplasmic reticulum (ER) in moss protonemata as an example. Then, out of the confocal z-stacks, we create 3D constructions of the membranes and their alterations to present a holistic, structural view from different angles.

A rapid freezing method to determine tissue layer thickness in drought-stressed leaves.

Plants have been affected by water stress ever since they settled on dry land. In severe and persisting drought, plant leaves are wilting. However, a documentation at the anatomical level of the minute changes that occur before wilting is challenging. On the other hand, understanding the anatomical alteration in plant leaves with respect to water stress provides a stronger basis to study molecular and submolecular processes through which plants enhance drought tolerance. In this work, we applied an affordable method to visualise mesophyll layers of Arabidopsis thaliana cell lines without preparation steps that would alter the volume of the cells. We rapidly plunge-froze the leaves in liquid nitrogen, cut them while in the N2 bath, and immediately imaged the mesophyll cross sections in a scanning electron microscope. We applied a reduction of watering from 60 to 40 to 20 mL per day and investigated two time points, 7 and 12 days, respectively. Interestingly, the overall thickness of leaves increased in water stress conditions. Our results showed that the palisade and spongy layers behaved differently under varying watering regimes. Moreover, the results showed that this method can be used to image leaf sections after drought stress without the risk of artefacts or swelling caused by contact to liquids as during chemical fixation.

Confocal microscopy reveals alterations of thylakoids in Limnospira fusiformis during prophage induction.

The alkaliphilic cyanobacterium Limnospira fusiformis is an integral part in food webs of tropical soda lakes. Recently, sudden breakdowns of Limnospira sp. blooms in their natural environment have been linked to cyanophage infections. We studied ultrastructural details and prophage components in the laboratory by means of confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). For a comparison at the subcellular level, we included transmission electron microscopy (TEM) material of infected cells collected during a field survey. Compared to TEM, CLSM has the advantage to rapidly providing results for whole, intact cells. Moreover, many cells can be studied at once. We chemically induced lysogenic cyanophages by means of mitomycin C (MMC) treatments and studied the ultrastructural alterations of host cells. In parallel, the number of cyanophages was obtained by flow cytometry. After treatment of the culture with MMC, flow cytometry showed a strong increase in viral counts, i.e., prophage induction. CLSM reflected the re-organization of L. fusiformis with remarkable alterations of thylakoid arrangements after prophage induction. Our study provides a first step towards 3D visualization of ultrastructure of cyanobacteria and showed the high potential of CLSM to investigate viral-mediated modifications in these groups.