My detailed notes:

Cyanobacteria analysed
- Cyanobacterial harmful algal blooms of the genera Microcystis aeruginosa and
Planktothrix agardhii
- These genera produce high intracellular amounts of the toxic peptides microcystins
(MCs) and anabaenopeptides (APs)
- Intracellular toxic peptides can be released into the environment by cell lysis or by
active transport mechanisms across the membrane
- Investigation of the structural variants of microcystins (MCs) and anabaenopeptides
(APs)

Methods

- Chemically modified microcystins (MCs) and anabaenopeptides (APs) in

Microcystis and Planktothrix can be localised at the subcellular level based on
peptide storage and transport mechanisms using copper-catalysed alkyne azide
cycloaddition (CuACC), the so-called click chemistry methodology (molecular
imaging technique by in vivo peptide detection with bioorthogonal labelling)
- Comparison of numerous isolates that differ significantly from intra- and
extracellular toxin content under physiological stress conditions
- Different non-natural amino acids carrying alkyl and azide side chains were fed to
individual Planktothrix strains (CYA126 and No371) and Microcystis strains (Maer)
- Labelling of these alkyne-modified peptides with two azide-modified fluorophores
Alexa 405 (blue fluorescence) and Alexa 488 (green fluorescence, incorporation of
these amino acid substrates occurs during non-ribosomal peptide synthesis (NRPS))
- Use of confocal laser scanning microscopy (three-dimensional 30 nm) for qualitative
and quantitative analysis of cyano and peptide distribution at inter- and intracellular
level (secondary analysis), data set consists of several components, evaluation is
carried out at the physical limit of 100 nm
- Image processing analysis using Huygens software (Huygens Essential 22.10, new
version 23.10)
- 432 images (Gas vesicle disruption) have already been processed in Huygens
(named as Maer Disrupted/No Disrupted (Microcystis strains) and CYA126 and
No371 Disrupted/No Disrupted (Planktothrix strains)
- "Disrupted" images: Cells that contain gas vesicles and affect the size of the cell,
gas-filled structures in the cytoplasm surrounded by a protein layer that give the cells
buoyancy (vertical migration) [Brock Microbiology, 13th edition, 2013], there are
thousands of vesicles in a cell
- There are another 2880 images (signal build-up) being processed in Huygens (Maer
and No371)
- During image processing in Huygens, the cells are categorised into three
compartments: intact cell, cytoplasm and periplasmic membrane
- A single, intact cell and then the periplasmic membrane (about 10% of the intact
cell) were cut out of the respective images. The cytoplasm and the periplasmic
membrane should again result in the intact cell. The tolerance range is between 1.5
and 24% of the excised periplasmic membrane from the total cell of all processed
images of gas vesicle disruption
- Exact procedure for measuring the spherical intact cell (Maer) and then the
cytoplasm, the so-called "black hole" with the help of 2D visualisation, Ortho Slicer
and slice in Huygens. Here, only the width is measured in the upper left window xy
based on the shape of the cell and is required for the calculations
- The same procedure is used for the cylindrical cells (CYA126 and No371). First the
height and width of the intact cell is determined, then the width of the cytoplasm, as
both will be required for the calculations. It is not necessary to measure the height of
the cytoplasm as it is the same height as the intact cell
- The values of the dimensions are entered in the Excel list provided for this purpose
using the formulae provided
- The volume calculations in µm3 and in per cent are based on the formula of the
sphere (Maer, Microcystis aeruginosa) and that of the cylinder (CYA126 and No371,
Planktothrix agardhii)
- The dimensions of the cells should generally be taken after the images have been
cut out so that any inaccuracies in the measurements can be ruled out immediately.
Values below 1% and above 24% volume fraction of the periplasmic membrane were
discarded
- Statistical values of channel 0 and 1 of the cropped images in Huygens (Image
statistics for channel 0 and 1, Sum  Sum of signal intensities) are transferred to the
Excel list
- The Pearson correlation coefficients of the intact cell, the cytoplasm and the
periplasmic membrane are calculated in Huygens with these determining factors:
Theshold, R/G channel settings: Selection of the channel combination Ch0/Ch1,
Threshold estimation: Gaussian minimum, Colocalisation coefficients: Select all,
Colocalization map (Pearson) and compute. Finally, the results of the individual
calculations are obtained (Colocalisation results)

First results

- The percentage values of the periplasmic membrane vary between 5 and 23.5% of
the total cell in the Microcystis strains (Maer). In contrast to Microcystis, the
Planktothrix strains (CYA126 and No371) have slightly lower values. These are
between 1.5 and 20% for Planktothrix CYA126 and between 4 and 22.5% for No371
- The disadvantage of this method is that the values are sometimes subject to large
fluctuations due to the manual measurement in Huygens using the Ortho Sclicer and
sometimes very different values were determined
- The statistical values have shown that the signal intensities within a cell decrease
from the intact cell to the periplasmic membrane
- When comparing the signal intensities between the Microcystis strains and the
Planktothrix strains, no difference is recognisable; all of them decrease within the cell
from the intact cell to the periplasmic membrane
- The Pearson correlation coefficients of the modified samples (intact cell, cytoplasm
and periplasmic membrane) differ on average between +0.2 and +0.9. These r-
values indicate a positive correlation in which the values of both variables (channel 0
and 1) tend to increase together