TITLE: Quantification of fluorescence intensity of antisense constructs within Bodo saltans and its intracellular symbiont
AUTHORS: Marie Held, Mastaneh Ahrar, Gregory DD Hurst, Ewa Chrostek

[DESCRIPTION]
The purpose of this Protocol is to quantify the intensity of fluorescence resulting from antisense molecules within a microeukaryote Bodo saltans and its intracellular symbiont Candidatus Bodocaedibacter vickermanii1 following incubation. This protocol is specific to two-channel fluorescent images of objects contained within larger objects. It can be used directly to analyse images of objects of similar size.  

The images were recorded using a confocal laser scanning microscope as single focal plane images, containing three channels: 1) fluorescent antisense molecule, 2) DAPI, 3) transmitted light. 

The antisense molecule intensity in channel 1 is to be measured. The DAPI channel allows the segmentation of both, the B. saltans nucleus and the symbionts. The transmitted light channel is used for general reference. 

This Protocol.io contains two sample files (.czi), which open in Fiji and can be re-analyzed using the steps described here. A separate word file with all the steps listed can also be found attached to this Protocol.io.  

[STEPS]
SECTION: Incubation of B. saltans with antisense molecules
1. Filter B. saltans culture through 100 and 8 µm filter.
SECTION: Incubation of B. saltans with antisense molecules
2. Harvest the cells by centrifugation at 1200 × g for 12 mins at 19 °C.
SECTION: Incubation of B. saltans with antisense molecules
3. Wash the cells with 10-15 ml sterile filtered (SF) 1×PBS and centrifuge as above.
SECTION: Incubation of B. saltans with antisense molecules
4. Re-suspend the cells in 5 ml SF 1×PBS, count the cells using hemacytometer and take the volume of cells which contains 5×106 cells for every 10 samples (5×105 cells per sample is required). Preparing at least duplicates for each treatment and including the sense, scrambled and no-treatment control as well as 20% excess in case of pipetting errors means that at least 10 samples have to be prepared for each experiment.
SECTION: Incubation of B. saltans with antisense molecules
5. Centrifuge at 1200 × g for 12 mins at 19°C.
SECTION: Incubation of B. saltans with antisense molecules
6. Remove the PBS and resuspend the cells in SF cerophyll medium so that the volume of the medium and the molecules tested is 100 µl per sample. E.g. if you add 10 µl of molecules under investigation per sample you have to use 90 µl of medium per sample. For ten samples, resuspend the cells in 900 µl of medium.
SECTION: Incubation of B. saltans with antisense molecules
7. Move 90 µl of the cell suspension to a sterile 2ml tube.
SECTION: Incubation of B. saltans with antisense molecules
8. Add the tested molecules to the B. saltans cells in 2 ml tube. To have final concentration of 50 µM of the molecule we use 10 µl from the500 µM stock. Mix well the tested molecule and cells by pipetting and then place the tubes in the incubator.
SECTION: Incubation of B. saltans with antisense molecules
9. After 24 h prepare samples for imaging. Centrifuge the 2 ml tube containing the cells (1200 × g for 12 mins at 19°C), remove the supernatant and leave 30 µl of liquid in the tube. Resuspend the cells and divide the volume in two wells of a 96 well plate, 15 µl in each well. Add 15 µl of  low melting temperature agarose (e.g. Thermo Fisher Scientific) to each well and mix. Let it set for a few seconds. The following steps will be done similarly for both wells in order to make two slides for each treatment.
SECTION: Incubation of B. saltans with antisense molecules
10. Add Hoechest 33342 (Thermo Fisher, 1:2000 in PBS) to the well for 10 minutes.
SECTION: Incubation of B. saltans with antisense molecules
11. Rinse and wash 1x with PBS.
SECTION: Incubation of B. saltans with antisense molecules
12. Remove the agarose from the well with clean forceps and place it on a microscope slide.
SECTION: Incubation of B. saltans with antisense molecules
13. Add a drop of a mounting medium (eg. Vectashield, Vector Laboratories), and flatten the agarose as much possible using the coverslip.
SECTION: Imaging B. saltans using an LSM 880 Laser Scanning Confocal (Zeiss)
14. When using a Confocal microscope, always follow local facility rules. Microscopes are expensive, sensitive, and choosing wrong settings can lead to artifacts and non-processable results. 
SECTION: Imaging B. saltans using an LSM 880 Laser Scanning Confocal (Zeiss)
15. Turn the microscope and all the systems on according to local rules. The order of turning individual components on matters. 
SECTION: Imaging B. saltans using an LSM 880 Laser Scanning Confocal (Zeiss)
16. View your samples using the eyepiece and light source, to make sure you can find your samples and
focus. Due to its small size, B. saltans is best visualized using a high numerical aperture (e.g. 63×, 1.40 NA) objective.
SECTION: Imaging B. saltans using an LSM 880 Laser Scanning Confocal (Zeiss)
17. Switch to scanning mode, acquire images of all of your samples and negative controls using the same settings. 
SECTION: Image preprocessing
18. Open raw image in Fiji2
SECTION: Image preprocessing
19. Duplicate DAPI channel [Shift + D].
SECTION: Image preprocessing
20. Subtract the background (out-of-focus light and/or autofluorescence) via rolling ball Background subtraction with a radius of 25 pixels.
SECTION: Image segmentation
21. Duplicate background subtracted DAPI channel.
SECTION: Image segmentation
22. Use global thresholding algorithm [Fiji > Image > Adjust > Threshold > IsoData]3 to segment the areas
positive for DNA staining, which are the symbionts and the B. saltans nucleus, from the background and convert image to a binary mask. 
SECTION: Image segmentation
23. Run [Fiji > Process > Binary > Watershed] operation to separate touching objects. 
SECTION: Image segmentation
24. Run connected component analysis [Analyze Particles] with a size thresholding of 0-300 pixels, the [Add to manager] and [Exclude on Edges] options enabled. The size exclusion should ensure that the identified objects are only the symbiont and not the B. saltans nucleus. his generates a set of regions of interest (ROIs). 
SECTION: Image segmentation
25. Save the bacteria ROIs.
SECTION: Image segmentation
26. Select raw image and duplicate the fluorescent antisense molecule channel [Shift + D]. 
SECTION: Image segmentation
27. Use global thresholding algorithm [Fiji > Image > Adjust > Threshold > Mean] 4 to segment the areas
positive for antisense staining from background and convert image to a binary mask.
SECTION: Image segmentation
28. Run connected component analysis [Analyze Particles] with a size thresholding of 1000-Infinity pixels and the [Add to manager] and [Exclude on Edges] options enabled. The size exclusion should ensure that the identified objects are only the symbiont and not the B. saltans nucleus. This generates a set of regions of interest (ROIs), usually only one. 
SECTION: Image segmentation
29. Rename the ROI, e.g. to “cell”. 
SECTION: Image segmentation
30. The B. saltans ROI and the individual symbiont ROIs are combined using the XOR operator to generate a single ROI containing the B. saltans cytoplasm only, excluding the symbiont. With two inputs, XOR is true if and only if the inputs differ (i.e. one is true, one is false). 
SECTION: Image segmentation
31. Rename the ROI to “cell-minus-bacteria”. 
SECTION: Measurements
32. Activate the following parameters to be measured via [Fiji > Analyze > Set Measurements…]:
1. Area 
2. Mean gray value
3. Modal gray value
4. Integrated density
5. Median 
6. Activate Display Label option
 
SECTION: Measurements
33. Select raw image.
SECTION: Measurements
34. Set channel to antisense channel.
 
SECTION: Measurements
35. Deselect ROIs in the ROI Manager (or select all).
SECTION: Measurements
36. Measure set parameters by [ROI Manager > More > Multi Measure]. 
SECTION: Measurements
36.1. Inactivate [Measure all 3 slices] and [One row per slice]. 
SECTION: Measurements
37. Save results table.
SECTION: Automation
38. The whole process can be automated using a Fiji macro script:  https://github.com/Marien-kaefer/General_Fiji_macros/tree/main/BodoSaltans
SECTION: References
39.  Midha S, et al. Bodo saltans (Kinetoplastida) is dependent on a novel Paracaedibacter-like endosymbiont that possesses multiple putative toxin-antitoxin systems. ISME J. 15(6):1680-1694 (2021 Jun). doi: 10.1038/s41396-020-00879-6. 
Schindelin J, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676–682 (2012). https://doi.org/10.1038/nmeth.2019.
Ridler TW & Calvard S. Picture Thresholding Using an Iterative Selection Method in IEEE Transactions on Systems, Man and Cybernetics, Volume 8, Issue 8, pp. 630-632 (1978).  doi: 10.1109/TSMC.1978.4310039.
 Glasbey CA. An Analysis of Histogram-Based Thresholding Algorithms, CVGIP: Graphical Models and Image Processing, Volume 55, Issue 6, pp. 532-537 (2021) https://doi.org/10.1006/cgip.1993.1040. 
SECTION: Acknowledgements
40. We thank the Centre for Cell Imaging (CCI) at the University of Liverpool for the assistance with live Bodo imaging. Zeiss 880 BioAFM at the CCI was funded by BBSRC grant number BB/M012441/1. 
This work was funded by Gordon and Betty Moore Foundation’s Symbiosis in Aquatic Systems Initiative, Grant ID: #9357 (https://doi.org/10.37807/GBMF9357).