TITLE: LifeWatch Belgium: FlowCam sampling and lab protocol for imaging microphytoplankton in the Belgian part of the North Sea.
AUTHORS: Rune Lagaisse

[DESCRIPTION]
In the framework of the Belgian Lifewatch RI, a number of fixed stations in the Belgian Part of the North Sea (BPNS) are visited during mulitdisciplinary campaigns. A grid of nine stations covers the coastal zone and are sampled monthly. Eight additional stations, located more offshore, are sampled on a seasonal basis. For the phytoplankton monitoring, samples are taken using a 55µm mesh size Apstein net, fixed in Lugol's iodine solution and preserved in dark and refrigerated conditions. In the lab, the samples are processed using a FlowCAM VS-4 benchtop model, equipped with a Sony XCD SC90 digital gray-scale camera and VisualSpreadsheet software version 4.2.52. For the monitoring, the FlowCam device is mounted with the 4X magnification and associated hardware to image a particles size range of 55-300µm. The identification of the images data is done with a CNN and followed by a manual validation step to check each predicated image. Since May 2017, this dataset provides micro- and phytoplankton observations, mainly covering diatoms, dinoflagellates and cilliates, for the Belgian Part of the North Sea (BPNS).
This published protocol descibes sampling and lab SOPs in detail. 

[STEPS]
SECTION: Sample collection
1. 9 onshore stations are visited on a monthly frequency and 8 offshore stations are visited on a seasonal frequency during the LifeWatch muldisisciplinary campaigns in the Belgian part of the Nort Sea. 

 


 

SECTION: Presample run
26. For each sample we perform a small presample run to determine particle load and appropriate dilution of the sample to avoid particles overlapping in a single frame.
SECTION: Sample collection
2. 1 replicate sample per station is taken while logging the action in the Research Vessels’ information system (MIDAS). Choose actiontype 'Planktonnet Apstein', fill in reporter, select the correct station and press start and stop before and after taking the sample. 

SECTION: FlowCAM hardware set up
9. Wear lab coat and gloves, Lugol can stain. 
SECTION: FlowCAM hardware set up
14. Place FC with FC holder onto the FC holder mount on the rail of the FlowCam. Centre the flow cell in the holder if necessary using the screws on the FC holder. Make sure the flow cell is vertical (straight edges) and the edges are not in the field of view of the camera.
SECTION: FlowCAM hardware set up
13. Clean the 300µm disposable flow cell (FC) and place in the correct FC holder with the short tube end at the top. Screw the holder ring to attach the FC in to the holder, don't put too much pressure to avoid breaking the glass photo chamber. The tubes of FCs are cut to 20cm (bottom, direction of syringe) and 10cm (top, direction of pipette tip) length, these measurements are saved in the context file and important to calculate the dead volume.  
SECTION: FlowCAM hardware set up
15. Attach the short tube end (10cm) to the pipette tip and the longer tube end (20cm) to the inlet of the syringe pump. Ensure that the tubing from the FC to the pipette tip is as straight as possible. 
SECTION: FlowCAM hardware set up
16. Attach an extra tube at the outlet of the syringe and put the end in a small beaker to collect the waste outflow. 
SECTION: FlowCAM hardware set up
19. Select Setup > Pump > Change syringe and follow the appropriate steps to install the 5mL syringe. Select the correct syringe size mounted when you're done.
SECTION: FlowCAM hardware set up
10. Turn on FlowCAM using the power switches located on the rear and front (UPS system should never be turned off in order to ensure a continuous power supply).
SECTION: FlowCAM hardware set up
11. Clean and screw in 4X objective.
SECTION: FlowCAM hardware set up
17. Open VisualSpreadsheet on the computer.
SECTION: FlowCAM hardware set up
18. Select the correct objective. Ensure that the status bar colour (if displayed) at the bottom of the main window matches the coloured stripe on the installed objective.
SECTION: Load sample and focus
20. Select Setup and Focus.
SECTION: Load sample and focus
21. Adjust flow rate: Choose a flow rate within the range of the mounted syringe. We use a max. flow rate of 5mL/min for the 4X magnification. 
SECTION: Load sample and focus
22. Rinse FC thoroughly:
Setup and focus > Start pump.
Alternate rinsing with 5mL Milli-Q water and 5mL 70% ethanol and leave air bubbles in between, do this 3 times each and finish by rinsing with Milli-Q water.
SECTION: Load sample and focus
23. Measure the volume of the sample sample and note the measurement on the sample recipient cap.
SECTION: Load sample and focus
24. Load some sample into the flow cell using a Pasteur pipette, gently mix the sample before pipetting to avoid sedimentation of cells. Leave some air between the sample and Milli-Q water from the last flush. Go to the Setup and Focus menu and press Start Pump, pause the pump when the sample is visible in the field of view, pinch the flow cell tubing if air bubbles would be present in the tubing. 
SECTION: Load sample and focus
25. Focusing is done manually on the sample before the run as this allows to focus on the main cell shape in a sample as opposed to spherical manufacturers beads. Focus on the cells in the field of view by first manually moving the FC holder on the rail using the screws, and in a second step by fine scale manual focus using Focus > Tools > Enable manual focus and choose + or - to finetune focus. Close the setup and focus menu when done.

SECTION: Presample run
27. Load the sample context file and check the settings: context > load >load context file: ‘VLIZ_FC300_Focus_sample.ctx'. This context file will process 1.5mL sample volume in order to estimate cell load in the sample.
SECTION: Presample run
28. Press AutoImage Mode.
Nomenclature for LifeWatch sample run:   
YYYYMMDD_station_samplevolume_dilution_sample
e.g. 20220929_230_SV113_sub1_3
SECTION: Presample run
29. Press start. 
SECTION: Presample run
30. After the presample run, navigate to the run output in file explorer, open the run_summary.txt file and check the Particles Per Used Image (PPUI). Ideally, the PPUI should be between 1,00 and 1,20. If the PPUI exceeds a value of 1.20, the sample should be diluted according to; 
DilutionPPUI1.20 =(0.87055*ln(1.20) - 0.03325)/(0.87055*ln(PPUI) - 0.03325)
DilutionPPUI1.10 =(0.87055*ln(1.10) - 0.03325)/(0.87055*ln(PPUI) - 0.03325)
 Choose a dilution between these two values, the formulas have been derived from an in-house dilution experiment.
SECTION: Presample run
31. Flush the sample out via Setup and Focus > Start pump. Alternate rinsing the flow cell with 5mL Milli-Q water and 5mL 70% ethanol  and leave air bubbles in between, do this 3 times each and finish by rinsing with Milli-Q water. Keep flushing until all cells are gone.
SECTION: Presample run
32. Make appropriate dilution of sample using artificial seawater of 0.22 prefiltered seawater.
SECTION: Sample runs
35. Go to Setup >  Context > Load > Load a Context file: VLIZ_FC300_standard_context_file.ctx
SECTION: Post analysis
40. Store samples in the fridge in the dark to slow down degradation of the sample.
41. Rinse FC thoroughly. Alternate rinsing with 5mL Milli-Q water and 5mL 70% ethanol and leave air bubbles in between, dothis 3 times each and finish by rinsing with Milli-Q water. Keep flushing until all cells are gone. Dirty FCs need to be replaced. 
42. Leave Milli-Q water in the flow cell, never let it dry. 
43. Robocopy data to the VLIZ archiving server in the folder of your project.
44. Shut down the FlowCam. 
45. Raw FlowCam data is processed using in-house Python packages. Single images are cut from the collages using image coordinates, height and width pulled from the .lst files, background is not removed. Desired metadata is pulled from the run summary, .ctx file and .lst file. Sample directory name is parsed, and the associated sampling metadata is pulled from MIDAS. All data is formatted and uploaded to the in-house BioSense MongoDB data, hosting images, metadata, trained classifiers, classifier metadata and training data splits. Images are predicted using Convolutional Neural Networks (CNNs), trained on the Belgian LifeWatch data. All predictions are checked and corrected if necessary. Data is aggregated tot cell densities and taxon abundances and made available through https://rshiny.lifewatch.be/flowcam-data/.  For a full description of data processing and publication we refer you to the associated data paper. 
SECTION: Sample runs
33. Take some diluted sample with a Pasteur pipette. Do not shake the sample, but gently mix to avoid sedimentation of cells in the sample.  Insert the sample in the pipette tip. Leave some air between the sample and Milli-Q water from the last flush.
SECTION: Sample runs
34. Use Setup and focus > Start pump to load the sample into the field of view again. Stop pump when the sample comes into view. Make sure no air bubbles are present in the tube, if so remove by pinching. 
SECTION: Sample runs
36. Select AutoImage Mode. Name the sample run appropriately, this is crucial for data pipelines!
Nomenclature for LW: YYYYMMDD_station_SVsamplevolume_subdilution_replicaterun
Eg. 20220716_120_SV278_sub0.01_1  -> change the sub value to the dilution you
performed, and put 1, 2 or 3 at the end for the replicate run you’re at
SECTION: Sample runs
37. Press start and keep pipetting sample into the pipette for the duration of the run, keep the sample gently mixed.
SECTION: Sample runs
38. Repeat the steps in the sample run three times to perform three technical replicate runs for each sample. Flush between each replicate run by alternate rinsing with 5mL Milli-Q water and 5mL 70% ethanol and leave air bubbles in between, do this 3 times each and finish by rinsing with Milli-Q water. 
SECTION: Sample runs
39. After performing 3 replicate tuns, rinse FC and filter thoroughly inbetween samples. Alternate rinsing with 5mL Milli-Q water and 5mL 70% ethanol and leave air bubbles in between, do this 3 times each and finish by rinsing with Milli-Q water. Keep flushing until all cells are gone.
SECTION: Presample run
27.1. 
 

SECTION: Sample runs
35.1. 

 


SECTION: Sample collection
4. Filter the 50L through a 55µm Apstein net (1.2 m long, 0.5 m diameter).
SECTION: Sample collection
5. Transfer the sample from the cod-end of the net into a sample recipient.

SECTION: Sample collection
6. Note down station, date, project and PI on the sample recipient.

SECTION: Sample collection
7. Fix the phytoplankton sample at max 1% final concentration of acid Lugol Iodine’s solution. 
SECTION: Sample collection
8. Store at 4°C in the dark until analysis in the lab (as fast as possible).
SECTION: Sample collection
3. Collect 50L of surface water with a stainless-steel bucket and rope from the back of the starboard side of the ship.

SECTION: FlowCAM hardware set up
12. Attach sample introduction stand and 5mL pipette tip. Put funnel on top of pipette tip and put 300µm prefilter in place.