Lombard Fabien

Fabien Lombard

Thibaut Pollina

Planktoscope protocol for plankton imaging V. (bytypwpw)

Keywords: Planktoscope protocol for plankton imaging, protocols, io protocols, io

bytypwpw 1 this protocols applies to the version 2.1 to 2.3 of the planktoscope and the 2.3 version of software. it is optimised to image 40µm-200µm organisms using the 25mm lens (as tube lens) and 16mm one as objective one and may be inaccurate with other configurations or light. Please note that the segmenter in currently also optimised for this and may need to be recoded (or adjusted) for other configurations, notably the size threshold but also the intensity threshold this protocol is for using planktoscope and collect usable result for quantitative imaging of plankton see also https://www.planktoscope.org/

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Planktoscope is an optical instrument. As it optical elements (camera, lenses, flowcell) are highly sensible to dust and dirt. we recommend that you never touch any of those component with fingers and store the planktoscope in a dust free and humidity free area (or in a box when not used) complete manual of assembly and software could be found at https://planktonscope.readthedocs.io/en/latest/ Plankton net 200µm sieve Squizing bottle micrometer slide (or millimetric ruller) Optical paper Dry gas dispenser Planktoscope is an electronic device, powered with electricity. It is therefore sensible to water.

-Place it in an environment where water can not enter in contact with the instrument and secure its electrical part.

-Be careful when manipulating samples, take care of having the exhaust tube in a "trash" contained to avoid spillage -glass parts are present (flowcell) and should be manipulated with caution (can break and injure you), but also should be kept clean (avoid touching it with fingers) this protocols applies to the version 2.1 to 2.3 of the planktoscope and the 2.3 version of software. it is optimised to image 40µm-200µm organisms using the 25mm lens (as tube lens) and 16mm one as objective one and may be inaccurate with other configurations or light. Please note that the segmenter in currently also optimised for this and may need to be recoded (or adjusted) for other configurations, notably the size threshold but also the intensity threshold -Test the protocol before acquisition of your first sample -Calibrate your instruments to ensure coherent measures -Create an Ecotaxa account and request the right to create project way before -Collect a plankton sample using a net first use and basic check 1 Make sure the different lenses are in place (tube lens on the camera side is a 25mm/ objective lens on the light side is a 16mm one). Insert the Ibidi flowcell on the flowcell stage. Assemble the fluidic part tube / objective lenses in place. Ibidi flowcell on its stage assemble the fludic parts put everything in place -Try to get the shortest path as possible between the sample syringe and the flowcell -Try to not get horizontal path between the syringe and the flowcell -Orient the flowcell in order that it flows vertically from up to down If not followed properly, plankton have the tendency to sediment and will progressively creates form clogs in the fluidic system which may severely lead to undersampling of plankton -Choose tubing which is the smallest as possible (eg. if flowcell is 200µm wide, tubing with internal dimensions >1mm is not needed and will creates larger stagnation of the plankton, larger risks of sinking in the fluidic path and also larger chance that plankton can swim against the current) example of optimised setup to avoid long path between sample and flowcell 1.1 power you planktoscope by connecting the different cables if the usb-c cable powering the planktoscope hare a switch, put it on.

1.2 wait few minutes and you should see a wifi appearing with the name "planktoscope_baba...." Connect to it (password "copepode") it is possible to connect by ethernet instead if you do have a cable 1.3 connect to the planktoscope using the following webpage https://planktoscope.local:1880/ui alternative url http://192.168.4.1:1880/ui alternative url to debug (no camera return) http://192.168.4.1:1880/ui/ For software versions previous to 2.3 -For windows users, you may need to download the "bonjour" printing system to communicate with the planktoscope: see here : https://support.apple.com/kb/dl999 -For mac users, this shouldn't be a problem -for linux, we don't have tested it and may not have a solution 1.4 Check that your planktoscope is properly operating: on the optic configuration page:

-turn the light on/off (you should see the light and the camera overview got white) -check that adjusting the focus makes the focus motor to turn -check that the pump motor is functioning by pumping a little -check that changing ISO/ white balance/ shutter speed leads to changes on the camera optical configuration window 1.5 adjust manually the white balance in order that it looks about similar to the automatic white balance (AWB) this latter should be desactivated once you obtain correct results (it will continue to correct depending on the images and may leads to incorrect results) 1.6 Locate how to shut down the planktoscope (main Planktoscope page) calibration 2 Pump calibration: Pump calibration:

Peristaltic pump tubes flexibility varies with age, care and type of liquid used (e.g. lugol may age it quicker), calibrating the pump regularly could be needed but is not highly important to get good quantitative count since this is the number of image (therefore the volume imaged) which is important (not the pumped volume) 2.1 -prepare a large volume of tap water and put in in the syringe targeting a total volume of e.g. 20ml -on the optic configuration tab: tell him that you want to pass 10ml and record the exact volume it finally ends to pass (eg. by looking on the graduation of the syringe) note X= final volume passed for a 10ml instruction final volume (here X=20-12.2) 2.2 -then on the hardware setting, note the "pump step per ml" parameter (old step) -calculate the "calibrated" pump step per ml such as = 10*old step/ X -replace the "pump step per ml" parameter with this value hardware settings page (pump per step is at the bottom) 3 Size calibration: Size calibration:

Size calibration is an important process to get good data and should be absolutely done and noted. Please however note that currently calibrations is currently bugged and you need to manually replace the comma by a point in the hardware setting at every start manually replace the comma by a point in the hardware setting at every start 3.1 -tilt the planktoscope on the side (camera on the bottom) tilted position for calibration 3.2 -remove the flowcell and place a micro metric ruler (or a millimetric one) on the sample stage such as the ruler is either vertical or horizontal but not in diagonal but not in diagonal (using the 20mm/16mm combo of lenses the camera field of view should be about 3mm by 4mm). Make the focus on the scale placing the calibration slide (once removing the flowcell) focusing on the scale 3.3 -take few images (enter sample name, positions don't matters but enter 90S, 0W to easily spot the sample as a test, date and time, these are the only mandatory fields; see section 5.4 for filling metadata see section 5.4 for filling metadata ) example of metadata entered in "sample" page here two images are acquired (and random information entered on the pumping one) 3.4 -download images on a computer and measure how much pixels are needed to obtain the longest path possible on the image (e.g. using imageJ https://imagej.nih.gov/ij/index.html) (see section 7 for communicating with your ee section 7 for communicating with your planktoscope and downloading the raw images planktoscope and downloading the raw images) downloading resulting images (using Filezilla) using imageJ open your image (File/open), draw a line as long as possible (here 3mm on the scale) and measure it (analyse/measure). The line is 3476 pixel length (i.e. one pixel is 0.86 with this example)

3.5 -calculate how much micron is represented by each pixels (should not be strongly different from 1.01 which is the default value for 25/16mm lenses combo) 3.6 Enter the calibrated pixel size value in the hardware setting (note here that the note here that the comma should be replaced by point at every restart of the system comma should be replaced by point at every restart of the system ) Get your sample not agitating your sample will let plankton to sediment and could even block the fluidic part. More importantly, the organisms concentration will be inhomogeneous, and because you will first get the sinking plankton, will lead your measurements to over-estimate true concentrations 5.3 adjust the concentration of the sample: ideally not more than 20-30 objects ideally not more than 20-30 objects should be present per frame. If the sample is over-concentrated, dilute it by a factor 2 (add in a jar 1/2 of the sample -after agitating it-and 1/2 of seawater). Note dilution Note dilution xxx in metadata in 5.4 xxx in metadata in 5.4

Having too much object per frame will create : 1. increase probability that objects are touching (making them impossible to count or identify) 2. increased probability to clog the fluidic system 3. artefacts during the segmentation step 5.4 Go to "sample" page and fill metadata This step is critical because those data are the ones that will make your sample usable or not -fill the sample identification fill the sample identification (project, name, boat used, your name and the station number) -tells how you sampled the plankton tells how you sampled the plankton (recording mesh size with "minimal minimal fraction size fraction size " (will be used afterwards in the segmentation process, object smaller than this wont be segmented; "Maximal fraction size Maximal fraction size " is the size of the mesh used in step go to step # go to step #4.2 4.2 ; Filtered Filtered volume is important if you recorded it but could be calculated from other parameters . Make sure to either have filled it or to have filled either min and max depth min and max depth if using a vertical net; initial/final positions initial/final positions, speed speed and length (min) of deployment and length (min) of deployment if using an horizontal towed net in all cases the diameter of the net opening diameter of the net opening will be needed to calculate the filtered volume Dilution factor if dilution have been done in go to step # go to step #5.3 5.3

Fill the net initial and final position (if towed horizontally) remember to validated both of them (readings disappear after validation, but are recorded)

5.5 Go to fluidic acquisition and set parameters -number of image to acquire (to be chosen depending on the desired final object number and the observed concentration on images) pump significantly between two images will help to :

1. not let plankton to sediment in the fluidic system 2. not image two time the same plankton Target a sample size (by setting the number of images to acquire) that finally have something like 1000-2000 final objects (e.g. if you do have 10 object per images, imaging 100-200 frames would be enough)

5.6 go to optic configuration and pump with high flow rate a good amount of water (objective, remove plankton that have sunk in the fluidic system)

5.7

Go to fluidic acquisition and start the acquisition. Awaits that the acquisition is done Results could be consulted by passing through the gallery img If your fluidic system is not optimised to avoid plankton sedimentation, some plankton could accumulate in the fluidic system. This could be check by pinching on the tube half way in between the flowcell and the pump during 1-2 seconds (to accumulate suction pressure) and releasing it. If a large quantity of plankton pass suddenly then plankton have sedimented between the syringe and the flowcell.

segment the acquisition 6

Go on segmentation and clic on the "update acquisition's folder list" select the samples you wish to segment Setup the different options of the segmenter 1. Recursive folder means that it will segment all samples within a selected sample 2. Ecotaxa archive: it will create a zip file containing all files needed for a easy importation within ecotaxa 3. Force rework: if yes it will re-segment samples already segmented 4. Keep objects: it will keeps the final segmented images visible in the planktoscope (that could be accessed by the gallery in the objects folder)

scroll down and clic on start segmentation awaits that the segmenter status turn to "done" download the results

You will need a computer connected to the planktoscope together with free software FileZilla Needs to be done only once: basic rights don't include the "create project". As a protection against bots, To create a new project and upload images in it, please contact the user manager(s): PlQv (piqv@imev-mer.fr) The rights to create projects will be activated (by a humain, please be patient few days) soon and you will see the following right appearing without the right to create projects with the right to create projects 9.3 You can now create your own project on which you will be able to import, visualise and classify images 9.4 upload the ecotaxa archives (see step 6-7) on the EcoTaxa ftp Please think to regularly remove those temporary files from the ftp, at this point they are not secured at all and everybody can access them (and disk space is not free)

9.5 In your project/ on your project options button, select import images and metadata 9.6 locate your file on the ecotaxa ftp folders and import it (only works one zip file at a time for now)

How to use efficiently ecotaxa 10 Configure your project efficiently: in Project/project settings Configure your project efficiently: in Project/project settings Select a data sharing license (we recommend one of the CC-BY one or CC-0 if you want data to have a future use for science) Define if the project is visible for visitors (only "validated" images will be visible) Add a preset list of taxa for manual sorting (could be copied from any other project, or taxa added manually): those will be present in the taxonomic filter (see 10.1) Add useful sorting variables : in "Fields available for sorting": add at least those parameters that are pretty useful and will be added to the Quickfilters (see 10. The different validation "states" in ecotaxa and how to validate image arrives in EcoTaxa with the status "unclassified" (grey surrounding of the image)

However they could be also set as "predicted" (blue surrounding; classified automatically by taking as example one pre-existing project), "validated" (green surrounding; checked and annotated by a human), or dubious (orange surrounding; checked and annotated as dubious by a human) -validating consist in selecting one or several picture and attributing them a taxonomic or morphological identity by either displacing them in the list of taxa present in the « taxonomic filter » tab (in which you can force some categories to be present by using the « preset » in the project settings… or just by typing the name using the keyboard (which should use right away the research on top of the »taxonomic filter ». Whatever happens you need to save (ctrl.S or save button at the bottom of the page) before your action gets finally implemented.

photo here to illustrate typing "copepo" brings several results once validated the name appears in red bellow the images -validation could be tedious and requires large taxonomic expertise, however there is plenty of tools to help you! Filters are one of those tools, but the more interesting one is to use previous project to « predict » some taxonomic identity on your new images, in best cases you will face thousands of rightly predicted images and will be able to validate thousands per minutes! sliding into existing categories does also works dont forget to save your validations example of (well) predicted objects which would be easily validated 10.3 Do not hesitate to "predict" your project right away Do not hesitate to "predict" your project right away (even with a project/instrument that have nothing to do)

-how to do prediction (and helps you to boost your validation ability)

In the project (or "Filtered", in this case only the filtered vignettes would be used), select "train and Predict identification"

You can select any of the pre-existing project (including your own project) as a template for image recognition. By experience, using another project is only a firstaid, but won't replace prediction on images that you acquired with the same instrument/ same location / same plankton communities Note that currently, only few "sorted" planktoscope projects do exists (especially acquired with the same segmentation procedure than here), we therefore strongly encourage you after a first trial of prediction to quickly validate to re-predict on your Learning features (if not available see step 10). Inactivate variables that are not relevant for prediction and relates to position of the vignette into the initial images (bx, by, depth min/max, label, local centroid col/row, x, y) Once done images are now "predicted" (blue) but still awaits validation. While classifying the different objects, the classifier also gave a classification "score score " which determine if the label is attributed with high or low confidence. Using this score as a quick-filter is usually a good idea to be able to validate quickly well recognised images (and quickly start new predictions) Doing repeated predictions on your own samples is better than doing some Doing repeated predictions on your own samples is better than doing some global one on random example project global one on random example project Quickly validate objects to start to predict on your own plankton composition: the classifier is quite efficient and starts to give reasonable results starting from 30-50 images as example. Ecotaxa is then optimised to operate regular prediction rounds which could be heavily guided by the human (e.g. by doing prediction only on selections, stopping to predict some organisms etc).

-once fully validated, export your results (lots of different solutions does exist, the more easy to understand being the summary export with count per sample) example of images sorted by score of prediction Maintenance of your planktoscope 11 clean tubing and flowcell from inside clean tubing and flowcell from inside imaging plankton will lead to have a lot of organic material and seawater in the fluidic system. Some may clog or accumulates in some parts of the fluidic system. 1. Don't let it dry and try to get rid of it as soon as possible (if its occurs during sample acquisition, even abort this latter, take care of the clog, maybe dilute the sample go to step # go to step #5.3 5.3 and restart acquisition while noting that the sample get diluted in the metadata go to step # go to step #5.4 5.4 ) 2. Pump tap or distilled water with high pumping rates helps to unclog the system. make sure no plankton organisms remain in the fluidic system and especially on the internal walls of the flowcell.

If it is the case don't hesitate to pinch (during 1-2 second) and release the tubing between the flowcell and the pump while pumping to creates sudden variation of pressure (e.g.

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  Use a net to collect plankton Record Latitude Longitude Latitude Longitude (taking photo of the GPS when launching and recovering the net could serves, if UTC time could be present on the GPS this could aslo be interesting) -if vertical net vertical net, record min and max depth min and max depth -if horizontal records initial/final positions initial/final positions, speed and length (min) of deployment speed and length (min) of deployment -if you have flowmeter flowmeter, record the initial/final digits of the flowmeter initial/final digits of the flowmeter and calculate the filtered volume filtered volume in all cases the diameter of the net opening diameter of the net opening will be needed Those are critical informations to get to quantitative sampling (see step 5.4) Those are critical informations to get to quantitative sampling (see step 5.4) 4.1 Get the content of the collector. 4.2 Pass the volume through a 200µm sieve larger organisms may clog the flowcell filtration through a 200µm mesh detail of the (home made) filter 4.3 Rinse the sieve using seawater and a squeezing bottle (helps to pass small object) 4.4 Recover the fluid / measure it volume/ record it on logsheets (will be entered latter as "concentrated sample volume concentrated sample volume ") Those are critical informations to get to quantitative sampling Those are critical informations to get to quantitative sampling concentrated final volume of plankton Pass the sample on planktoscope 5 assemble and start the planktoscope (see go to step # go to step #1 1 go to step # go to step #1.1 1.1 go to step # go to step #1.2 1.2 go to step # go to step #1.3 1.3 ) 5.1 check for lenses alignment : remove the objective lens, start the light and check if the light source is centred. -if yes place back the objective lens and flowcell -if no adjust the position of the tube lens to center the light source (magnets allows for 1-2mm adjustments) objective lens and flowcell removed unaligned lens centered lens unaligned tube lens could create strong inhomogeneous background in final images, leading to creating lots or artefact during segmentation of the different plankton objects. Unfortunately the magnets lets a 1-2 mm degree of freedom which is responsible from this severely unaligned lens final results and and final artefact object created 5.2 Go to optic configuration. turn the light on verify focus on dry slide (tip, if the slide is slightly wet, you can check for the focus simultaneously on the water traces on the two sides of the flowcell) Add your sample in the syringe (and keep it suspended by agitating it manually regularly or even gently insert an air bubbler with 1 bubble/second in it) pump until you see your sample passing by and flowing through the peristaltic pump Eventually get rid of air bubble by pinching the tubing half way between the flowcell and the pump while pumping (see also 5.7). finely adjust the focus on the organisms passing by (tip: start using the "1mm" buttons, then the 100µm buttons and finalise by typing 25 or 50µm adjustments in the middle box (and pressing external arrows of focus)

  (https://filezilla-project.org/) Open FileZilla either clic on the top right create a new connection or use the quick-connection fields bellow Enter the following informations: Host: sftp://planktoscope.local (192.168.4.1 should also works) Username: pi Password: copepode Port: 22 clic on connect on the bottom panels you have (at left) the access to what is in your computer and (at right) the access to what is in the planktoscope (clic and slide to transfer data in between both) Exports file for EcoTaxa are in /home/pi/data/export/ecotaxa Raw images files are in /home/pi/data/img Different control files to check the segmentation process (images after background substraction, masks of the different objects etc) are in /home/pi/data/clean Final vignettes are in /home/pi/data/objects Clean the planktoscope 8 1. Drain the sample out of the syringe 2. Disconnect the syringe and clean it with tap water (or even distilled water) 3. Pump (at high speed!) at high speed!) the full content of the fluidic system to remove any liquid 4. Reconnect the syringe 5. fill it with tap water (or distilled water) 6. pump (at high speed!) at high speed!) while regularly pinch the tubing to unstuck any plankton in the system (see go to step # go to step #5.7 5.7 ) 7. Drain again the syringe (repeat steps 7.2-7.7 at least 2 times repeat steps 7.2-7.7 at least 2 times more until no plankton is visible on the camera) 8. Finally drain the system Upload your images on EcoTaxa 9 At First connection: At First connection: Create an account on EcoTaxa (https://ecotaxa.obs-vlfr.fr/) by clicking on the top right "log in/register" Put your real name and a valid mail to contact you 9.1 Once logged you can consult the project on which you are registered (e.g. your own projects + the ones you have been invited by the different data owners) by clicking onto "contribute to a project contribute to a project" on the main page 9.2 Needs to be done only once:

  using Filezilla (see go to step # go to step #7 7 ) create a connection by using the following informations: Select File > Site Manager... Create a New Site called : Ecotaxa_VLFR In General tag : Host : plankton.obs-vlfr.fr Protocol : FTP -File Transfer Protocol Encryption : Only use plain FTP (insecure) Logon Type : Normal User : ftp_plankton Password : Pl@nkt0n4Ecotaxa this done you could use FileZilla to load the Zip files downloaded from the Planktoscope onto the EcoTaxa ftp server (e.g. /Ecotaxa_Data_to_import/PLANKTONSCOPE) Please eventually create your own folder to "try" to keep it clean and tied

  of contact (mandatory) define what pre-trained Deep Learning features to use on your project (we recommend to use «flowcam » unless we start to have some trained on planktoscope image And invite people to manage the project with you (will have the same rights than you) helps you to annotate images (won't have options below "export" in the project see go to step # go to step #9.4 9.4 ) or just viewing the project (both validated and non validated) 10.1 Use filters wisely Use filters wisely there is three layers of filters in EcoTaxa: the quick access filters (top bar) The taxonomic filter tab (allows to filter by taxonomic groups) and the other filter tabs Filters are additive, so you can addition filers on geography, date, who validated them, taxonomic group and every numeric fields/ text fields entered in ecotaxa to search for specific things (and you can get rid of them easily too, see grey fields on to of the next image) taxonomic filters other filters quickfilters are pretty useful since you can sort object by specific values (eg. mean saturation bellow) to quickly observe object that have here lot of chlorophyll (ps. you can revert the sorting order of those filter to get by ascending or descending order) 10.2 The different validation "states" in ecotaxa

  own project. what could be used for first prediction : #4605 -Planktoscope NOAA WCOA21 rita-net (coastal US West coast from Vancouver to San Diego; Processed with current segmenter; Partly validated; Contains lots of Contains lots of artefacts due to lens mis-alignment artefacts due to lens mis-alignment see 5.1) #4356 -Planktoscope Tara Microbiomes P2/miniHSN : Lorient > Punta Arenas V3 (Trans-atlantic transect from France to Ushuaia; Processed with other segmenter (works only with adding Deep Learning features into play works only with adding Deep Learning features into play); Fully validated) Push the button "select project bellow then click me. You then have the possibility to select what types of object to consider. It is recommended to try to avoid selecting too much object to partly correct the usual strong imbalance between categories (here as an example limited to 100 example per groups) (If you use project #4605 as an example, please remove artefacts) click on "continues to the classifier option screen ". Activate the pre-trained Deep

  time, wet conditions and organic mater may create favorable condition for the growth of a bacterial film. The flowcell and tubing will looks dirty from the inside. You can avoid this by pumping diluted bleach sometimes, let it acts for 1-2 hours and carefully rinsing the whole system go to step # go to step #8 8 4. Water, bacteria, and bleach together may favour the apparition of a calcium carbonate film inside the tubing and flowcell. It may either appears as dispersed cristals attached inside the flowcell or a white coating inside the tubing. To remove and clean this pumps some acidic solution (vinegar, citrus juice or other kind of other acids), let it rest for few hours and rinse the system go to step # go to step #8 8 clean flowcell outside: clean flowcell outside: The flowcell is an optical critical component, keeping it clean is an absolute necessity. Don't touch it with fingers or other kind of dirty material. If dirty: 1. if dry dusts only are present, gently blow the flowcell (ideally with dry gas dispenser at a large distance -dry gas dispenser are also creating thermal chocs if used from too close, test it on other material before) 2. if dirt in not only dry dusts it could be cleaned with optical paper and ethanol. DO NOT USE DO NOT USE CLASSICAL WIPING PAPER CLASSICAL WIPING PAPER which are usually enriched in silica fibers for solidity ... and may create scratches on the flowcell. (disposable nose tissue are better alternative if optical paper is not available)clean optical lenses clean optical lenses as for the flowcell, optical lenses are critical elements of your planktoscope and should be kept as clean as possible. It starts by never touching them with fingers (cleaning those would requires lot of patience, efforts and may even lead to unexpected disappointments) 1. dry dust: dry gas (with even more caution than previously 2. others: only used optical paper clean the camera sensor clean the camera sensor ... critical part if any, NEVER touch it, only use dry gas regularly calibrate the pump regularly calibrate the pump go to step # go to step #