TITLE: Planktoscope protocol for plankton imaging
AUTHORS: Pierre Kostyrka, Lombard Fabien

[DESCRIPTION]
This protocol is for using PlanktoScope and collecting usable results for quantitative imaging of plankton. 
This project has received funding from the European Union’s Horizon 2020 research and innovation programme “Atlantic Ecosystems Assessment, Forecasting and Sustainability” (AtlantECO GA#862923)
This research was co-funded by the European Union (GA#101059915 - BIOcean5D).

See also https://www.planktoscope.org/

[BEFORE_START]
-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

[GUIDELINES]
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/

[STEPS]
SECTION: Table of Contents
1. Table of Contents  
Introduction  
Quick usage version  
Assemble the PlanktoScope  
User Interface and initial connexion  
How to export data  
White Balance calibration  
Pump calibration  
Size calibration  
Get your sample  
Pass the sample on the PlanktoScope  
Segment the Acquisition  
Clean the PlanktoScope  
Upload your images on EcoTaxa  
How to use efficiently EcoTaxa  
How to compute biovolumes  
Maintenance of your PlanktoScope  
Update the software or reset the PlanktoScope  
Troubleshooting  
External links  


SECTION: Introduction
2. The PlanktoScope is a frugal, microfluidic microscope designed with an open-hardware and open-software approach. It was conceived with the idea of providing the thousands of scientists and sailors exploring the oceans with a high-quality instrument suitable for deepening our knowledge of the sea around us. In this manual you will learn how to operate the Planktoscope and take images of plankton.
SECTION: Quick usage version
3. This part is a quick version of the protocol, where you will find the essential steps to follow. If you're using your PlanktoScope for the first time or need to calibrate it, please read the full protocol. Don't wait too long between the sampling and the processing of your sample through the PlanktoScope to avoid sedimentation and aggregation.
SECTION: Quick usage version
3.1. Preparation

Assemble and plug in the PlanktoScope   
Connect to PlanktoScope’s Wi-Fi  
Go to  http://192.168.4.1:1880/ui/ (v2023: http://home.pkscope/ > Node-RED Dashboard) 
Fill ALL the sample details (in SAMPLE) [Critical]   
Put the light on (in OPTIC CONFIGURATION) 
Check the WB parameters (you should have already done it during the initial connection)   
Check flow cell alignment and check the focus    
Put 20 mL sample   
Turn on the bubbler  
Check the focus (in OPTIC CONFIGURATION)   


SECTION: Quick usage version
3.2. Acquisition

Fill acquisition parameters (in FLUIDIC ACQUISITION)   
Pump to drain sedimented organisms before acquisition (in OPTIC CONFIGURATION)
Start the acquisition (in FLUIDIC ACQUISITION)
SECTION: Quick usage version
3.3. Segmentation and data export 

Fill in segmentation parameters (in SEGMENTATION)   
Start segmentation 
Back-up data (in GALLERY or by using FileZilla)   
Import on Ecotaxa   
SECTION: Quick usage version
3.4. Cleaning   

Drain the syringe (disconnect your system)
Drain the content 
Replace with fresh water and drain several times (blowing in the syringe may helps)
Replace the system and drain first with tap water and then air
Empty waste container

If not used immediately

Put 20 mL diluted bleach
Leave 15’
Drain the content (high pump speed)
Put 10 mL fresh water
Drain the content (high pump speed)
SECTION: Quick usage version
3.5. Shut down

Turn OFF virtually (in HOME) and wait one minute
Unplug the PlanktoScope
SECTION: Assemble the PlanktoScope
4. The PlanktoScope kit

In this part you will learn :

What is inside the PlanktoScope kit
How to assemble the fluidic path with the flowcell
How to assemble the bubbler
How to assemble all the components together 

Open the PlanktoScope box and check that everything is in it. The PlanktoScope kit also includes a bubbler, power cable, waste container, falcon tube of tap water, syringe, sample holder, flowcell holder and flowcell (fig.1; fig.2).

 



 



 


 


SECTION: Assemble the PlanktoScope
4.1. The flow cell 

 

It is recommended to remove the upper screwed parts used to maintain the syringe (F3 in fig.2).
Take a flowcell and plug the short part to the syringe with the help of a male clip.
Install it like in the image on the left of the fig.5. 
Then, press the tube where the arrows point (fig.5). 
Screw softly the two parts together. 
Place the receptacle on the magnetic flowcell holder. You can put some tape on the magnetic parts to reduce the "impact" between the flowcell holder and the receptacle.

 

SECTION: Assemble the PlanktoScope
4.2. The fluidic path

Now that you have fixed the flowcell (see last section), assemble all the fluidic system as below (fig.6). Don't forget to install the "tube clip" on the matching tube (figure below). It will allow you to stop the flow when it is necessary.

 

SECTION: Assemble the PlanktoScope
4.3. The bubbler 

Planktoscope image fluid at low speed. You will need a "bubbler" to avoid sedimentation.
 

Assemble the bubbler like in the image below (fig.7). It is recommended to use a cut glass pipette or something similar instead of the needle provided (the needle can be clogged and does not allow a good control on the air flux).
Plug the Bubbler into one of the USB ports on the PlanktoScope. 
Place the tubing into the syringe so that it reaches the bottom. 
Affix the tubing to the syringe using an elastic band, string or similar. 
Setup the 'Bubbler': the flow of air needs to be adjusted to 1 bubble/sec approximately, bubbling the bottom of the syringe. You will need water to check this. 
Do not put the tip of the bubbler in the center of the syringe or it will inject bubbles into the flowcell.



 


SECTION: Assemble the PlanktoScope
4.4. Assemble the PlanktoScope 

The fluidic path, the flowcell and the bubbler should be all assembled now.
Now you just need to (if not already done) :

Place the flask holder on the magnetic patches (B in fig.1)
Place the trash in the outer hole of the flask holder.
Place the sample tube in the inner hole.
Place the flowcell receptacle on the magnetic flowcell holder 
Plug the bubbler   
Plug the power cable


 


SECTION: User Interface and initial connexion
5. Initial connection

You can access to the user interface of the PlanktoScope by connecting to the Wi-Fi generated by the PlanktoScope. Your computer will be only a projection of the software of the PlanktoScope. It means that even if you disconnect from the Wi-Fi of the PlanktoScope, it will still be running.

Power your Planktoscope by connecting the power cable to the power input and turning on the wall switch. 
Within 1 minute of turning on your PlanktoScope, you should see the LED flash once. This means that the PlanktoScope is ready to be connected by Wi-Fi. 
You should see a new option for Wi-fi appearing on your computer. Connect to it with the password: "copepode".

For more information and alternative methods of connection, see the designer's Connectivity Tutorial here:   

 

SECTION: User Interface and initial connexion
5.1. The User Interface (Node-RED dashboard, UI)

Open the PlanktoScope's User Interface (UI) on your web browser (Chrome, Firefox, Edge etc.) using the following webpage link (either click on the link or copy and paste into your browser) and go to the Node-RED dashboard :

http://192.168.4.1:1880/ui/ (v.2022)
http://home.pkscope/ > Node-RED dashboard (v.2023)

There are several tabs on the User Interface (UI, fig.1) that can be used to adjust setting, run samples and take images. To navigate around the UI, all tabs are available from the Home tab, including the Shutdown button which we will use when we have finished using the PlanktoScope. We can also use the 'Hamburger Menu', situated in the top-left corner of the UI, to navigate between tabs.

 

 
You will find :
The "SAMPLE" page, to fill all the metadata of your sample.
The "OPTIC CONFIGURATION" page allows you to control the various features of PlanktoScope. You can focus, turn on the LED or start the pump.
The "SEGMENTATION" page is used to start the segmentation of the images taken in the previous phase. The images will then be processed to extract only the plankton thumbnails.
In the "GALLERY" you can find the different files of the Planktoscope: the exports for EcoTaxa, the original images and the extracted thumbnails.
The "SYSTEM MONITORING" page allows you to check the correct operation of the device. You will not use this step in standard use.
The "WIFI" page gives you access to the characteristics of the wifi generated by the PlanktoScope to which you will connect in order to control the device. You will not have to modify anything on this page. The procedure for connecting will be detailed later in this manual.
The "HARDWARE SETTINGS" page. You will not need it and it is strongly advised to not change anything else.

SECTION: User Interface and initial connexion
5.2. The Optic Configuration tab

Once the UI has loaded on your browser, navigate to the Optic Configuration tab and we will make sure the PlanktoScope is operating correctly (fig.2).
 

a) Under Optic Characterisation, switch on the Light by clicking 'On' (fig.3). You should see the Preview image turning from dark to light. The Preview image could be any color so do not worry if yours show blue, red, green, etc. ; it will be adjusted later during the White Balance (WB) calibration.
 

b) Under Focus Adjustment (fig.4), click 'UP 1MM' and 'DOWN 1MM' to ensure focus buttons turn the focus motor (same for the 100 microns version of the button). You should see the mount moving further from (UP) or closer to (DOWN) the camera. 
You can also use the ">>" button to move your focus according to a personalized value ("Focus distance").
 

c) Under Fluidic Manual Manipulation (fig.5), click clockwise arrow to check that the Peristaltic Pump is working. You should see the pump rotating in an clockwise direction. 
You can choose to pass a volume, but keep in mind that this parameter will not impact the acquisition. On the other hand, the flowrate parameter will also be effective during an acquisition.
 

d) Under Camera Settings, you can see a button to change the ISO value (fig.6). Set the ISO to 100. 
The ISO is the light sensitivity of the camera. A low ISO will be less sensitive to light and then darker. A low ISO tends to provide images of better quality.
 

 

SECTION: How to export data
6. 
 

With Filezilla

You will need a computer connected to the planktoscope together with the free software FileZilla (https://filezilla-project.org/).

Open FileZilla
Either click on the top left to create a new connexion or use the quick-connection fields below (it will not save the ftp site for later, fig.1)
To create a new connexion "file > site manager > new site"
 
Enter the following information: 
Host: sftp://192.168.4.1 (note images were taken with a previous version, the address does not correspond to images)
Username: pi
Password: copepode
Port: 22

 

Click on connect
On the bottom panels you have (on the left) the access to what is in your computer and (on the right) the access to what is in the planktoscope (click 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 subtraction, masks of the different objects etc) are in /home/pi/data/clean
Final vignettes are in /home/pi/data/objects

With the gallery

Go to the Gallery tab (fig.3):
For raw images: img 
For segmented images used in EcoTaxa: export > ecotaxa
For detailed process: clean
For segmented images without metadat: objects

 

SECTION: White Balance calibration
7. White Balance (WB) calibration

Stay in the Optic Configuration tab. For this step, you will need the software ImageJ https://imagej.nih.gov/ij/index.html. To check the WB, do not put the flowcell or anything between the camera and the LED.

 


 

Try pressing the Auto White Balance button to its 'on' and 'off' positions on the Optic Configuration tab; you will likely see the Preview image changing color (fig.1). 
 

The Auto White Balance feature is currently not optimized. In addition, not using Auto White Balance enhances the performance of the PlanktoScope over time (the camera will try to adjust it in between every image). It is then recommended to manually adjust the white balance of your PlanktoScope. Set the AWB button to 'off'.

To manually set to the White Balance:
Turn off the Auto White Balance. You will need to adjust WB: Red and WB: Blue until it looks white/grey. The WB should never be perfect white (but grey), as it imply over-exposition during the segmentation step. Never set it to 0 or less, or it will create a bug in the software.
Take one image in the acquisition tab (you should not use the preview image as it will not show the real colors taken by the camera).
Download the raw image in the "gallery" tab or by using filezilla.
Open the software ImageJ. The corrected WB Red or Blue is the ratio between the green value (G) on the corresponding color value (Red = R ; Blue = B) multiplied by the old configuration. You can extract the RGB mean values on a software like ImageJ.
File > open.
Plugins > analyze > RGB. If you do not have the plugin or if it is not working go to Analyze > Histogram > Click on RGB until you see the mean red, green and blue values. 
Once the values are extracted, calculate the ratio G/R and G/B like in the example below.
multiply it by the old one to get the new one.
Change the configuration.

Example : 
If the default configuration is:
  
  
With this actual configuration, we measure the RGB values:
  
  
  
So the new configuration should be:
  
  


 


SECTION: Pump calibration
8. Pump calibration
 

SECTION: Pump calibration
8.1. Calculate pump step

Prepare a large volume of tap water and put in in the syringe targeting a total volume A of 20ml
On the optic configuration tab: choose to pass a volume Y = 10 mL and record the exact volume X it finally ends to pass (eg. by looking on the graduation B of the syringe). X = final volume passed for a 10 mL instruction = A - B 
If the pump calibration is correct, X should be equal to Y = 10 mL.
If it is not correct, see next step.

 
 
   

SECTION: Pump calibration
8.2. Update pump step

Go to the hardware setting (fig.2).
Note the initial calibration “pump step per ml” here : ........
Calculate the "calibrated" pump step per ml such as New Step = Y/ X*old step
Replace the “pump step per ml” parameter with this value.

 


SECTION: Size calibration
9. Size calibration
 

SECTION: Size calibration
9.1. Set up the scale

Tilt the planktoscope on the side (camera on the bottom)
Remove the flowcell and place a micro metric ruler (or a millimetric one) on the sample stage such that the ruler is either vertical or horizontal but not in diagonal (fig.1). 
Make the focus on the scale (fig.2).

 

 

SECTION: Size calibration
9.2. Take images of the scale

Fill metadata in the test mode in the sample tab (fig.3).
Take a few images, go on "acquisition" and put 1 or 2 images (fig.4). You should not use only acquired images to measure the real size of the pixels, and not the preview image as it will give you wrong results.

 

 

SECTION: Size calibration
9.3. Measure the number of pixels

Download the images on a computer using filezilla, the datafile manager or the gallery according to your software version.
Open ImageJ.
File > Open. Choose the image.
Click on the line button (see fig.5) and trace a line.
Analyze > Measure.
Check the length value (in pixel).

 

SECTION: Size calibration
9.4. Calculate the micron/pixel ratio

Calculate how much microns are represented by each pixel (The expected value for the default camera is expected to be around 0.75). You will need to do Length(micron)/Length(pixels).
SECTION: Size calibration
9.5. Update the micron/pixel ratio

Enter the calibrated pixel size value in the hardware setting (fig.6).

 



SECTION: Get your sample
10. This protocol considers that a plankton net is used to collect the sample. 
In this section, you will:
Collect your sample 
Fill a log sheet
Filter your sample with a 200µm sieve
SECTION: Get your sample
10.1. Collect your sample and the metadata

 

You can find here a logsheet example (based on the datas needed in the SAMPLE Tab).

Operator: ........
Station ID: ........
Sampling gear: ........
Process time: ........

Net throw Lat: ........
Net throw Lon: ........
Net throw date/time: ........

(If it a horizontal sampling)
Net retrieval Lat: ........
Net retrieval Lon: ........
Net retrieval date/time: ........

Minimal fraction (μm): ........
Maximal fraction (μm): ........
Min depth (m): ........
Max depth (m): ........

/!\ Critical information
Filtered Volume (L): ........
Concentrated sample volume (mL): ........
Dilution factor: ........ (<1 if there has been a dilution)

Net opening dimension (mm): ........
Speed Through Water (kts): ........
SECTION: Get your sample
10.2. Filter your sample

 

 

SECTION: Pass the sample on PlanktoScope
11. Assemble and start the planktoscope before (see   )

In this section you will:

Fill the metadata of the logsheet in the sample tab
Check the focus and the flowcell (image quality)
Put the light on and the bubbler
Put your sample and check the concentration of your sample
Dilute your sample if necessary 
Pass sedimented organisms
Launch an acquisition
SECTION: Pass the sample on PlanktoScope
11.1. Fill the metadata

Go to the "sample" page and fill the metadata. This step is critical because those data are the ones that will make your sample usable or not.

 


Fill the sample identification (project, name, boat used, your name and the station number)

 
Note how you sampled the plankton (recording mesh size with "minimal fraction size" (will be used afterwards in the segmentation process, objects smaller than this won't be segmented
"Maximal fraction size" is the size of the mesh used (to remove too large organisms) 
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 if using a vertical net
initial/final positions, speed and length (min) of deployment if using a horizontal towed net.

 

In all cases the diameter of the net opening will be needed to calculate the filtered volume (only if you used a plankton net).

Note the mesh size used for collection in "minimal fraction size " (it will be used afterwards in the segmentation process, object smaller than this won’t be segmented);
The "Maximal fraction size " is the mesh size used to filter the sample during preparation (It must have been done at 200µm so as not to block the fluidic circuit); 
The “Filtered volume” is the volume passed through the net during sampling. It is better if you recorded it but could be calculated from other parameters. So, make sure to either have filled it or to have filled either min and max depth if using a vertical net; initial/final positions, speed and length (min) of deployment if using an horizontal towed net; and in all cases the diameter of the net opening (to be able to calculate the volume afterwards).
“Concentrated sample volume” is the volume recovered by the net.
If a dilution has been done,  note the “dilution factor” (if not, write “1”). <1 if it is diluted, >1 if it has been concentrated. 

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

 


 

SECTION: Pass the sample on PlanktoScope
11.2. Check flow cell alignment

Turn the light on. 
Check for lens alignment with the flow cell. Move slowly the flow cell receptacle until there is no black background like in the image below.
 
 

SECTION: Pass the sample on PlanktoScope
11.3. Do the focus 

 
Go to the OPTIC CONFIGURATION tab:

Turn the light on if not already done.
Check focus on the two sides of the flow cell, and try to have a focus between the two sides. In the figure above, you can see that on a dry flow cell that has been exposed to water, there are still water droplets on both sides. Use them as an indicator to do the focus. 
You can also do the focus on one side, and then move the focus by the size of the flowcell divided by two. For a flowcell of 300 microns, you should move the focus by 150 microns in the opposite direction.

 
 


SECTION: Pass the sample on PlanktoScope
11.4. Put your sample in the PlanktoScope

Close the stopper
Fill the sample in the syringe. For this you can just remove the full sample holder (and fill it on top of a sink (to not risk spills on-top of the planktoscope). 
Reconnect the syringe to the pump
Open the stopper
Place the bubbler and adjust the flow to 1 bubble/second.

 


 


 

SECTION: Pass the sample on PlanktoScope
11.6. Pump sedimented particles

In the OPTIC CONFIGURATION tab, pump with high flow rate a good amount of water (goal: remove plankton that have sunk in the fluidic system). You do not need to pump a large amount of your sample, 1 mL is sufficient.

 
 
 
SECTION: Pass the sample on PlanktoScope
11.5. Dilute your sample if necessary

Adjust the concentration of the sample: 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  in metadata !
 


SECTION: Pass the sample on PlanktoScope
11.7. Acquisition

Go to fluidic acquisition and set parameters.

- Number of images to acquire (to be chosen depending on the desired final object number and the observed concentration on images)

 


 

- Volume to pump is the volume pumped in between two images: it should be large enough to : avoid taking twice the same object in photo; avoid large sedimentation in the fluidic system; avoid objects to stick on the flowcell. It is recommended testing it in order that the volume passed between two images correspond at least to 5-10 times to the volume imaged (see here the discrepancy between imaged volume/pumped volume).

- Delay to stabilise image is the time lag in between the stop of the pump and the acquisition of the image. It should be large enough to avoid objects moving while imaged.

Go to fluidic acquisition and start the acquisition.
First take two or three images to check if the focus is good. If it is not, try to do it again directly on the plankton.
Change the acquisition ID (the old one is used by the focus test).
Launch the real acquisition.
Wait for the acquisition to be done.
Results can be consulted by consulting the gallery or the file manager (data > export).

 
  
SECTION: Segment the acquisition
12. Segmentation

Go on segmentation and click on the "update acquisition's folder list".
Select the samples you wish to segment. Note that you can select either the parent folder or select the file itself. 
Setup the different options of the segmenter.

 

Recursive folder means that it will segment all samples within a selected sample
Ecotaxa archive: it will create a zip file containing all files needed for a easy importation within ecotaxa
Force rework: if yes it will re-segment samples already segmented
Keep objects: it will keep the final segmented images visible in the planktoscope (that could be accessed by the gallery in the objects folder)

 

scroll down and click on start segmentation
Wait for the segmenter status to turn to "done"

 

Export your data on your computer
  

SECTION: Clean the planktoscope
13. Cleaning

Drain the sample out of the syringe
Disconnect the syringe and clean it with tap water (or even distilled water)
Pump (at high speed!) the full content of the fluidic system to remove any liquid
Reconnect the syringe
Fill it with tap water (or distilled water)
Pump (at high speed!) while regularly pinch the tubing to detach any plankton in the system (see   )
Drain again the syringe (repeat steps 2-7 at least 2 more times until no plankton is visible on the camera)
Finally drain the system

If not used immediately

Put 20 mL diluted bleach
Leave 15’
Drain the content (high pump speed)
Put 10 mL fresh water
Drain the content (high pump speed)
If there are traces of calcification, use a diluted acid solution (like HCl diluted by 6).
SECTION: Upload your images on EcoTaxa
14. In this section you will learn to :

Do your first connexion on EcoTaxa
Create a new project 
Connect to EcoTaxa with filezilla
Import data of the PlanktoScope to EcoTaxa

First connexion

Create an account on EcoTaxa  (https://ecotaxa.obs-vlfr.fr/) by clicking on the top right "log in/register" then on "Create your EcoTaxa account".

 

Put your real name and a valid mail so that you can be contacted

 

SECTION: Upload your images on EcoTaxa
14.1. Projects

Once logged in, 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 on "contribute to a project" on the main page.

 

SECTION: Upload your images on EcoTaxa
14.2. Create a project

Go to "Contribute to a project > Create a new project". You can create your own project on which you will be able to import, visualise and classify images.

In the creation panel, you can (fig.3):
Add the title of your project
Describe your project
Comment your project
Define the instrument used (here, the PlanktoScope)
Choose if you want to annotate (define taxonomy) or only explore images, etc. and who can see your project
Define what pre-trained Deep Learning features to use on your project (it is recommended to use «Planktoscope_2022-09 » unless you see a more recently trained model on planktoscope image)
Choose a license for your images (it is recommended to use one of the CC-BY one or CC-0 if you want data to have a future use for science)
Define a list of taxa to help you classify your sample (in the "Taxonomy" field)
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 :
area=area
meanhue=meanhue
meansaturation=meansaturation
meanvalue=meanvalue
Invite new contributors/viewer/manager

If you want to edit the settings later, go to "edit project settings" (fig.4).

 



 

SECTION: Upload your images on EcoTaxa
14.3. Connect to EcoTaxa ftp

 

Upload the ecotaxa archives (see step 6-7) on the EcoTaxa ftp
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

Once this is 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/PLANKTOSCOPE).

 

SECTION: Upload your images on EcoTaxa
14.4. Import data in your project

In your project/ on your project options button, select import images and metadata (fig.6).
Locate your file on the ecotaxa ftp folders and import it (only works for one zip file at a time for now; fig.7). 
Check the quality of your images and the quality of the segmentation once the images are imported.

 


 



SECTION: How to use efficiently EcoTaxa
15. In this section, you will learn to:
Use filters
Validate taxonomy
Do a prediction
Export the results

 

SECTION: How to use efficiently EcoTaxa
15.1. Use filters wisely

There are three layers of filters in EcoTaxa: 

The taxonomic filter tab (allows to filter by taxonomic groups) and the other filter tabs.
Filters on sample parameters (date, location, etc).
The quick access filters (top bar). Quick filters are pretty useful since you can sort objects by specific values (eg. mean saturation in fig.3, to quickly observe objects that have lots of chlorophyll). You can revert the sorting order of those filters by ascending or descending order.

Filters are additive (fig.2), so you can add filters 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).
 


 


 
 
SECTION: How to use efficiently EcoTaxa
15.2. 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.

 
 
 
 
Validation could be tedious and requires large taxonomic expertise, however there are 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!

 

SECTION: How to use efficiently EcoTaxa
15.3. Prediction 

Do not hesitate to "predict" your project right away (even with a project/instrument that has nothing to do, even if it is recommended). 

 

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

 

You can select any of the pre-existing projects (including your own project) as a template for image recognition. By experience, using another project is only a first-aid, 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 exist (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 own project.

Projects that could be used for first prediction : 
#6818 - MOOSE-GE-2022_tests_ID_vignettes (Med sea; Processed with current segmenter; Fully validated) https://ecotaxa.obs-vlfr.fr/prj/6818
#9621 -Planktoscope APERO_PP_Phytonet_35mu https://ecotaxa.obs-vlfr.fr/prj/9621
#9942 Planktoscope APERO_TH_Phytonet_35m (PlanktoScope; Fully validated) https://ecotaxa.obs-vlfr.fr/prj/9942
#10 056 - AtlantECO_P2_Ada_2023 (PlanktoScope; 60% validated) https://ecotaxa.obs-vlfr.fr/prj/10056
#6765 - Planktoscope Reference (Processed with other segmenter,  works only with adding Deep Learning features into play); Fully validated) https://ecotaxa.obs-vlfr.fr/prj/6765

 
Push the  button "Next: Choose objects in selected projects". You then have the possibility to select what types and quantity of objects to consider. It is recommended to try to avoid selecting too much objects in one category to partly correct the usual strong imbalance between categories (In the fig.13, it is limited to 500 objects per group).

 
Click on "Next: Choose features in selected objects". Activate the pre-trained Deep Learning features (if not available see step 10). 
Inactivate variables that are not relevant for prediction and relate to position of the vignette in the initial images (bx, by, depth min/max, label, local centroid col/row, x, y)
Click on "Start prediction task"

 

Once done, images are now "predicted" (blue) but still wait for validation. Note that while classifying the different objects, the classifier also gives a classification "score" which determines 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).

 
 

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 optimized 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). 
SECTION: How to use efficiently EcoTaxa
15.4. Export your results from EcoTaxa

Once fully validated, export your results (lots of different solutions exist, the easiest to understand being the summary export with count per sample).

 


 

SECTION: How to compute biovolumes
16. Calibrate your data

Ecotaxa generate a lot of interesting variables to analyse your sample. Only a few of them are depicted here, but don't hesitate to explore the other ones. 
Before any analysis, it is important to relate all the parameters computed by Ecotaxa with the acquisition parameters. You will need :


 
It will allow you to compute the conversion factor "conver". The dilution is a factor that should be < 1 if a dilution has been operated and > 1 if the sample has been concentrated (it is not the sample concentrated volume, which is the original volume taken from the collector) :

  

 

Compute Biovolume

There is three methods to calculate the biovolume (BV) of an object.  

Ellipsoid:
  
  

Plain:
  
  
 

Riddled:
  
  
 

SECTION: Maintenance of your PlanktoScope
17. 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.

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 and restart acquisition while noting that the sample got diluted in the metadata).
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 create a sudden variation of pressure.
Over time, wet conditions and organic matter may create favorable condition for the growth of a bacterial film. The flowcell and tubing will look dirty from the inside. You can avoid this by pumping diluted bleach sometimes, let it act for 1-2 hours and carefully rinse the whole system.
Water, bacteria, and bleach together may favour the apparition of a calcium carbonate film inside the tubing and flowcell. It may either appear as dispersed crystals attached inside the flowcell or a white coating inside the tubing. To remove and clean this, pump some acidic solution (vinegar, citrus juice or other kind of other acids), let it rest for a few hours and rinse the system.

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:

if only dry dusts 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) 
if dirt in not only dry dusts it could be cleaned with optical paper and ethanol. DO NOT USE CLASSICAL WIPING PAPER which are usually enriched in silica fibers for solidity ... and may create scratches on the flowcell. (disposable nose tissues are a better alternative if optical paper is not available)

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 require a lot of patience, efforts and may even lead to unexpected disappointments).

dry dust: dry gas (with even more caution than previously)
others: only used optical paper

Clean the camera sensor
Critical part ! NEVER touch it, only use dry gas.

Regularly calibrate the pump and the WB.
SECTION: Update the software or reset the PlanktoScope
18. If you need to update the software, or if there are a lot of bugs and you want to reset it, follow this procedure.

Withdraw the micro SD card at the bottom of the PlanktoScope with a fine forceps (see fig.1). The fine forceps is not provided.
Connect the SD card to your computer with a SD card adapter if needed (you can find one in the supplementary materials box).

 
Open BalenaEtcher > Flash from file 
Choose the desired version of the software (.img file). You can find the actual one on the github of the PlanktoScope in the "external links" section of the protocol. 
Choose the SD card you want to flash.
Click on "flash" and wait until it is flashed.
Eject it from your computer and put it back in the PlanktoScope with a fine forceps.

 

SECTION: Troubleshooting
19. The flow cell is clogged with plankton

Why this happens: 
First this may happen if your sample does not have been pre-filtered (it is recommended to do a pre-filtration to 200µm). 
It may aslo happen if your sample is too concentrated. If you got more than 20 plankton objects per image this may already be the case, dilute your sample and fill the dilution factor in the sample metadata.
If you forget to agitate your sample using a bubble or if you let your sample to stagnate for too long in the fluidic system.

Unclogging the flowcell:
Try to pinch the tube in between the flowcell and the pump (while the pump is running). 
Try to do the same while pumping in the reverse direction (eventually at high speed- see pump controls in optical configuration page).
Dismount the flowcell but keeping the luer lock connectors on it. on the side which was connected with the pump, either blow in it or connect a syringe and pass air/water to chase the blocked plankton.


SECTION: Troubleshooting
19.1. The image is partly blurred

Why this happens:
The focus is correctly done, but the flowcell is not well positioned.

How to correct it:
Try to adjust the position of the flowcell with tape. 


 


SECTION: Troubleshooting
19.2. The software is not working

Why this happens:
The python code encountered a bug. 
There is a segmentation error because the number of objects is too important.
The optical configuration tab does not work (black screen, impossible to change the WB, etc...).

Solutions:
Try to restart the PlanktoScope
Try to change values of the WB, check if you use commas or dots and restart the PlanktoScope
Try to stand by a number of final objects around 2000~3000 per sample.
Ask questions on the PlanktoScope Slack (see External links section)
If you do not find any solution, flash the SD card of the PlanktoScope with the software by using BalenaEtcher 

SECTION: Troubleshooting
19.3. The pump is not working

If there is any problem with the pump, check that it is properly positioned. If it's not the problem, remove it and check that the pump tube is correctly positioned.

If you need to change the internal pump tube, you can take the pump off by turning it counter-clockwise. 
Position the tube correctly.
To install it again, place it in the position shown in the image below until it clips, push it in and turn it clockwise until it snaps into the white socket. 
Be careful to clean the grease afterward.


 

 

SECTION: External links
20. Planktoscope website
https://www.planktoscope.org/

Planktoscope github
https://github.com/PlanktonPlanet/PlanktoScope

Planktoscope complete assembly guide and complete documentations
https://planktoscope.curious.bio/ (v2.5)

Planktoscope Slack channel (to exchange ideas/protocols/solutions/questions)
https://forms.gle/qvh5jwuMvmyBKMQC7

Plankton Planet website
https://planktonplanet.org/

EcoTaxa tutorials:
https://sites.google.com/view/piqv/piqv-manuals/ecotaxaecopart-manuals?authuser=0
https://www.youtube.com/watch?v=PSO6ZS765tk
https://www.youtube.com/watch?v=RaWUqIoKk0E