planktoscope/docs/protocols/basic_primer.md

Plankton imaging with the PlanktoScope: A setup and sampling guide Forked from Planktoscope protocol for plankton imaging

Lombard Fabien, Will Major 1 Works for me Will Major

ABSTRACT

This is a instructional manual for setting up the PlanktoScope (V2.5) for regular data collection and processing, as well as a step-by-step guide for running samples. ATTACHMENTS Pollina et al 2020 2020.04.23.056978v1.full. pdf

EXTERNAL LINK

https://www.planktoscope.org/ PROTOCOL INFO

Lombard Fabien, Will Major . Plankton imaging with the PlanktoScope: A setup and sampling guide. protocols.io https://protocols.io/view/plankton-imaging-with-the-planktoscope-a-setup-andb5znq75e MANUSCRIPT CITATION please remember to cite the following publication along with this protocol

Pollina T, Larson A, Lombard F, Li H, Colin S, Vargas C de, Prakash M (2020) PlanktonScope: Affordable modular imaging platform for citizen oceanography. bioRxiv 2020.04.23.056978. doi: 10.1101/2020.04.23.056978 FORK FROM

Forked from Planktoscope protocol for plankton imaging, Lombard Fabien KEYWORDS

plankton, PlanktoScope, imaging, microscope IMAGE ATTRIBUTION

Fabien Lombard, Thibaut Pollina, Will Major CREATED

Mar 07, 2022 LAST MODIFIED

Jul 14, 2022 1

PROTOCOL INTEGER ID

59150 GUIDELINES

PlanktoScope is an optical instrument. Its optical elements (camera, lenses, flowcell) are highly sensitive 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). MATERIALS TEXT

PlanktoScope Plankton Net 200 µm sieve Optical paper Pine Pollen in Water and Pine Pollen dry SAFETY WARNINGS

Planktoscope is an electronic device, powered with electricity. It is therefore sensitive 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 handled with caution (to avoid injury) and kept clean (avoid touching it with fingers). BEFORE STARTING

Make sure to read through an entire subsection before taking any action. Initial connection and setup

1

Powering, connecting to and configuring the PlanktoScope. This section will introduce you to using your PlanktoScope:

• subsections 1.1 and 1.2 cover turning on and connection to the machine;
• subsections 1.3 - 1.7 will cover running the necessary tests to make sure your PlanktoScope is working as we expect. Please ensure you carefully read through each subsection before you take any action.

1.1

Power your Planktoscope by connecting 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. After a few minutes, you should see a new option for Wi-fi appearing on your 2

computer. Connect to it using the password: "copepode". For more information and alternative methods of connection, see the designer's Connectivity Tutorial here:

1.2

PlanktoScope - Connectivity Tutorial.pdf

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): http://192.168.4.1:1880/ui/ There are several tabs on the UI 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.

The 'Home' tab of PlanktoScope's User Interface.

The 'Hamburger Menu' icon, situated in the top-left corner of the screen, can be used to navigate around the User Interface

1.3

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

To test the PlanktoScope, navigate to the Optic Configuration tab:

3

The Optic Configuration tab which can be used to adjust the camera settings. If only Preview is visible on your screen, the other options should be available below by scrolling down or by adjusting 'Zoom' on your browser (usually Ctrl + scroll UP or DOWN on Windows or command + scroll UP or DOWN on Mac).

a) Under Optic Characterisation, switch on the Light by clicking 'On'. You should see the Preview image turning from dark to light. The Preview image could be any colour so do not worry if yours does not show blue; it will be adjusted later.

The red box highlights the location for turning on the LED. You will need to do this every time you use your PlanktoScope.

b) Under Focus Adjustment, click 'UP 1MM' and 'DOWN 1MM' to ensure focus buttons turn the focus motor. You should see the mount moving further from (UP) or closer to (DOWN) the camera.

4

Red boxes highlight 'UP 1MM' and 'DOWN 1MM' that will move the Mount (pictured below).

Green arrows highlight the movement of the Mount when using Focus Adjustment buttons on the Optic Configuration tab. 'UP 1MM' will move the Mount towards the right-hand side of the picture, while 'DOWN 1MM' will move the Mount to the left-hand side of the picture.

c) Under Fluidic Manual Manipulation, click clockwise arrow to check that the Peristaltic Pump is working. You should see the pump rotating in an clockwise direction.

5

The red square highlights the location of the clockwise arrow that will rotate your Peristaltic Pump in the same direction.

The clockwise arrow from the Fluidic Manual Manipulation section should rotate your PlanktoScope's Peristaltic Pump in a clockwise direction.

d) Under Camera Settings, change the ISO value. You should see changes to the Preview image. After this test, Set ISO to 100 . 6

The red box highlights the location of the ISO setting. You should see your Preview image change colour when you adjust this setting. Make sure it is set to 100 once you have tested this.

1.4

Now we will align the lenses in your PlanktoScope . To do this: a) Remove the Fluidic Path from the mount and gently lay to the side.

Remove the Fluidic Path and lay to the side

7

Remove the Fluidic Path and lay to the side

b) Make sure we have enough space to remove the lenses by clicking the 'UP 1MM' button under Focus Adjustment.

Red box highlights 'UP 1MM' button that will move the Mount away from the lenses.

8

Move the Mount away from the lenses using the 'UP 1MM' button to allow for outer lens removal.

c) Remove the outermost lens (16MM).

9

Remove the outermost (16MM) lens.

d) On the Preview image on the Optic Configuration tab, you should see a light spot surrounded by darkness. By moving the 25MM lens on your PlanktoScope, you can move the light spot on the Preview image. Try to get the light spot as close to the centre of the Preview image as possible.

With the 16MM lens removed, your Preview image should resemble this picture. If the light spot is not centred, gently reposition the 25MM lens until it is as close to centre as you can get it.

10

Gently reposition the inner (25MM) lens.

e) Once centred, place the outermost lens back to where you removed it from in step 3.

Place the outer (16MM) lens back into position.

11

f) On the Preview, you may see darker areas in the corners. To get rid of the darker areas in the corner, reposition the outermost lens (16MM) while holding the innermost lens (25MM) steady; the darker corners should disappear. Try to achieve homogenous light across the Preview image. g) Place the Fluidic Path back into position.

1.5

Manually adjust the white balance of your PlanktoScope . 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 colour. We need to achieve the Preview image colour that the Auto White Balance feature provides, without using the Auto White Balance. Not using Auto White Balance enhances the performance of the PlanktoScope over time. To manually set to the White Balance, turn off the Auto White Balance and adjust WB: Red and WB: Blue until it looks white. Then switch AWB back on to see if it matches. Repeat this process until there is no colour change when clicking the AWB button. Set the AWB button to 'off' once you have completed this step.

12

The red box highlights how to manually adjust the white balance of the Preview image. In this example, the correct setting was WB: Red = 4 and WB: Blue = 1.21. The AWB button should be set to 'off' once you have completed this step.

1.6

Setup the 'Bubbler' :

Locate the Bubbler.

13

Tie a knot in the Bubbler.

Plug the Bubbler into one of the USB ports on the PlanktoScope.

14

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.

15

1.7

Load in a practice sample and manually adjust the focus of the camera . a) Locate the Pine Pollen in Water sample and a Pasteur Pipette.

b) Shake the Pine Pollen in Water sample, remove the lid, and use the Pasteur Pipette to transfer roughly 10 mL into the Syringe. Take this opportunity to check that your Bubbler is working appropriately: you should see no more than 4-5 bubbles per second. Tighten the knot in the tubing to reduce the airflow if you need to.

16

c) From the Optic Configuration tab, run the Peristaltic Pump so that the Flow System is filled with the sample. Double-check that the tubing from your Peristaltic Pump runs into the Waste (Test Tube with dark blue lid).

d) On the Optic Configuration tab under Focus Adjustment, use the 'UP 1MM' and 'DOWN 1MM' to adjust course focus , and 'UP 100UM' and 'DOWN 100UM' to 17

adjust fine focus . You will likely need to use the 'DOWN 1MM' to move the Mount close to the lenses before using the fine focus buttons. You should see small dark particles on the Preview image; these particles need to be in perfect focus, adjust until correct.

Pine pollen particles in perfect focus in the Preview image.

You may see the edge of the flow system in your Preview image (see the red box). To fix this, simply reposition the outer lens (16MM) as we did in step 1 .5 f .

NB: if you do not see any particles, try adding a very small amount more of Pine Pollen to your Syringe by dipping the handle of a teaspoon into your dry Pine Pollen sample, and then into your Syringe sample. Then run the Peristaltic Pump once more to pass the sample with a higher concentration of Pine Pollen in front of the camera. Wash the spoon thoroughly after use.

18

The handle of a teaspoon dipped into dry Pine Pollen sample. Less is mo re!

1.8

To finish the setup , run the Pump from the Optic Configuration tab until all of the Pine Pollen sample has passed into the Waste. Fill the Syringe with tap water and run it through into the Waste to clean the PlanktoScope (you may need to empty the Waste into the sink/toilet before you do this). Shut down the PlanktoScope by navigating to 'Home', click the 'Unlock Button' and then click 'Shutdown'. Leave for one minute before turning off your PlanktoScope at the wall plug.

Get your sample

2

Using a net to collect a sample

2.1

Remove the net from its bag and set aside the 200 µm sieve. 19

Within the net bag you will find the net (left of photo) and 200 µm sieve (right of photo).

• Position yourself close to but a safe distance from the water's edge and ensure a stable footing.
• Unspool the rope.
• Hold the lead weight on the rim of the net along with the cod end in your throwing hand while holding the plastic handle tightly with your other hand, throw the net into the water.
• Gently pull the net back towards you just before it hits the water to orientate it. Pull the net back towards to at a speed so that it is submerged but does not sink. For areas of fast flow, you may only have to perform one cast. With areas of sluggish or no flow, two or three casts may be necessary.

2.2

To get the sample from the net:

• remove the cod end and spray its mesh sides with water from the Pasteur Pipette.

20

Remove the cod end from the net by twisting in an anti-clockwise direction. Ensure to keep the cod end upright.

Spray mesh sides of the cod end with water from the Pasteur Pipette to ensure all particles move down into your sample.

• Once you are satisfied that any particles are no longer stuck to the mesh sides, 21

you are ready to transfer your sample into a Falcon Tube. While holding the 200µm sieve above a Falcon tube, slowly pour your sample so that it passes through the sieve and into the tube. Label your Sample and keep a record of its date, time and location.

Position the 200 µm sieve above the Falcon Tube

Pour your sample from the cod end, through the sieve and into your Falcon Tube.

• Make sure you label your sample and keep a log of when and where you have collected it. 22

- Ensure to rinse your net and sieve in fresh water before the next use. Running a sample

3

This section covers your regular use of the PlanktoScope, and the setup and procedure you will undertake each time you want to run a sample.

3.1

Navigate to the Sample tab to set up running a sample through the PlanktoScope.

The Sample tab on the PlanktoScope's UI

a) Begin inputting Sample Identification information.

Fill the entries shown by the red box: Project name = MAPPS Name of the operator = Your Name Station ID = Your sample location e.g River Thames or Southampton Water.

b) Input the 'Net Throw Location' information including Latitude, Longitude, Date and Time before clicking the 'VALIDATE' button. The format for this section is strict and can be a bit of a fiddle; if you have not input the information perfectly 23

when you click 'VALIDATE', you will have to input the information from scratch. The next few images will show you how to input these pieces of information.

b) i. Latitude and Longitude Use your web browser and navigate to https://www.google.com/maps

ii. Click on your sample collection location on Google Maps and you should see a grey symbol that matches that in the red circle. If you do not see this symbol, try to adjust the position of your click slightly. iii. You will see coordinates in the red square (above), click on these numbers.

24

iv. The relevant information is the numbers in the red box (above) that are separated by a comma. Copy (Ctrl + C for Windows or command + C for Mac) the first number and paste (Ctrl + V for Windows or command + V for Mac) it into the Latitude entry on the Sample tab. The second number may have a minus sign before it. Do not include this. Copy the second number (without the minus sign) and paste it into the Longitude entry on the Sample tab. You then need to include °N in the Latitude box, and °E or °W depending on whether there was a minus sign before the number (minus = °W; no minus = °E). The easiest way to enter the degree symbol will be to copy and paste it from this document. Alternatively, on Windows you can press Alt + 0176 and on Mac you can press Shift + Option + 8.

v. Then enter the Date (and time) of your data collection (as in, the date and time you had your net in the water) in the format listed e.g. 2022-03-11 12:00 vi. Then enter Time e.g. 12:00. Please ensure that the time in the Date section and Time sections match. vii. Click 'VALIDATE'

3.2

Run a sample by navigating to the Fluidic Acquisition tab. In the red box pictured below, update the following parameters: a) Acquisition Unique ID should be a three-digit number starting with 001 and 25

increasing with each sample you run. b) Number of images to acquire should be 100 c) Pumped Volume (mL) should be around 0.1 d) Delay to stabilise image should be 0.5 e) Flowcell should be 300 μm Capillary.

Once you have updated the parameters correctly, click 'UPDATE CONFIG'. Then, click 'START ACQUISITION' and the Pump and imager should start.

3.3

Once you have run your sample and collected the images, you will need to segment them (separate each image into images of individual particles). Navigate to the Segmentation tab.

1. Click on the "update acquisition's folder list"
2. Select the samples you wish to segment
3. Setup the different options of the segmenter (see below for idealised setup). 26

- 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)

The idealised Control setup for segmenting images within the PlanktoScope User Interface

4. Scroll down and clic on start segmentation

5. Wait for the segmenter status to turn to "Done"

6. Finally, download your segmented images from the PlanktoScope using FileZilla (described in section 4) Download your Plankton images

4 You will need a computer connected to the planktoscope together with free software FileZilla (https://filezilla-project.org/) Open FileZilla Either click on the top right to create a new connection or use the quick-connection fields below Enter the following informations: Host: sftp://planktoscope.local (192.168.4.1 should also works) 27

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 substraction, masks of the different objects etc) are in /home/pi/data/clean Final vignettes are in /home/pi/data/objects Once you have downloaded the images, you can clean and shutdown your PlanktoScope (see the next section), and reconnect to the internet. Clean the planktoscope

5

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!) 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!) while regularly pinch the tubing to detach any plankton in the system (see

)

7. Drain again the syringe (repeat steps 7.2-7.7 at least 2 more times until no plankton is visible on the camera)
8. Finally drain the system Shut down the PlanktoScope by navigating to 'Home', click the 'Unlock Button' and then clicking 'Shutdown'. Leave for one minute before turning off your PlanktoScope at the wall plug. 28

Upload your images on EcoTaxa

6 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 so that you can be contacted

29

6.1

Once logged you can consult the project on which you are registered by clicking onto "contribute to a project " on the main page. Our project is called #4970 PlanktoScope - MAPPS - 2021 You must first notify a project manager who will assign you permissions

6.2

upload the ecotaxa archives (see step 4) on the EcoTaxa ftp using Filezilla (see

) create a connection by using the following information:

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 30

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/PLANKTONSCOPE)

Please eventually create your own folder to "try" to keep it clean and tidy 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)

6.3

In the project on your 'Project' options button, select import images and metadata

6.4

locate your file on the ecotaxa ftp folders and import it (only works for one zip file at a time for now)

31

Maintenance of your planktoscope

7

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 acquisition while noting that the sample got diluted in the metadata

and restart )

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 create a sudden variation of pressure (e.g.

)

3. 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
4. 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 cristals 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:
5. 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)
6. 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 tissue are 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 requires a 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 ... critical part if any, NEVER touch it, only use dry gas regularly calibrate the pump External links

8

Planktoscope website https://www.planktoscope.org/ 32

Planktoscope github https://github.com/PlanktonPlanet/PlanktoScope Planktoscope complete assembly guide and complete documentations https://planktonscope.readthedocs.io/en/latest/ Planktoscope Slack channel (to exchange ideas/protocols/solutions) https://forms.gle/qvh5jwuMvmyBKMQC7 Plankton Planet website https://planktonplanet.org/ EcoTaxa tutorials: https://sites.google.com/view/piqv/ecotaxa?authuser=0 https://www.youtube.com/watch?v=PSO6ZS765tk https://www.youtube.com/watch?v=RaWUqIoKk0E

33