planktoscope/docs/protocol.md

### FEB 14, 2023

# Planktoscope protocol for plankton imaging V.

## Lombard Fabien ,Will Major,Anna Oddone ,

## Clémence Clausse

```
Sorbonne Université, Centre National de la Recherche Scientifique,
Laboratoire d’Océanographie de Villefranche (LOV), Villefranche-sur-Mer,
France;
Institut Universitaire de France, 75231 Paris, France;
National Oceanography Centre, European Way, Southampton SO14 3ZH;
Plankton Planet
```

```
LOVComplex
```

Lombard Fabien
**DOI:**
dx.doi.org/10.17504/protocol
s.io.bp2l6bq3zgqe/v

**External link:**
https://www.planktoscope.org
/

**Protocol Citation:** Lombard
Fabien, Will Major, Anna
Oddone, Clémence Clausse 2023. Planktoscope protocol
for plankton imaging.
**protocols.io**
https://dx.doi.org/10.17504/p
rotocols.io.bp2l6bq3zgqe/v2V
ersion created by Lombard
Fabien

```
1,2 3 4
4
```

```
1
```

```
2
3
4
```

### DISCLAIMER

```
this protocols applies to the version 2.5 of the planktoscope and the 2.
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
```

### ABSTRACT

```
this protocol is for using planktoscope and collect usable result for quantitative
imaging of plankton
see also https://www.planktoscope.org/
```

```
IMAGE ATTRIBUTION
Fabien Lombard, Thibaut Pollina, Karine Leblanc, Will Major
```

```
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/
```

## VERSION 2

```
protocols.io |
```

**MANUSCRIPT CITATION:**
Pollina T, Larson AG, Lombard
F, Li H, Le Guen D, Colin S, de
Vargas C, Prakash M (2022)
PlanktoScope: Affordable
Modular Quantitative Imaging
Platform for Citizen
Oceanography. Frontiers in
Marine Science 9. doi:
10.3389/fmars.2022.

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.

Mériguet Z, Oddone A, Le
Guen D, Pollina T, Bazile R,
Moulin C, Troublé R, Prakash
M, de Vargas C, Lombard F
(2022) Basin-Scale Underway
Quantitative Survey of Surface
Microplankton Using Affordable
Collection and Imaging Tools
Deployed From Tara. Frontiers
in Marine Science 9. doi:
10.3389/fmars.2022.

de Vargas C, Le Bescot N,
Pollina T, Henry N, Romac S,
Colin S, Haëntjens N,
Carmichael M, Berger C, Le
Guen D, Decelle J, Mahé F,
Poulain J, Malpot E, Beaumont
C, Hardy M, Guiffant D,
Probert I, Gruber DF, Allen AE,
Gorsky G, Follows MJ, Pochon
X, Troublé R, Cael BB,
Lombard F, Boss E, Prakash
M, the Plankton Planet core
team, Bazile R, Boss E,
Bourdin G, Cael B, Casati R,
Colin S, Vargas C de, Gorsky
G, Guiffant D, Haentjens N,
Henry N, Larson A, Bescot NL,
Lombard F, Mirambeau G,
Moulin C, Oddone A, Prakash
M, Prazuck C, Raimbault V,
Trellu C, Troublé R (2022)
Plankton Planet: A frugal,
cooperative measure of
aquatic life at the planetary
scale. Frontiers in Marine
Science 9

### MATERIALS

```
Plankton net
200μm sieve
Squizing bottle
micrometer slide (or millimetric ruller)
Optical paper
Dry gas dispenser
```

```
SAFETY WARNINGS
```

```
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)

### BEFORE START INSTRUCTIONS

```
-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
```

**License:** This is an open
access protocol distributed
under the terms of
the Creative Commons
Attribution License, which
permits unrestricted use,
distribution, and reproduction
in any medium, provided the
original author and source are
credited

**Protocol status:** Working
We use this protocol and it's
working

**Created:** Oct 06, 2022

**Last Modified:** Feb 14, 2023

**PROTOCOL integer ID:**
70911

**Keywords:** Planktoscope,
plankton, microscopy,
quantitative imaging,
microplankton

# 1 PreparationPreparation

```
1. Plug in the Planktoscope
2. Connect to Planktoscope’s Wi-Fi go to step #3.
3. Type Planktoscope URL http://192.168.4.1:1880/ui/
4. Check WB (in OPTIC CONFIGURATION) go to step #3.
5. Put 20 mL sample, add the air pump go to step #7.
6. Check Focus (in OPTIC CONFIGURATION) go to step #7.
```

# 1.1 AcquisitionAcquisition

```
1. Fill sample details (in SAMPLE) go to step #7.
2. Fill acquisition parameters (in FLUIDIC ACQUISITION) go to step #7.
3. pump to drain sedimented organisms go to step #7.
4. START! go to step #7.
```

# Quick usage version

```
protocols.io |
```

# 1.2 Cleaning Cleaning go to step

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

```
Plus, if not used immediately:
1. Put 20 mL diluted bleach
2. Leave 15’
3. Drain the content (high pump speed)
```

```
1. Put 10 mL fresh water
2. Drain the content (high pump speed)
```

# 1.3 AnalysisAnalysis

```
1. Fill segmentation parameters (in SEGMENTATION) go to step #
2. Start segmentation
3. Back-up data go to step #
4. Import on ecotaxa go to step #
```

# 1.4 Shut downShut down

```
1. Turn OFF (HOME)
2. Unplug the Planktoscope
```

# 2

```
The Planktoscope is a frugal, microfluidic microscope designed with an open-hardware, open-
software approach. It was conceived within the idea of equipping the thousands of 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.
```

```
Material:Material:
```

# Planktoscope, overview.

protocols.io |

```
The device and its different parts are shown in the following figure.
```

```
The PlanktoScope kit also includes a bubbler, power cable, waste container, falcon tube of tap
water, syringe (containing the sample), sample holder, flowcell holder and flowcell.
```

```
The User InterfaceThe User Interface
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
```

protocols.io |

```
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.
```

protocols.io |

```
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.
```

protocols.io |

```
You will only need the "Hardware settings" page to replace the comma with a dot in the "pixel
size calibration" box. Do not change anything else.
```

# 3 Open the planktoscope box and check all the part first

# 3.1 if not installed, place the pump tube in place (the pump could be turned to be open). caution,

```
clean the grease afterward
```

# 3.2 assemble the fluidic system

# Initial connection and setup

protocols.io |

# 3.3 assemble the flow cell with care and step by step, note that a short length of the tube may

```
need to be cut to get the right distance from the syringe to the flowcell
```

# 3.

```
Safety information
```

```
to protect the flow cell during assemblage, first be gentle with it, and second place a
spacer between magnets to avoid accidental breakage (here 3 layers of duck tape)
```

protocols.io |

# 3.5 finally assemble all the system

# 3.6 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 computer.
Connect to it using the password: "copepode".
```

```
For more information and alternative methods of connection, see the designer's Connectivity
Tutorial here: PlanktoScope - Connectivity Tutorial (1).pdf
```

# 3.7 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
```

protocols.io |

```
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
```

# 3.8 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:
```

protocols.io |

```
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 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).
```

protocols.io |

```
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.
```

```
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.
```

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

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

protocols.io |

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

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

```
The red box highlights the location of the ISO setting. You should see your Preview image
change colour when you adjust this setting.
```

protocols.io |

```
Safety information
```

```
Make sure it is set to 100 once you have tested this.
```

# 3.

```
Safety information
```

```
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
```

```
Mis-aligned lenses will create an inhomogeneous illumination and will create lots of
artefacts if not corrected. We advise to check this every times you set back your
planktoscope to use (closing the box may move the lenses)
```

protocols.io |

```
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.
```

protocols.io |

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

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

protocols.io |

```
c) Remove the outermost lens (16MM).
```

protocols.io |

```
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.
```

protocols.io |

```
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.
```

```
Gently reposition the inner (25MM) lens.
```

protocols.io |

```
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.
```

```
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.
```

protocols.io |

# 3.10

```
Safety information
```

```
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 (the camera will try to adjust it in between every
images...)
```

```
Planktoscope are normally cross-calibrated for white balance initially, this information
could be recovered from the provider. We strongly encourage you to note the initial values
before trying to change those and this procedure should not be done without reasons
(incorrect image with initial calibration; reboot or update of the software.
Note your calibration here:
WB RED:
WB blue:
```

protocols.io |

```
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.
```

```
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.
```

# 3.11

```
Safety information
```

```
Setup the 'Bubbler': the flow of air needs to be adjusted to 1 bubble/sec approximately,
bubbling the bottom of the syringe. We encourage adapting a rigid tube at the end to ensure
the flexible part does not get aspired by the water flow
```

```
Planktoscope image fluid at low speed.
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.
```

protocols.io |

```
Plug the Bubbler into one of the USB ports on the PlanktoScope. Place the tubing into the
```

```
the bubbler
```

```
Tie knot to adjust air flow (basic way,
not recommended)
```

```
examples of other possibilities to regulate air
(choose preferred one) and adaptation at the
end of tubing
```

protocols.io |

```
Syringe so that it reaches the bottom. Affix the tubing to the Syringe using an elastic band,
string or similar.
```

# 4 Pump calibration:Pump calibration:

```
Safety information
```

```
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 it is the number of images (therefore the volume imaged)
which is important (not the pumped volume)
```

# 4.1 -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)
```

# Calibration

protocols.io |

# 4.2 -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)
```

# 5 Size calibration:Size calibration:

```
Safety information
```

```
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 startmanually replace the comma by a point in the hardware setting at every start
```

# 5.1 - 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 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

protocols.io |

```
the scale
```

```
tilted position for calibration
```

```
focusing on the scale
```

# 5.2 -take few images (select the test or culture mode in sample tab), goes on acquisition and put 1

```
or 2 images
```

protocols.io |

```
example of metadata entered in "sample" page
```

```
here two images are acquired (and random information entered on the pumping one)
```

# 5.3 -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 planktoscope and downloading the rawee section 7 for communicating with your planktoscope and downloading the raw
imagesimages)
```

protocols.io |

```
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)
```

# 5.4 -calculate how much microns are represented by each pixels (should not be strongly different

```
from 1.01 which is the default value for 25/16mm lenses combo)
```

# 5.5 Enter the calibrated pixel size value in the hardware setting

protocols.io |

```
Safety information
```

```
Known bug: note here that the comma should be replaced by point at everynote here that the comma should be replaced by point at every
restart of the systemrestart of the system
```

# 6 Use a net to collect plankton

# Get your sample

protocols.io |

```
Safety information
```

```
Using logsheets:
-Record Latitude Longitude Latitude Longitude (taking photos of the GPS when launching and recovering the
net could serve, if UTC time is on the GPS this could also be interesting)
-if vertical netvertical net, record min and max depthmin and max depth
-if horizontal records initial/final positionsinitial/final positions, speed and length (min) of deploymentspeed and length (min) of deployment
-if you have flowmeterflowmeter, record the initial/final digits of the flowmeter initial/final digits of the flowmeter and calculate the
filtered volumefiltered 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)
```

# 6.1

```
Get the content of the collector.
Pass the volume through a 200μm sieve
```

```
Safety information
```

```
larger organisms may clog the flowcell
```

protocols.io |

```
filtration through a 200μm mesh
```

```
detail of the (home made) filter
```

protocols.io |

# 6.2 Rinse the sieve using seawater and a squeezing bottle (helps to pass small objects)

# 6.3 Recover the fluid / measure its volume/ record it on logsheets (will be entered latter as

```
"concentrated sample volumeconcentrated sample volume")
```

```
Safety information
```

```
Those are critical informations to get to quantitative samplingThose are critical informations to get to quantitative sampling
```

```
concentrated final volume of plankton
```

# Pass the sample on planktoscope

protocols.io |

# 7 assemble and start the planktoscope (see go to step #3 )

# 7.1 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)

```
Safety information
```

```
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 for this
```

```
severely unaligned lens final results and final artefact object created
```

# 7.2 Fill the sample on the sample holder. 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).
Replace the full sample holder and reconnect it to the pump, open the stopper, place the
bubbler and adjusts it flow.
```

protocols.io |

# 7.3 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
by gently inserting 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#1tip#1: start using the "1mm" buttons,
then the 100μm buttons and finish by typing 25 or 50μm adjustments in the middle box
(and pressing external arrows of focus; tip#2tip#2: you can connect your phone or a tablet to
the planktoscope to have controls on the focus while checking a zoomed portion on the
streamed image on another device)
```

```
Safety information
```

```
not agitating your sample will let plankton 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. you should agitate your sample using bubbling and use pumping rate
enough to avoid sinking/clogging of sample
```

# 7.4 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 in metadata in 7.5Note dilution in metadata in 7.5
```

protocols.io |

```
Safety information
```

```
Having too much object per frame will :
1. increase the probability that objects are touching (making them impossible to count or
identify)
2. increased the probability of clogging the fluidic system
3. create artefacts during the segmentation step
```

# 7.5 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)

```
-note how you sampled the plankton how you sampled the plankton (recording mesh size with "minimal fraction sizeminimal fraction size"
(will be used afterwards in the segmentation process, objects smaller than this won't be
segmented; "Maximal fraction sizeMaximal fraction size" is the size of the mesh used in step
go to step #6.1 ; 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 maxmin and max
depth depth if using a vertical net; initial/final positionsinitial/final positions, speed and length (min) ofspeed and length (min) of
deployment deployment if using a horizontal towed net
```

```
known bug:known bug: if filtered volume is provided but also initial/final size, calculation from this latter
may replace the measured filtered volume
```

```
in all cases the diameter of the net opening diameter of the net opening will be needed to calculate the filtered volume
```

- 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);

protocols.io |

```
-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”Concentrated sample volume” is the volume of sample recovered after all the steps of
concentration or dilution.
```

- If dilution have been done, note the “dilution factor” (if not, write “1”).

```
Dilution factor if a dilution has been done in go to step #7.4
```

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

protocols.io |

# 7.6 Go to fluidic acquisition and set parameters

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

```
Safety information
```

```
pump significantly between two images will help to :
```

```
1. avoid plankton sedimentation in the fluidic system
2. avoid imaging two times the same plankton
```

```
Target a sample size (by setting the number of images to acquire) that finallyTarget a sample size (by setting the number of images to acquire) that finally
have something like 1000-2000 final objectshave something like 1000-2000 final objects (e.g. if you have 10 objects per image,
imaging 100-200 frames would be enough)
Getting lower numbers
```

protocols.io |

- Volume to pumpVolume to pump is the volume pumped in between two images: it should be large enough to
  : avoid taking two time the same object in photo; avoid large sedimentation in the fluidic
  system; avoid objects to stick on the flowcell. We recommand to test 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 imageDelay 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 object moving while imaged.

```
Safety information
```

```
The Planktoscope is operation using a "rolling shutter camera" which means that there is
a small delay in between the first line of pixel imaged and the last line of pixel imaged. To
overcome this, it use a "stop and go" strategy where the imaging only takes place when
the flow of the pump is stopped. Not setting this properly will generate artefacts,
swimming organisms will also suffer from this (example bellow)
```

```
diatom imaged when moving
```

protocols.io |

```
copepode nauplius moving while imaged
```

# 7.7 go to optic configuration and pump with high flow rate a good amount of water (goal: remove

```
plankton that have sunk in the fluidic system)
```

# 7.8 Go to fluidic acquisition and start the acquisition.

```
Wait for the acquisition to be done
Results can be consulted by consulting the gallery
```

```
Safety information
```

```
If your fluidic system is not optimised to avoid plankton sedimentation, some plankton
could accumulate in the fluidic system. This can be checked by pinching 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 this means that
plankton have sedimented between the syringe and the flowcell.
```

protocols.io |

# 8 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 keep the final segmented images visible in the planktoscope (that could be
accessed by the gallery in the objects folder)
```

# Segment the acquisition

protocols.io |

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

# 9 You will need a computer connected to the planktoscope together with free software FileZilla

```
(https://filezilla-project.org/)
```

```
Open FileZilla
Either clic on the top right to create a new connection or use the quick-connection fields below
```

```
Enter the following informations:
Host: sftp://192.168.4.1 (note images were taken with a previous version, the adress does not
correspond to images)
Username: pi
Password: copepode
Port: 22
```

# Download the results

protocols.io |

```
clic 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 (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
```

# 10 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 detach any plankton in the system
(see go to step #7.8 )
7. Drain again the syringe (repeat steps 2-7 at least 2 more timesrepeat steps 2-7 at least 2 more times until no plankton is visible
on the camera)
8. Finally drain the system
```

# Clean the planktoscope

# Upload your images on EcoTaxa

protocols.io |

# 11 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 so that you can be contacted
```

protocols.io |

# 11.1 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 acontribute to a
projectproject" on the main page
```

# 11.2 Needs to be done only once: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 human, please be patient few days) soon
and you will see the following right appearing
```

protocols.io |

```
without the right to create projects
```

```
with the right to create projects
```

# 11.3 You can now create your own project on which you will be able to import, visualise and classify

```
images
```

# 11.4 upload the ecotaxa archives (see step 6-7) on the EcoTaxa ftp

```
using Filezilla (see ) create a connection by using the following informations:
Select File > Site Manager...
```

protocols.io |

```
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/PLANKTONSCOPE)
```

```
Safety information
```

```
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)
```

# 11.5 In your project/ on your project options button, select import images and metadata

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

protocols.io |

```
now)
```

# 12 Configure your project efficiently: in Project/project settingsConfigure 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 10.1)
area=area
meanhue=meanhue
meansaturation=meansaturation
meanvalue=meanvalue
```

# How to use efficiently ecotaxa

protocols.io |

```
Define a person of contact (mandatory)
Define what pre-trained Deep Learning features to use on your project (we recommend to use
«Planktoscope_2022-09 » unless you see a more recently trained model on planktoscope
```

```
image)
```

```
And invite people to manage the project with you (will have the same rights than you), to help
you to annotate images (won't have options below "export" in the project see
go to step #11.5 ), or just view the project (both validated and non validated)
```

# 12.1 Use filters wiselyUse filters wisely

protocols.io |

```
there are 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
```

```
taxonomic filters
```

protocols.io |

```
Filters are additive, 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)
```

```
other filters
```

protocols.io |

```
quickfilters are pretty useful since you can sort objects by specific values (eg. mean saturation
below) to quickly observe objects that have here lots of chlorophyll (ps. you can revert the
sorting order of those filters by ascending or descending order)
```

# 12.2 The different validation "states" in ecotaxaThe different validation "states" in ecotaxa and how to validate

```
image arrives in EcoTaxa with the status "unclassified" (grey surrounding of the image)
```

protocols.io |

```
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
```

protocols.io |

```
-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
```

```
once validated the name appears in red below the images
```

```
sliding into existing categories also works
```

```
dont forget to save your validations
```

protocols.io |

```
minutes!
```

```
example of (well) predicted objects which would be easily validated
```

# 12.3 Do not hesitate to "predict" your project right awayDo not hesitate to "predict" your project right away (even with a project/instrument that

```
has nothing to do)
```

```
-How to do prediction (and help 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 classifications"
```

```
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 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
```

protocols.io |

```
first trial of prediction to quickly validate to re-predict on your own project.
what could be used for first prediction :
```

```
#6818 - MOOSE-GE-2022_tests_ID_vignettes (Med sea; Processed with current segmenter;
Fully validated)
#4605 - Planktoscope NOAA WCOA21 rita-net (coastal US West coast from Vancouver to
San Diego; Processed with current segmenter; Partly validated; Contains lots ofContains lots of
artefacts due to lens mis-alignmentartefacts due to lens mis-alignment )
#6765 - _Planktoscope Reference (Processed with other segmenter, works only withworks only with
adding Deep Learning features into playadding Deep Learning features into play); Fully validated)
```

```
Push the button "select project below 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
objects to partly correct the usual strong imbalance between categories (here as an
example limited to 100 example per group) (If you use project #4605 as an example, please
remove artefacts)
```

```
click on "continue to the classifier option screen ". 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)
```

protocols.io |

```
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 "scorescore" 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 globalDoing repeated predictions on your own samples is better than doing some global
one on random example projectone 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.
```

```
example of images sorted by score of prediction
```

protocols.io |

```
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 exist, the easiest to
understand being the summary export with count per sample)
```

# 13 Clean tubing and flowcell from insideClean 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,
```

# Maintenance of your planktoscope

protocols.io |

```
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 )
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: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 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)
2. if dirt in not only dry dusts it could be cleaned with optical paper and ethanol. DO NOT USEDO 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 lensesClean 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 sensorclean the camera sensor
... critical part if any, NEVER touch it, only use dry gas
```

```
regularly calibrate the pumpregularly calibrate the pump
```

# Troubleshooting

protocols.io |

# 14 My flow cell is clogged with plankton, what to do?

```
Why this happens (preventive solutions):Why this happens (preventive solutions):
```

- first this may happens if your sample does not have been "pre-filtered (we recommand pre-
  filtration to 200μm). Make sure to do this. go to step #6.1
- It may aslo happens 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 go to step #7.5
- it may also happens if you forget to agitate your sample using a bubble ( go to step #3.11 )
  or if you let sample to stagnate for too long in the fluidic system.

```
unclogging the flowcell:unclogging the flowcell:
-try to pinch the tube in between the flowcell and the pump (while the pump is running). see
go to step #7.8^
-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 luerlock 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.
```

# 15 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)
https://forms.gle/qvh5jwuMvmyBKMQC7
```

```
Plankton Planet website
https://planktonplanet.org/
```

```
EcoTaxa tutorials:
https://sites.google.com/view/piqv/ecotaxa?authuser=0
```

# External links

protocols.io |

```
https://www.youtube.com/watch?v=PSO6ZS765tk
https://www.youtube.com/watch?v=RaWUqIoKk0E
```

protocols.io |