single-cell printer user manual - Molecular Devices

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single-cell printer

user manual

cytena GmbH

SCP user manual (Version 3.2)

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Content

1 Electrical and chemical risks and safety.................................................................................. 6

2 Introduction.......................................................................................................................... 8

2.1 Overview and naming conventions ......................................................................................... 8

3 Delivery and Installation ....................................................................................................... 9

3.1 Scope of delivery and required peripherals ............................................................................ 9

3.2 Installation ............................................................................................................................. 10

3.2.1 Connection overview ..................................................................................................... 10

3.2.2 Vacuum connection ....................................................................................................... 12

4 SCP Hardware ..................................................................................................................... 13

4.1 Overview ................................................................................................................................ 13

4.2 Robot workstation ................................................................................................................. 14

4.3 P9 dispensing unit ................................................................................................................. 14

4.4 Cartridge ................................................................................................................................ 15

4.5 Droplet quality control (DQC) system ................................................................................... 16

4.6 Substrate carrier .................................................................................................................... 16

4.7 Reservoir Agitation ................................................................................................................ 17

4.8 Pneumatic shutter system ..................................................................................................... 19

4.9 Control PC unit ...................................................................................................................... 21

4.10 LED front panel and emergency switch ................................................................................. 21

5 SCP utilization ..................................................................................................................... 23

5.1 Starting sequence .................................................................................................................. 23

5.2 Unpacking a cartridge ............................................................................................................ 23

5.3 Filling sample into a cartridge ............................................................................................... 24

5.4 Mounting a cartridge ............................................................................................................. 25

5.5 Applying agitation.................................................................................................................. 26

5.6 Mounting substrate ............................................................................................................... 27

5.7 Cleaning protocol .................................................................................................................. 29

6 SCP Software ...................................................................................................................... 30

6.1 Software initialization ............................................................................................................ 30

6.2 Cell detection parameters ..................................................................................................... 31

6.3 Starting experiments ............................................................................................................. 32

6.4 Printing single cells in micro-well plates ............................................................................... 33


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7 FAQ & Troubleshooting ....................................................................................................... 36

7.1 System start ........................................................................................................................... 36

7.1.1 Device manager ............................................................................................................. 36

7.1.2 Drivers ........................................................................................................................... 36

7.1.3 Error messages in software ........................................................................................... 36

7.2 DQC mode ............................................................................................................................. 37

7.2.1 Interpreting droplet images .......................................................................................... 37

7.2.2 Tuning droplets .............................................................................................................. 37

7.2.3 Vacuum shutter ............................................................................................................. 38

7.3 Agitation system .................................................................................................................... 39

7.3.1 Tip compatibility ............................................................................................................ 39

7.3.2 Correct work flow .......................................................................................................... 39

7.3.3 Problems with tight seal ................................................................................................ 39

7.3.4 Problems with filter tips and disk filters ........................................................................ 40

7.4 Piston Guide .......................................................................................................................... 40

7.4.1 Why cleaning is important............................................................................................. 40

7.4.2 Problems with disk filters .............................................................................................. 40

7.5 Moving the SCP ..................................................................................................................... 40

7.5.1 Disconnecting cables and tubing ................................................................................... 40

7.5.2 Vacuum tubing connection ........................................................................................... 41

7.5.3 Transportation ............................................................................................................... 42

7.5.4 Fitting in biosafety cabinets .......................................................................................... 43

7.5.5 Laminar flow .................................................................................................................. 43

7.6 IT topics ................................................................................................................................. 43

7.6.1 Network connection ...................................................................................................... 43

7.6.2 Domain .......................................................................................................................... 43

7.6.3 Data storage and access rights ...................................................................................... 43

7.6.4 Windows Updates ......................................................................................................... 44

7.6.5 Driver Updates ............................................................................................................... 44

7.6.6 Anti-virus software and firewalls ................................................................................... 44

7.7 Cleaning and disinfection ...................................................................................................... 45

7.7.1 List of compatibilities .................................................................................................... 45


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About this manual

Before putting the SCP instrument into operation, please read the instructions given within

this manual carefully and keep this document safe.

This manual contains information about the cytena SCP instrument. Besides the content

about technical data, explanations, handling issues, etc. the manual is containing warnings

and hints in each chapter helping to guide the user and show him/her where to pay special

attention.

WARNING: warning notes should prevent harming the user and/or damaging

the device due to inappropriate use. Warnings are marked in red and are

indicated by an exclamation mark. Please read them carefully.

HINT: Hint notes should provide additional help and support understanding in

certain situations. Hints are marked in blue and are indicated by the letter “i”. It

is not mandatory but recommended to read them.


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Technical support contact

Andre Gross (Dipl.-Ing. | CTO)

Tel: +49 (0)761 / 203 732 57

Email: [email protected]

Disclaimer of warranty

The SCP instrument is designed for research and may not be used for diagnostic

applications.

This is the operation manual of the SCP instrument. Please note that no warranty of any kind

will be given. The cytena GmbH assumes no liability for improper handling.

The SCP instrument must only be operated by trained personnel. The cytena GmbH

assumes no liability for damages incurred due to improper handling of the device and the

device components (cartridge, etc.).

SCP instrument:

cytena GmbH

Georges-Koehler-Allee 103 79110 Freiburg

Germany

Type: Single Cell Printer Robot Serial: CYT-SCP-01-2017-xxx 100–240 V~; 50/60 Hz; 1,34 – 0,55 A; max. 135 W Build 20xx; Quality control xx/20xx

For the SCP control computer:

cytena GmbH

Georges-Koehler-Allee 103 79110 Freiburg

Germany

Type: Single Cell Printer Computer Serial: CYT-SCP-01-2017-xxx 100–240 V~; 50/60 Hz; 0,41-0,21 A; max. 41 W Build 20xx; Quality control xx/20xx

mailto:[email protected]


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1 Electrical and chemical risks and safety

WARNING:

High voltage hazard!

Do open neither the dispenser unit nor the device electronics! The device contains circuits

which carry power supply voltage. Opening is only allowed to skilled personnel while the

power plug is disconnected.

CAUTION: After switching off the electronics, a DC voltage of 170 V remains in the circuits

for several minutes due to a capacitance in the circuits!

The device must be used with insulated piezo actuators only, where the piezo ground signal

is discharged separately and not by the piezo actuator housing. Also, do not attempt to

measure the piezo voltage directly at the piezo connector port. This will most likely destroy

both your measurement equipment and the driving electronics. Use the supplied piezo

monitor port instead!

Service and maintenance of the NanoJet electronics is only allowed to skilled personnel!

Do not immerse the dispenser into liquid! Keep dispenser dry at all times! If the dispenser

gets into contact with liquids disconnect from voltage supply and make sure it has dried

completely before next use. In doubt, contact cytena for further assistance.

WARNING:

Danger of fire, explosion and injury by use of

aggressive liquids!

Do not use or clean electrical parts of the device with flammable or chemical aggressive

media or solvents. Always wear protective gloves, eye protection and lab coat when

operating the device. Always consult the safety data sheets of the liquids used for dispensing

and ensure that the required safety measures are taken.


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WARNING:

Injury and safety risks related to unprotected

moving parts! Hand injuries!

The SCP instrument has to be installed in such a way that a free travel of the axes is

ensured. Never let foreign objects or materials such as screws or liquids enter into the

device.

It is forbidden to place objects of any kind onto the device except the designated (substrate

in the substrate holder). When operating the SCP instrument hands or other body parts are

to be brought to a safe distance from all moving parts (axes, etc.).

Do not touch any moving parts during the SCP instrument is running. In an emergency, the

drive can be stopped by pressing the emergency stop button on the right hand side of the

device.

Load and unload components such as substrates or printing cartridges only when the SCP

instrument is not in operation.

WARNING:

Environmental issues!

Do not expose the device to extreme environmental conditions. Normal environmental issues

are:

Temperature: 10°C - 30°C

Humidity: 10 - 80 % (non-condensing)


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2 Introduction

This user manual is divided into four main sections. These are:

Delivery and installation notes

Overview over SCP hardware

Overview and introduction to SCP software

Troubleshooting and FAQ

2.1 Overview and naming conventions

The SCP instrument is consisting of three major hardware parts (cf. Figure 1):

SCP instrument

control PC

peripherals (monitor, keyboard, computer mouse)

Figure 1: Overview on SCP instrument major hardware parts.


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3 Delivery and Installation

For delivery of the SCP instrument a cytena staff member will be present to set up the

instrument, test and explain it.

3.1 Scope of delivery and required peripherals

The SCP instrument will be delivered in a special transport box.

Please check the condition of the transport box directly at its delivery and before unpacking

the scp instrument. It is strictly recommended to claim any damage immediately to the

transport company! Any damage done to the scp instrument during transport may then be

charged to the transport company.

Content of the transport box:

1x SCP instrument

1x control computer

1x monitor (24’’ DVI, HD wide screen)

3x power supply cable (black, 2 m, 110-230 V AC, 50/60 Hz)

1x DVI / HDMI cable (2 m)

1x USB 3.0 cable (2 m, type A/B)

1x keyboard (QUERTY or QUERTZ)

1x computer mouse

1x vacuum tubing (2 m, ID: 4 mm)

Required space:

● Instrument footprint: ca. 550x430 mm

● Instrument weight: ca. 30 kg

● Control PC footprint: ca. 300x300 mm

● Control PC weight: ca. 9 kg

● Peripherals footprint: ca. 600x600 mm

Additional surrounding space:

To the back (movement of cable ducts): ca. 70 mm

To the right (accessibility of emergency stop): ca. 70 mm

Figure 2: Dimensions and space requirements of the SCP instrument.


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Power consumption:

● Instrument: 100-240V AC, 50/60 Hz, 1.5 A

● Control PC: 100-240V AC, 50/60 Hz, 1.2 A

Hint: All connectors and cables are labelled. Please use the respectively

marked cable-connector combination to install the device.

3.2 Installation

3.2.1 Connection overview

The SCP instrument can be placed on a laboratory table or inside a biosafety cabinet. The

latter must provide sufficient space to insert and run the SCP instrument.

Connect all required supplies and peripherals according to the following guideline. Figure 3

illustrates the rear side connectors on the SCP instrument. Figure 5 illustrates how to

connect the instrument to the control PC. Please follow these steps and closely stick to the

figures:

1. Connect “system USB” cable to PC (red label) 2. Connect “cell cam USB” cable to PC (green label) 3. Connect keyboard & computer mouse to PC (blue label) 4. Connect monitor via DVI cable to PC (yellow label) 5. Connect power cable to “230 V power supply” connector on instrument 6. Connect power cable to “230 V power supply” connector on PC 7. Connect power cable to “230 V power supply” connector on monitor

Figure 3: rear view of the instrument with main switch, 230 V power supply, USB connectors and serial / license stickers.


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Figure 4: control PC front and rear view with connectors.

Figure 5: rear view of the instrument and PC. Color codes show how devices must be connected.

As soon as all connectors are properly installed switch on the screen. In case the emergency

button in the scp instrument is pressed, release it. Switch on the instrument by pressing the

main switch. The system is booting, green status LED lights up and blue detection light is

turning on. The instrument is operable after appearance of the Windows 7 desktop.

WARNING: Please do not change the USB-ports. Stick to the order shown

here. In exceptional cases a cytena technical engineer may change the order.


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3.2.2 Vacuum connection

The blue Festo vacuum connectors are so called quick connectors. To attach the tubing

simply insert into the connector until you feel a strong resistance. The connector is self-

sealing.

To release the blue vacuum tubing from the connector, please press in blue ring on the

connector, keep it pressed in and remove the tubing. It should detach without force. Please

do not use brute force, do not cut the tubing or dismount the connector with a tool.

Figure 6: Festo vacuum quick connector. Press in the blue ring to detach the tubing.


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4 SCP Hardware

4.1 Overview

Figure 7 illustrates the major components of the SCP instrument.

Figure 7: Overview of the SCP instrument with major components.

Figure 8: Detailed overview of the SCP instrument printhead with major components.


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4.2 Robot workstation

The robotic stage is a high precision, three axes benchtop platform. It provides the basis for

precise positioning of the printhead and allows for easy and flexible access to a variety of

different substrates. The BT600 carries the driver and control electronics for the printhead.

Key features are:

Footprint: 550x430 mm

Weight: 28 kg

Z-axis load: max. 3.5 kg

Axes speed: 50 mm/s

Accuracy X: +/- 22.5 µm

Accuracy Y: +/- 37.5 µm

Accuracy Z: +/- 2.0 µm

4.3 P9 dispensing unit

The P9 dispenser unit is a piezo-electric driver for inkjet applications. It is specifically

modified to drive cytena’s cartridges including the dispenser chip (see Figure 9).

Key features are:

System: piezo-electric

Principle: drop-on-demand

Piezo stroke: 1-15 µm

Actuation frequency: max. 1 kHz

Figure 9: P9 dispensing unit front and side view. The chassis contains the piezo driver. The attached piston guide connects the piezo plunger to the disposable dispenser chip.


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4.4 Cartridge

The cartridge is a combination of cell suspension reservoir and dispenser chip (see Figure

10). The cartridge contains all (fluidic) elements that are in direct contact to the cell

suspension. It is designed as disposable to avoid any cross-contamination.

Figure 10: Overview of the cartridge. Cell suspension is loaded into the reservoir. The dispenser chip is actuated by the P9 piezo driver.

The cartridge is mounted to the P9 dispensing unit as shown in Figure 11 via two screws.

Interconnection and alignment between P9 chassis and cartridge is established by the piston

guide. The latter also contains the droplet shutter system. Since the shutter system may get

in contact with cell suspension it can be detached and cleaned.

Figure 11: Overview over the main components of the dispensing unit.


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4.5 Droplet quality control (DQC) system

The DQC stroboscopic camera is responsible for quality control of the droplets created by

the P9 dispensing unit. It consists of a 1.3 MP ultra-fast sensor with shutter times of 9 µs.

Coupled to a white high-power LED the stroboscopic setup provides images showing the

droplets in flight.

Figure 12: Overview of the stroboscopic system. The droplet waste vessel collects dispensed liquid.

The stroboscopic mode further allows for adaptation of piezo parameters and thus to

manipulate droplet volume, speed and reproducibility. It is especially useful to avoid

formation of satellites and to calibrate the system for non-standard liquids.

4.6 Substrate carrier

The SCP instrument is compatible to a variety of different substrates. Since the dispensing

technology is contact-free, virtually any top-open substrate can be filled with single cells

using the scp instrument. To allow a maximum in flexibility regarding substrates a

changeable carrier system is mounted. The default standard system is the SLAS (former

ANSI / SBS) standard well plate format inlet shown in Figure 13.

Figure 13: Substrate carrier mounted with SLAS (former ANSI / SBS) standard well plate inlet.


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Hint: The SCP is delivered with one substrate carrier including ANSI / SBS

well plate inlet. On demand it can be equipped with other inlet types and with

up to four substrate carriers simultaneously. Please contact cytena technical

support for further information.

WARNING: Never change substrates while a batch is running or during

movement of the axes! You may injure yourself and damage the device!

4.7 Reservoir Agitation

To provide homogeneous cell distribution within the sample loaded in the cartridge reservoir

a support system called reservoir agitation is available. It consists of a small peristaltic pump

aspirating and dispensing small sample volume (few microliters only) from the reservoir. The

pump is coupled to a tip handle that allows for picking up standard 200 µl pipette tips1. The

picked up pipette tip is placed inside the cartridge reservoir. Handle thereby is held in

position by tiny magnets allowing for quick plug and play connection (cf. Figure 14).

Figure 14: detailed view on the reservoir agitation system. The magnetic tip handle is located on the front of the printhead while the power switch for agitation start/stop is located on the right side of the printhead.

1 For specific models / types please refer to section “FAQ”


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The agitation working principle is illustrated in Figure 15. Once the pipette tip is attached to

the tip handle, the tip is placed inside the cartridge reservoir. The ventilation valve is open

and liquid from the reservoir can enter the tip. Filling level will be determined by capillary

pressure. The peristaltic pump is switched on, the valve closes automatically and the pump

smoothly aspirates some more liquid (few microliters only) and directly dispenses this volume

back into the reservoir. After this, the valve is opened again to reset the liquid level inside the

tip by capillary pressure. This process is repeated every 5 sec and ensures a smooth,

constant mixing of the sample inside the cartridge reservoir.

Figure 15: schematic of the working principle of the agitation setup.

The system can either be used with filter tips and without an extra filter in the tubing or with

non-filter tips and extra filter in the tubing (as shown in Figure 15).

Hint: Please ensure that all Luer-Lock connectors in the tubing are certainly

correctly locked. The agitation system will not work when connection is not

established and/or not properly (air tight) connected!

Please note that the agitation system must be switched on (manually by using

the power switch on the right side of the printhead) in order to work.

Do not use a disk filter and a filter tip at the same time. Resistance in the

system will likely be too high to ensure proper working.


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4.8 Pneumatic shutter system

The pneumatic shutter system takes of unwanted droplets generated by the dispensing unit.

Unwanted in this context means void droplets and droplets populated with more than one

cell. It consists of a vacuum pump (located inside the control PC case) connected to the

piston guide (located front of the dispensing unit). In between them a high-speed valve

allows for fast switching on and off the vacuum.

Figure 16: detailed view on pneumatic shutter system. The vacuum pump is connected to a high-speed valve inside the printhead. The valve controls vacuum presence at the shutter out in the piston guide. A sterile filter can be placed in the tubing to avoid cross-contamination.

Figure 17 shows the working principle of the shutter system. An external pump generates

vacuum which is connected with tubing to the piston guide vacuum inlet. In between there is

a sterile filter and a high-speed valve.

Figure 17: schematic of the working principle of the shutter system.


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In standard operation the valve is open and vacuum is permanently present at the piston

guide inlet. All generated droplets are taken off. In single-cell printing mode, the valve is

switched whenever a single-cell appears in the droplet to be generated. Vacuum is blocked

such that this specific droplet can pass the inlet towards the substrate. After the droplet has

been ejected and has passed, valve is switched again and vacuum is reapplied.

Figure 18: schematic inner view of the piston guide with vacuum flow and droplet motion.

The piston guides internal structure is drafted in Figure 18. Droplets are ejected by the chip.

Below the chip nozzle the vacuum inlet sucks in the flying droplets. A sharp 90° angle

followed by a capture structure forces the droplets onto the center walls, where they dry out

immediately (evaporation time: ca. 1 sec). No liquid and thus no cells do leave the piston

guide towards the printhead. For further safety an additional sterile filter is applied after the

piston guide.

The metal piston guide including tubing can be taken off for cleaning and sterilization

purposes after experiments. Therefore disconnect the Luer connectors and slide off the

piston guide from the dispensing unit. The sterile filter can be changed.

Hint: Sterile filters must support Luer-Lock in and Luer-Lock out connection.

WARNING:

Do not use filters with pore sizes below 0.2 µm!

Do not use filters with Luer-slip connectors!

Please ensure that all Luer-Lock connectors in the tubing are certainly

correctly locked! The shutter system will not work when connection is not

established and/or not properly (air tight) connected!

Non-working shutter will result in all generated droplets entering your

substrate! Single-cell printing is not possible.


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4.9 Control PC unit

The SCP device includes a fully operable embedded PC:

SCP Revisions

CYT-SCP-04-2015-xxx

CYT-SCP-01-2017-xxx

SCP Revision

CYT-SCP-xx-2018-xxx

AMD Quad-Core @ 2.0 GHz Intel i7-6700 @ 3.4 GHz

4 GB DDR3 RAM 8 GB DDR4 RAM

128 GB SSD drive 256 GB SSD drive

OS: Win7 x64 Prof. OS: Win7 x64 Prof.

The SCP control software is completely installed and ready to use. The case also carries the

vacuum pump. The pump is permanent on while the control PC is powered on.

WARNING: Please do not use the embedded PC for any other purpose then

running the SCP device. Third party software that generates heavy CPU load

may compromise proper functionality of the SCP software while being

simultaneously in use.

4.10 LED front panel and emergency switch

The SCP device includes a LED front panel with three status LEDs. On the right side of the

instrument a red emergency switch is located. See also Figure 19.

The status LED’s have the following purpose:

● Green: shows power state of the device

● Yellow: indicates that piezo droplet generator is under operation

● Blue: indicates that axes are in motion

Steady state (non-operating) after powering up the device is:

● Green: permanent on

● Yellow: permanent off

● Blue: permanent off

Operational state is:

● Green: permanent on

● Yellow: blinks when piezo droplet generation is active

● Blue: lights up when axes are moving


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The red emergency switch is for any kind of emergency situation that can occur during

operation. Be it electrical, chemical or man-made critical situations or accidents do not

hesitate to press the button. It shuts down the complete system electronics and separates

them from power supply when pressed. This only excludes the embedded PC.

Figure 19: Overview of emergency switch (located right side) and status LED panel (located front).

Warning: In case of emergency press the emergency button to immediately

shut down 24V and 48V DC power supplies. Please note that 230V AC power

supply is kept active.

Warning: In case the emergency button was pushed, please wait at least 10 s

until releasing the switch to ensure that all system buffers are reliably deleted

and no unforeseen actions (e.g. axes motion) occur after release.

The following power and hardware errors are indicated by the LED front panel:

1) All LEDs off = no power on device switch on main power on backside of device check if power supply cable is correctly installed

2) Yellow LED permanent on = boot failure of piezo droplet generator electronics power off device (main switch on backside) for 3 sec. power on again yellow LED is shortly on (1-2 sec) then turn off. Green LED is permanent on.


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5 SCP utilization

5.1 Starting sequence

Please start the devices in the following order:

1) Start the control PC by pressing the “power on” button on top of the device. Hold the “power on” button until the status LED on the control PC bottom lights up (ca. 5 sec)

2) Wait until the system has booted to Win7 desktop

3) Start the SCP instrument by switching on the main power switch on the device rear side.

4) Wait until the device starting routine has completed and the LED front panel shows the green LED permanent on and the yellow and blue LED permanent off.

5) Start the SCP software by clicking the link on the desktop. Wait until starting routine has completed and GUI is present.

Warning: It is important to start the control PC as described. If you press the power on button not sufficiently long, the PC will not start.

It is important to first start the control PC and then the instrument. Otherwise the hardware is not correctly initiated in Win7 and software will fail on startup.

5.2 Unpacking a cartridge

Take the sealing foil bag with cartridge inside, peal transparent side open from the top side

and remove cartridge by hand (wear gloves!).

Figure 20: Schematic on how to unpack a cartridge from its sealing foil bag.


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5.3 Filling sample into a cartridge

Pipette 10 to 80 µl of your cell sample into the cartridge reservoir. The dispenser chip will

self-fill via capillary forces.

Figure 21: Schematic cartridge handling. Aspirate sample with pipette, place pipette tip into cartridge reservoir (to the very bottom) and dispense sample. Tilting the cartridge in your hand helps for more comfortable pipetting.

Figure 22: Schematic on how to dispense sample into the cartridge reservoir using a hand pipette. Dip the pipette tip inside the reservoir, until bottom is reached. Dispense slowly while moving the tip out of the reservoir. Once sample is in, make sure that no entrapped air bubbles are visible inside the reservoir.


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Figure 23: Schematic of false cartridge filling. Avoid placing the tip too high in the reservoir. Sample might stick to the walls. Avoid inducing air bubbles during dispensation. Don’t empty the tip at the reservoir bottom. The likelihood of forming a bubble at the chip inlet is high. Avoid dispensing fibers or other impurities together with your sample. Fibers are likely to clog the chip inlet.

5.4 Mounting a cartridge

The cartridge can then be mounted by clipping it with the chip facing inwards to the

positioning pin and fixing it with the two M2x13 screws. Screws should be tightened

alternately to avoid damaging the chip. Also screws must not be tighten too heavy to avoid

breaking the chip. Use the provided M2 hex key to screw.

Figure 24: Schematic of orientation of cartridge. Once having filled the sample in, turn the cartridge such that the chip (dark blue part) is facing towards the P9 dispenser front. Shift the cartridge on top of the single steel pin sticking out of the P9 dispenser.


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Figure 25: Schematic on correct screw placement when mounting a cartridge. Insert the M2x13 hex screws into the front top and rear bottom hole in the cartridge (blue arrows). Cartridge is hold by the pin (red arrow). Leave the front bottom hole empty. The eye indicates position of the operator and his/her view point.

5.5 Applying agitation

The agitation prevents sedimentation of cells inside the cartridge reservoir over time. This

ensures a continuously homogeneous distribution of cells inside the chip. Figure 26 shows

the single steps that have to be performed to enable agitation.

Figure 26: Schematic on how to use the agitation setup. (A) detach adapter from print head. (B) take up a pipette tip from the rack. (C) insert tip into mounted cartridge reservoir. (D) reattach the adapter to the print head.

Please follow the steps to ensure a proper start of the system:

1. Load the cartridge and mount it to the dispenser 2. Make sure the agitation is switched off (switch on right side of print head) 3. Take the agitation adapter and pick up a pipette tip 4. Make sure the tip is tight attached to the adapter 5. Place the tip inside the cartridge reservoir 6. Switch on the agitation (switch on right side of print head) 7. Make sure it visibly aspirates and dispenses (tip)


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Please follow the steps to ensure a proper stop of the system:

1. Switch off the agitation (switch on right side of print head) 2. Make sure aspiration and dispensing has visibly stopped 3. Remove tip from cartridge reservoir 4. Detach tip from adapter 5. Dispose tip and reattach adapter to the print head

5.6 Mounting substrate

All supported substrates can be mounted by simply clipping them in the foreseen carriers on

the baseplate. To learn how to mount a substrate correctly, please take a look at the

following Figure 27.

Figure 27: Schematic view on how to mount a well plate in one of the substrate carriers.

To mount the substrate (well plate), follow these steps:

1. Orientate the well plate with well A1 located to the top left corner (facing towards you). You can find a mark “A1” on the carrier as well

2. Place the plate like shown and slide it towards the rear right corner of the carrier

3. Press the plate against the spring clamps until it clips into the carrier.

4. Ensure that the well plate is properly clipped and sitting even in the carrier.

Ensure that the plate sits proper on the carrier and is clamped correctly. It should be even

and not tilted. Typical mistakes are:

Well A1 does not face front left side of the carrier


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Plate does not set even on the carrier

Plate sits on top of the spring clamps rather than being clamped by them

Plate is tilted

Please see the following schematic (Figure 29) as guideline.

Figure 28: Schematic view on wrong positioning of substrates.

Warning: Avoid incorrect placement of the substrate (typical situations shown in Figure 28). This will lead to hardware collision between dispenser and substrate and/or incorrect deposition of single cells in the wells!

cytena does not accept any warranty claims in case damages caused by incorrect mounting of substrates or use of non-compatible substrates!


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5.7 Cleaning protocol

Please follow the protocol listed below to clean the SCP after having completed your single-

cell printing experiment runs for the day. The protocol refers to Ethanol as the standard

cleaning and disinfection agent. You can also use your preferred liquid for cleaning and

disinfection. For a list of material compatibilities please refer to section “FAQ”.

1. Stop idle shooting mode (if activated) 2. Detach agitation adapter, unplug tip and dispose tip 3. Dismount cartridge and dispose it 4. Prepare a glass with 70% Ethanol 5. Unplug piston guide from dispenser 6. Unplug vacuum tubing (LuerLock) from printhead 7. Unplug waste plate from DQC system 8. Place piston guide incl. tubing and waste plate in glass with Ethanol for 5 min 9. Clean wipe agitation adapter with Ethanol 10. Place all parts on a clean wipe to dry inside biosafety cabinet 11. Reassemble system once all components are dry

WARNING: Regular cleaning is mandatory!


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6 SCP Software

Please refer to the cytena software manual for detailed instructions. The follow section is

only giving a brief overview.

6.1 Software initialization

If not already done: To start the software click on the “SCP software” icon on the desktop.

As soon as the software is started it will automatically connect the cameras and the core.

Automatic ROI detection is performed and the result is displayed to the user in a “ROI result”

window. After this the stage will automatically start to reference its axes. Until referencing is

done all software buttons will be disabled to ensure proper homing of all three axes. When

referencing is done, core and camera are successfully connected and the ROI is detected,

the three traffic lights will turn green and the software hands over control to the user. In case

anything is not properly started or connected the traffic light will indicate this with a red light

and a corresponding error message below (see picture below).

Figure 29: Hardware control panel with traffic lights during initialization (left) and after (right).

Red traffic lights indicate:

● Dispenser: Either the piezo or its driver electronics are not correctly initiated. To

solve the problem, shut down the software and press the emergency button to shut

down electronics power supply. Wait at least 10 sec before releasing the switch and

such reboot the electronics. Then restart the software.

● Cell Cam: If the software is started while no chip is mounted, the automatic ROI

detection fails and the traffic light stays red. Mount a chip and press “re-detect ROI”

button. For debugging / testing issues alternatively press “manually set ROI” and

place the ROI somewhere in the image to set the traffic light to green.

Another case could be that the camera is not correctly initiated and not properly

connected to the CORE doing the image processing. Restart the software.

● Axes: Either motor controllers or driver electronics are not correctly initiated. Perform

action listed in previous point “dispenser” to reboot electronics.

Incorrect or incomplete referencing could also be an issue. To restart referencing

procedure call expert mode and press button “start referencing”.


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Warning: Setting the ROI manually without having a chip mounted will turn the traffic light green and enable full user control of the software. However we cannot guarantee correct operation under such conditions!

6.2 Cell detection parameters

In order to correctly calibrate the detection parameters along which the algorithm recognizes

and classifies bypassing objects inside the chip, the available functions and parameters are

explained in detail.

Figure 30: Overview of cell detection parameters size and roundness and Detection Radius (ROI).

The two main input fields are:

Minimum / Maximum cell diameter (size)

The min./max. cell diameter allows the user to define the size range wherein the cells of

interest are. The algorithm takes these values as classification criteria, filters all bypassing

objects and classifies them in first instance by size.

Size in this context is calculated by a fitting ellipse approach (cf. Figure 31) whereby the

average of minor and major half axes is used as “average diameter” of the cell.

Roundness

There are two morphological parameters extracted and used to classify the roundness of

objects:

1. Aspect ratio AR: measures aspect ratio using fitting ellipse (cf. Figure 31)

2. Circularity CR: measures circularity using area and perimeter. (cf. Figure 32)

Roundness is then calculated as quotient of CR and AR.


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Figure 31: Description of aspect ratio measurement. An object (cell) is surrounded by the smallest possible ellipse. Major and minor half-axis of the ellipse is measured. Aspect ratio is calculated along given equation.

Figure 32: Description of circularity measurement. Area and perimeter of the object (cell) is measured. Circularity is calculated along given equation.

6.3 Starting experiments

The first actions required before starting experiments are listed below.

1. Set experiment name

2. Enter cartridge ID

3. Enter cell detection criteria (size, roundness)

4. Set ROI (if not already done)

5. Perform droplet quality control

6. Save droplet QC images (optional)

7. Leave droplet quality control

8. Program experiment (batch)

9. Remove lid from substrate (if there is one on)

10. Execute experiment


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6.4 Printing single cells in micro-well plates

Depositing single cells in micro well plate (MWP) formats is the standard operation for the

instrument. As standard formats 96-well and 384-well plate are pre-programmed as

executable batch commands. Specific wells can be addressed like shown below. The

number of cells per well is free to choose between 1 and 100.

Figure 33: Dispense 96 well plate batch with exemplary input values.

Hint: Please note that the SCP can be equipped with either two 96-well plate

carriers or one 96-well plate and one 384-well plate carrier.


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Please follow these instructions to program a MWP batch:

1. Click “add line” to add a new empty line in the batch.

2. Go to the drop down menu and choose “96-well plate” from the drop-down menu.

3. This action requires further user input therefore some input fields appear next to right

to the drop down menu. Here input fields “coordinates” and “cells per well” will pop

up.

4. In the “coordinates” field enter the well coordinates you want to address. There are

several options to do this:

a. Addressing single wells by typing in their coordinates like “A1” or “B4”. If you

would like to enter multiple single coordinates, use multiple lines.

b. Addressing rows like A1 to A8 (for a 96 well plate) you have to type start and

end coordinate separated by a dash e.g. “A1-A8”. Same for addressing

columns like C3 to H3 (for a 96 well plate) enter “C3-H3”. First coordinate

must be smaller than second.


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c. For addressing complete sections you type e.g. “B3-F7”. Whereas the first

coordinate defines the top left corner and the second coordinate the bottom

right corner of the section. First coordinate must be smaller than second.

5. In “cells per well” type in the number of single cells you want to be printed in each

well.

6. If you have finished all required input mark the empty line in the batch below click on

the “transfer” button to program the data into this line.

7. The empty line will be filled with a short description of your well plate like “96,A1-H12”

for a 96 well plate where all wells from A1 to B12 should contain a single cell.

8. Steps 1-9 can be repeated to add other batch lines with other parameters e.g. rows

with two cells per well.

Hint: Well plate batches can consist of multiple batch lines to perform complex

filling operations. For example you want to increase the number of single cells

in each row of your well plate you will have to program a separate batch line

for each row looking like this:

● print in 96 well plate, A1-A12, 1 cell per well

● print in 96 well plate, B1-B12, 2 cells per well

● print in 96 well plate, C1-C12, 3 cells per well

● …

● print in 96 well plate, G1-G12, 8 cells per well


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7 FAQ & Troubleshooting

7.1 System start

7.1.1 Device manager

After the SCP has been moved, installed or reinstalled, use device manager to check the

correct connection of hardware. Start the PC, login and start the SCP. Run device manager

from Windows system or via Start button. Use view devices by connection to open the

connection part of the manager. Unfold the connection tree until you see the PCI and USB-

controllers.

You should see (depending in their physical connection) the following devices:

UI3240x Cell cam

UI3240x DQC cam

Trinamic stepper device motor controller

USB serial converter Trigger board

USB serial converter (COM) Piezo electronics

7.1.2 Drivers

In case drivers need to be installed or reinstalled, please refer to the following list of driver

versions:

Hardware Firmware version Driver version

UI3240x -- 4.61.00_64

Trinamic stepper device 1.0 1.09

Trigger board 1.0 2.12.18

Piezo electronics 2.6 --

7.1.3 Error messages in software

When you start the software and you receive one or all of the following messages, there is

connection problem with the respective hardware parts

1. Piezo not found or COM port already open 2. Axis system not found or connection port already open 3. DQC camera not found 4. Cell camera not found. Connection to Core not established


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Please check the following list and make sure you follow the instructions

Errors appearing Cause Corrective action

1-4 SCP is not connected to

the PC.

Check USB-cables. Connect the two USB

cables from the SCP to the PC and make

sure SCP is powered on afterwards.

Restart cytena software

1-3 SCP system USB is not

connected or SCP is not

powered on

Check system USB-cable. Connect the

cable from the SCP to the PC and make

sure SCP is powered on afterwards.

Restart cytena software

4 SCP cell cam USB is not

connected or camera

failed to initialize

Check cell cam USB-cable (fix attached to

the SCP). Connect the cable from the SCP

to the PC.

If cam initialization failed, unplug cable for

10 sec from PC and plug in again.

1 Piezo driver board failed

to initialize

Power off SCP, disconnect power cord and

wait for 1 min. Plug in power cord and

power on SCP. Restart software.

2 Motor controller failed to

initialize

Power off SCP and wait for 30 sec. Power

on SCP. Restart software.

7.2 DQC mode

7.2.1 Interpreting droplet images

7.2.2 Tuning droplets

Droplets are optimally adjusted / tuned when they appear as one, stable droplet like

illustrated in the ideal case in Figure 34. To tune droplets the dispensing parameters “depth”

and “speed” are used.

Depth defines how deep the piezo penetrates the chip membrane (measured in µm). Stroke

such tunes droplet volume in first order.

Speed defines how fast the piezo penetrated the chip membrane (measured in µm/ms). It

such tunes droplet kinetic energy and velocity.

Although being independent parameters depth and speed require to be balanced against

each other. The lower depth is, the lower the droplet volume is, the less kinetic energy the

droplet requires to be generated.


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Figure 34: Illustration of different typical droplet states appearing during droplet / piezo tuning visible at droplet quality control. The more speed, the more likely instabilities in droplet break-up occur, resulting in satellite formation up to multiple droplets (spray).

Rules of thumb:

For each in- or decrease x of d correct s about 20*x. (d+x s+20*x)

In case of satellites decrease s in steps of 10 until satellite disappears.

In case of no droplets increase d and s stepwise The higher the droplet velocity the more accurate its deposition on the substrate

7.2.3 Vacuum shutter

Once having found a stable piezo configuration and having centered the droplet in the

camera live window, use the button in software to check if the vacuum shutter is working.

The droplet should disappear. You should hear the piezo click and see the yellow LED on the

SCP front panel flashing. If so, the system works fine.

If you hear the click and see the LED, but the droplet does not disappear, please check the

following list of actions:

is the vacuum shutter really turned on software? check status label on left bar

is the transparent vacuum tubing between piston guide and print head connected?

is the blue vacuum tubing between SCP and PC connected?

do you hear the vacuum sound?

do you hear the vacuum pump inside the PC?

does the droplet disappear when you reduce “speed” value in software DQC?

Please contact the technical support after checking the list and report your findings.


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7.3 Agitation system

7.3.1 Tip compatibility

The agitation system is compatible with multiple standard 200µl pipette tips. The following list

provides an overview and shows exemplary tips that fit the cytena standard adapter and

some which require the special MT adapter.

Tip Vendor Cytena adapter type

200 µl Eppendorf Standard

DF200ST Gilson Standard

ART 200 LR Thermo Scientific Standard

RT-L200F Rainin MT

ART 200L Molecular BioProducts MT

7.3.2 Correct work flow

Make sure that you follow the correct order of steps given in section 5.5. Otherwise you will

not see a homogeneous distribution of cells over time.

Make sure that the agitation adapter a properly aligned to your type of pipette tips. The tip of

the tip must be close to the reservoir bottom, when the adapter is mounted on the print head.

Ask the technical support for help if required.

First insert the tip into the reservoir, then clip the adapter on using the magnets. It will not

work vice versa.

Don’t forget to switch on the agitation to make it work.

7.3.3 Problems with tight seal

To make the agitation system work effectively, you have to make sure that the entire system

is tightly connected. That requires

1. Transparent tubing connected to the LuerLock at the print head 2. Transparent tubing connected to the top of the agitation adapater 3. Pipette tip connected tight to the agitation adapter 4. Pipette tip inserted into liquid inside the cartridge reservoir

Most important is a proper seal between adapter and tip. Apply sufficient pressure on tip

pickup to ensure this seal.


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7.3.4 Problems with filter tips and disk filters

Disk filter can be applied in the LuerLock connection part. Filter tips can be used. It depends

on the density of the filter membrane material if the agitation still works when a disk filter is

inserted or a filter tip is used.

cytena recommends using filters with a pore size of 0.45 µm or larger. Pore size of 0.2 µm

can already be critical. Pore sizes lower than 0.1 µm typically do not work.

Please note that also the thickness of the filter may cause trouble. Double filter tips typically

also do not work well.

Do not use a disk filter and a filter tip at the same time. The accumulated resistance of the

filters is typically too high to make the system work reliably. Resign one of the filters.

Make sure the system visibly aspirates and dispenses the liquid inside the reservoir in a

cycle of 5 sec. If you do not see this, disconnect all filters respectively use a non-filter tip and

check again.

7.4 Piston Guide

7.4.1 Why cleaning is important

The piston guide contains the capture structure that collects all unwanted droplets. Those

droplets may be void but may also contain cells (doublets, debris, rejected, etc.). Droplets

being captured evaporate quickly inside the piston guide leaving the cell material behind.

Thus, regular cleaning of the piston guide is mandatory.

Cleaning can be done by dipping in Ethanol or Isopropanol or a mixture of both. Steam

autoclaving can be applied as well. The piston guide is made of stainless steel. Do not use

any aggressive liquids or solvents that attack steel.

Please note: not cleaning the piston guide may lead to a decrease in vacuum shutter

efficiency or even to a clogging of the vacuum shutter inlet. It is mandatory to clean it and

check the vacuum shutter regularly in the DQC mode.

7.4.2 Problems with disk filters

Similar to the agitation system, you can apply a disk filter also in the vacuum shutter path.

cytena recommends using filters with a pore size of 0.45 µm or larger. Pore size of 0.2 µm

can already be critical and may compromise vacuum shutter efficiency. Pore sizes lower than

0.1 µm typically do not work.

Please note: When you apply a filter, carefully check the vacuum shutter in DQC mode. The

SCP is delivered with no disk filter by default. cytena is not responsible for and does not

cover failures that develop due to insertion of such filters.

7.5 Moving the SCP

When moving the SCP (e.g. out of the biosafety cabinet for disinfection) please refer to the

following notes and hints.

7.5.1 Disconnecting cables and tubing

Disconnect the following cables and tubing from the SCP


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System USB-cable

Vacuum tubing (blue)

Power cord

Disconnect the following cables and tubing from the PC

System USB-cable

Cell cam USB cable

Mouse and keyboard

Monitor

Vacuum tubing (blue)

Power cord

The fix attached cell cam USB cable remains on the SCP. Please roll it up carefully and store

it on the working deck. Make sure that you do not buckle or kink the cable during transport.

7.5.2 Vacuum tubing connection

To release the blue vacuum tubing from the connector, please press in blue ring on the

connector, keep it pressed in and remove the tubing. It should detach without force. Please

do not use brute force, do not cut the tubing or dismount the connector with a tool.


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7.5.3 Transportation

For lifting and transporting the SCP you require two persons. Please only lift the SCP

grabbing the base structure (green dashed line). Never grab and lift it on moving parts like

the axis system (red dashed line) or the portal structure.

Ideally you grab the SCP next to its feet from both sides using four hands to stabilize.

Please note the weight of the instrument is 30 kg. False handling and dropping the

instrument can cause severe damage to its mechanics, electronics, optics and other sensible

parts. cytena will not cover any damage done through misuse or disregard of transportation

instructions. Please contact technical support for detailed instructions.


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7.5.4 Fitting in biosafety cabinets

The SCP fits into standard biosafety cabinets (class II) of certain dimensions. The space

requirements of the instrument should be met.

Main points taken into consideration are

Internal width (work space) of the cabinet should be >= 600 mm o instrument width = 550 mm o min. free space for emergency button = 50 mm

Internal depth (work space) of the cabinet should be >= 500 mm o Instrument depth = 430 mm o Min. free space for axis motion and cable duct = 70 mm

Internal height (work space) of the cabinet should be >= 560 mm o Instrument height = 550 mm

Front window opening >= 450 mm o Instrument must fit through front window o Even tilted by 90° it requires > 430 mm

Please note: Rear space for safe travelling of axis and cable duct must be kept. When cable

ducts or axis collide with rear wall of the cabinet, it cannot be ensured that accuracy of

droplet deposition will be met.

7.5.5 Laminar flow

The laminar flow inside the cabinet may be influenced by the SCP geometry. In case you

have any concerns about the air flow in your specific cabinet, please contact the cabinet

vendors technical support and ask for a measurement and potential recalibration.

Typically, disturbance of the air flow by the SCP is not the case. However, cytena cannot

guarantee that for a specific cabinet type air flow may not be influenced.

7.6 IT topics

7.6.1 Network connection

The cytena PC has two separate GigE connectors that can be freely configured to include

the PC into a local network.

7.6.2 Domain

Bringing the PC into a domain is possible. However, the standard local users admin and

operator have specific access rights for the data generated by the SCP software. Using

domain users requires to apply these rights via the domain controller. SCP data folder is

located on drive D:\SCPdata\

Please contact the technical support in case more information is required.

7.6.3 Data storage and access rights

Data generated by the SCP software is stored on the local hard drive, partition D. Changing

the storage location is not possible.


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Local user “admin” has full access to all data being generated. Local user “operator” has

restricted access covering the following issues:

Granted: o Create files and folders o Read files and file content o View permissions

Denied: o Modify files, folders and file content o Delete files, folders o Move files, folders o Take ownership o Change permissions

Please note that file protection is only given for “operator” user, not for admin.

7.6.4 Windows Updates

Windows updates can be installed. Update service can be activated. To date, there are now

issues or bugs known related to Windows updates.

7.6.5 Driver Updates

It is not recommended to update drivers for SCP hardware until absolutely necessary. Please

contact technical support in case a driver update is forced by e.g. Windows.

7.6.6 Anti-virus software and firewalls

You can run third-party software on the PC. To date, there are now issues or bugs known

related to firewalls or anti-virus software. However, cytena has not specifically tested certain

products from this category with regard to compatibility. Installation and operation is at your

own risk.


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7.7 Cleaning and disinfection

7.7.1 List of compatibilities

The following table provides an overview of the main component / material compatibilities of

the SCP. Rating is:

+ = compatible

- = incompatible

nt = not tested

Aluminum

parts

Plastic

parts

Lenses /

optics

Screws LEDs Feet Cable

ducts

Water + + + + + + +

Ethanol + + + + + + +

Isopropanol + + + + + + +

Acetone + - - + - - -

DNA-Ex + nt nt + nt nt +

Vaporized

hydrogen

peroxide (VHP)

nt nt nt nt nt nt nt


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Disclaimer OpenCV

cytena software uses OpenCV libraries. By downloading, copying, installing or using the

software you agree to this license. If you do not agree to this license, do not download,

install, copy or use the software.

License Agreement

For Open Source Computer Vision Library

(3-clause BSD License)

Copyrights: cytena GmbH, 2014-2018

Georges-Koehler-Allee 103

D-79110 Freiburg

Germany

Email: [email protected]

Internet: www.cytena.com

Amtsgericht Freiburg

HRB 711600

VAT: DE295807841

Volksbank Freiburg

IBAN: DE79680900000036726202

BIC: GENODE61FR1

single-cell printer user manual - Molecular Devices

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