x.sight user manual - molecular devices

x.sight

user manual

cytena GmbH

Supporting models

c.sight

b.sight

f.sight

x.sight - user manual

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16th edition English 02/2019 Documentation © 2018-19 cytena GmbH, Freiburg, Germany All rights reserved. Without the written consent of the publisher this manual or any part of it may not be in any form reproduced or distributed using electronic, mechanical or chemical processes. It is possible that the present manual still contains typographical or printing errors. The information in this manual however, is regularly reviewed and corrections are made in the next output version. For technical or typographical errors, we provide no liability for the nature and consequences thereof. All trademarks and copyrights are acknowledged. Printed in Germany. Changes in terms of technical progress are subject to change without notice.


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Content

1 About this manual .......................................................................................................... 5

2 Electrical and chemical risks and safety ........................................................................... 6

3 Safety labels ................................................................................................................... 9

4 Technical information ................................................................................................... 10

4.1 General information ....................................................................................................... 10

4.2 Electrical information ..................................................................................................... 10

4.3 Detection camera and optics information ..................................................................... 10

4.4 Compliance information ................................................................................................. 11

4.5 Embedded PC information ............................................................................................. 11

4.6 Peripheral hardware ....................................................................................................... 11

4.7 Optional barcode scanner .............................................................................................. 12

4.8 Environmental conditions .............................................................................................. 12

4.9 Device materials ............................................................................................................. 12

4.10 Cartridge materials and properties ................................................................................ 13

4.11 Compatibilities ................................................................................................................ 13

4.12 f.sight - fluorescence details ........................................................................................... 13

4.13 Type labels ...................................................................................................................... 14

5 Specification and intended use ..................................................................................... 15

5.1 c.sight ............................................................................................................................. 15

5.2 b.sight ............................................................................................................................. 15

5.3 f.sight .............................................................................................................................. 15

6 Scope of delivery .......................................................................................................... 16

7 Hardware overview ...................................................................................................... 17

8 Installation requirements ............................................................................................. 18

8.1 Transport and lifting ....................................................................................................... 18

8.2 Space requirements ........................................................................................................ 18

8.3 Airflow and cooling ......................................................................................................... 19

8.4 Installation requirements ............................................................................................... 19

8.5 Connections .................................................................................................................... 20

9 Calibration ................................................................................................................... 21

9.1 b.sight calibration ........................................................................................................... 22

9.2 f.sight calibration ............................................................................................................ 23


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9.3 f.sight channel mapping ................................................................................................. 25

10 Use .............................................................................................................................. 28

10.1 Unpacking a cartridge .................................................................................................... 29

10.2 Sample properties .......................................................................................................... 29

10.3 Load sample into cartridge ............................................................................................ 30

10.4 Lid ................................................................................................................................... 32

10.5 Loading a cartridge......................................................................................................... 33

10.6 Mounting a cartridge ..................................................................................................... 33

10.7 Applying agitation .......................................................................................................... 35

10.8 Substrate carrier ............................................................................................................ 36

10.9 Loading a substrate ........................................................................................................ 37

10.10 Cleaning ...................................................................................................................... 39

10.11 Disposal ...................................................................................................................... 41

11 Maintenance ................................................................................................................ 42


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1 About this manual

Before putting the x.sight device into operation, please read the instructions given within

this manual carefully and keep this document safe. This manual contains information about

the device hardware and device usability. The manual is containing hints and warnings

(caution) helping to guide the user and show him/her where to pay special attention. These

are color coded like shown below:

HINT: Hints shall help the user to understand certain controls or situation better.

Hints can also contain practically relevant tips and tricks.

DANGER: this is a safety relevant text. Danger indicates an imminently

hazardous situation which, if not avoided, will result in death or serious injury. This

signal word is limited to the most extreme situations.

WARNING: this is a safety relevant text. Warning indicates a potentially

hazardous situation which, if not avoided, could result in death or serious injury.

CAUTION: this is a safety relevant text. Caution indicates a potentially hazardous

situation which, if not avoided, could result in minor or moderate injury. It may also

be used to alert against unsafe practices.

Safety statement

If the equipment is used in a manner not specified by the manufacturer, the protection

provided by the equipment may be impaired.

Disclaimer of warranty

The x.sight device is designed for research and may not be used for diagnostic

applications.

This is the user manual of the x.sight. Please note that no warranty of any kind will be

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

The x.sight device 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.).


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2 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 180 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!

Hand injuries!

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 carrier of the device except the designated

(substrate in the substrate holder). The Lid of the device needs to be closed during

operation. In an emergency, the drive can be stopped by opening the lid of the device.

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

x.sight instrument is not in operation.

In any case, DO NOT bypass the mechanical and electrical safety installations (safety

switches, etc.). Otherwise, the safety cannot be warrantied. You risk health and safety.

Do not use other power cords than those delivered with the device. If you want to use other

(e.g. longer) cables, make sure they meet the appropriate safety standards. Have cables

checked and approved by your local safety officer before connecting them to the device.

WARNING:

Biohazard! Environmental issues!

Only place the x.sight in a closed room, on a level surface. It is advisable to place the

device in a biological laboratory in a laboratory bench or in a biosafety cabinet.

The x.sight can be used to work with potentially dangerous biological samples. The

wearing of gloves and lab coats is therefore required. Inform yourself about the dangers

of the biological samples in advance. Contact your laboratory manager for respective

working instructions in your laboratory.

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

issues are:

• Temperature: 5°C - 40°C

• Humidity: 10 - 80 % (non-condensing)

• Max. altitude: 2’000 m (above normally zero)

The cytena GmbH does not guarantee the proper operation of the x.sight device, should

it be operated outside of the above ambient conditions.


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WARNING:

Laser radiation! – Laser class 1

Laser class 3B product inside!

wavelength: 473 ± 1 nm | output power: 100 mW ± 5%

The f.sight is declared laser class 1, but it contains a class 3B laser with the following

properties: wavelength 473 ± 1 nm, optical output power 100 mW ± 5%. The laser radiation

measured outside the protective housing is does not exceed radiation limits of class 1

under normal operation.

This embedded laser is subject to protection class 3B according to DIN EN 60825-1: 2015

and 21 CFR 1040.10. The accessible laser radiation is dangerous for the eye and in

special cases also for the skin. Diffused scattered light is generally considered harmless.

The f.sight contains appropriate security features to protect the user from the laser beam.

The laser is only switched on when the software is started and the lid of the device is

completely closed. Correct closure of the lid is monitored by a two redundant magnetic

Reed-switches coupled to a fail-safe relay. The lid is non-transparent at the laser

wavelength.

WARNING: Do not bypass the lid security switches.

WARNING: Never open the device while it is switched on. Opening the protective housing

and servicing the device and the laser must only be carried out by a qualified cytena

employee.

Become familiar with the safety regulations in dealing with lasers according to DIN EN

60825-1: 2015 and 21 CFR 1040.10 and 1040.11. Also contact your laboratory safety

officer for information.

WARNING:

Heavy loads (>30 kg)

The x.sight devices weigh between 30 kg and 35 kg. Lifting heavy loads can result in injury.

Do not lift the device yourself, but always with the help of a second person. Do not lift

and/or carry the device if you have health problems or disabilities. Inform yourself at your

responsible laboratory supervisor or safety officer about possible further risks.


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3 Safety labels

The following safety labels can be found on the rear side of the devices:

For the f.sight the following laser safety labels can be found on the lid:


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4 Technical information

4.1 General information

c.sight b.sight f.sight

Width [mm] 600 600 600

Depth [mm] 400 400 400

Height [mm] 240 240 240

Weight [kg] 30 32 35

Power sockets required 2x 2x 2x

4.2 Electrical information


Input Voltage [VAC] 100-240 100-240 100-240

Input voltage frequency [Hz] 50/60 50/60 50/60

Max. input current [A] 1.4 1.2 1.4

Max. power consumption [W] 140 120 135

Input fuses 2x 3.15 A

quick react

2x 3.15 A

quick react

2x 3.15 A

quick react

Internal voltage levels [VDC] 12, 24 12, 24 5, 12, 24

Piezo driver voltage [VDC] 180 180 180

LED source 1 (DQC) white

1 W

white

1 W

white

1 W

LED source 2 (Detection) blue

3.0 W

blue

4.0 W

red

3.0 W

Embedded Laser source 1 - - 473 ±1 nm

100 mW ±5 %

4.3 Detection camera and optics information


Camera type CMOS CMOS CMOS

Sensor type monochrome monochrome monochrome

Resolution [pixels] 1280x1024 2056x1542 1280x1024

1936x1216

Field of view [µm] 400x300 200x150 400x300

Optical magnification 11x 20x 10x

Numerical aperture 0.01 0.28 0.14

Depth of focus [µm] 55 10 30


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4.4 Compliance information


CE mark yes yes yes

CB mark yes yes yes

QPS mark yes yes yes

RoHS compliant yes yes yes

WEEE yes yes yes

Laser protection class

DIN EN 60825-1: 2015

FDA 21 CFR 1040.10

- - Class 1

4.5 Embedded PC information

OS Win7 (x64) Professional

CPU Intel Core i7 6700 @ 3.4 GHz

RAM 8 GB DDR-4

Hard drive 240 GB SSD, SATA

Partition C:\ (OS) 100 GB

Partition D:\ (data) 130 GB

External interfaces

1x HDMI

1x Displayport (DP++)

2x GigE LAN

4x USB-3.0

4.6 Peripheral hardware

The x.sight will be delivered including keyboard, computer mouse and monitor. Customers

may exchange this hardware by internal peripheral standards. The following conditions

shall be met:

Monitor resolution min. 1920x1080 (HD)

opt. 1920x1200 (full-HD)

Compatible monitor interfaces HDMI

DisplayPort (DP++)

Compatible keyboard interface USB-2 / USB-3

Compatible mouse interface USB-2 / USB-3

Handheld barcode scanner USB-2 / USB-3


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4.7 Optional barcode scanner

Model Inatek BCST-20

Handheld Yes

Cable interface USB-2.0 (charging + data transfer)

Wireless interface WLAN 2.4 GHz band

Integrated battery 1800mAh-Li-Ion

Supported code standards

Coda bar, Code 93, MSI, Code 128,

UCC/EAN-128, Code 39, EAN-8, EAN-13,

UPC-A, UPC-E, ISBN, Industrial 25,

Interleaved 25, Standard 25, 2/5 Matrix;

USPS, FedEx

4.8 Environmental conditions

Emission sound pressure level < 70 dB(A)

Min. environment temperature [°C] 5

Max. environment temperature [°C] 40

Min. environment rel. humidity [%] 10 (non-condensing)

Max. environment rel. humidity [%] 80 (non-condensing)

Max. altitude above normally zero 2’000 m

Indoor use yes

Outdoor use no

Dust-proof no

Wafer-proof no

4.9 Device materials


Housing Powder coated

stainless steel

Powder coated

stainless steel

Powder coated

stainless steel

Base frame Aluminum Aluminum Aluminum

Housing lid PMMA PMMA PMMA

Piston Guide Stainless steel Stainless steel Stainless steel

Agitation adapter Stainless steel Stainless steel Stainless steel

Cartridge reservoir PMMA PMMA/PEEK PMMA

Cartridge chip Si-Glass Si-Glass Si-Glass

Tubing vacuum shutter Tygon™ Tygon™ Tygon™

Tubing agitation Tygon™ Tygon™ Tygon™


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4.10 Cartridge materials and properties

Reservoir PMMA

Microchip Silicon, Glass

Glue USP class VI epoxy

Coating N.N.

Seal foil bag Tyvek® 1073B

Sterilization method UV light (CWL 254 nm)

TSE / BSE free yes

Animal component free yes

4.11 Compatibilities

+ Compatible (+) Conditionally compatible

- Not compatible nt Not tested

Stainless

steel Aluminum PMMA PEEK Tygon™

Water + + + + +

Ethanol + + + + +

Isopropanol + + + + -

Acetone + + - + -

UV (CWL 254 nm) + + + + +

Steam autoclave (121°C) + + - - +

Vaporized hydrogen

peroxide (VHP gas) + + nt nt nt

Bleach solutions (+) (+) - nt nt

4.12 f.sight - fluorescence details

Amount of lasers 1

Excitation wavelength(s) 473 ±1 nm

Emission wavelength(s) 505 - 555 nm

Optical laser power 0 - 100 % (free tunable)

Fluorescence exposure time 0.1 - 20 ms (free tunable)

Tested dyes Green GFP ; Cell-Tracker Green ; FITC ;

Calcein AM ; DyLight ;


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4.13 Type labels

The following type labels are applied on the devices, depending on their model:


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5 Specification and intended use

5.1 c.sight

The c.sight is a high-tech, laboratory instrument for handling of small liquid volumes of

biological samples. Its purpose is the isolation and deposition of single, living cells from a

cell suspension into various substrates. Substrates can either be used for cultivation of

those cells into colonies or for genetic analysis of the single-cell genomes. The c.sight

consists of a computer, driving electronics, a linear stage, optics and cameras and a piezo-

electric driven droplet generator. The benchtop instrument comes with a screen, keyboard

and mouse. It is used in laboratory environment and specifically inside biosafety cabinets.

5.2 b.sight

The b.sight is a high-tech, laboratory instrument for handling of small liquid volumes of

biological samples. Its purpose is the isolation and deposition of single, living bacterial

cells from a cell suspension into various substrates. Substrates can either be used for

cultivation of those bacterial cells into colonies or for genetic analysis of the single-bacteria

genomes. The b.sight consists of a computer, driving electronics, a linear stage, high-

resolution optics and cameras and a piezo-electric driven droplet generator. The benchtop

instrument comes with a screen, keyboard and mouse. It is used in laboratory environment

and specifically inside biosafety cabinets.

5.3 f.sight

The f.sight is a high-tech, laboratory instrument for handling of small liquid volumes of

fluorescent biological samples. Its purpose is the isolation and deposition of single, living

cells from a cell suspension into various substrates based on their fluorescence emission.

Substrates can either be used for cultivation of those cells into colonies or for genetic

analysis of the single-cell genomes. The f.sight consists of a computer, driving electronics,

a linear stage, optics, fluorescence excitation laser and filters, cameras and a piezo-

electric driven droplet generator. The benchtop instrument comes with a screen, keyboard

and mouse. It is used in laboratory environment and specifically inside biosafety cabinets.


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6 Scope of delivery

The following parts will be delivered (unless otherwise agreed):

1x x.sight device

1x Monitor (full-HD, DisplayPort / HMDI)

1x Computer mouse (USB-2, wired)

1x Keyboard (USB-2, wired, country specific layout)

1x Optional barcode scanner

2x Power cord (black, 1.5 m, country specific plug)

1x DP++/HDMI cable (2.0 m)

1x x.sight user manual

1x x.sight software manual

1x x.sight practical guide

1x Torque hex key (1.5 mm, stainless steel)

10x Hex screws (M2x13, stainless steel)

Hint: Please check the condition of the transport box directly at its delivery and

before unpacking the x.sight. It is strictly recommended to claim any transport

damage immediately to the transport company! Any damage done to the x.sight

during transport may then be charged to the transport company.


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7 Hardware overview

The following main components are relevant for the user.

Description:

A. Power button B. Lid C. Substrate carrier D. P9 dispenser module E. Vacuum shutter supply F. Agitation supply G. Security switch for lid


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8 Installation requirements

8.1 Transport and lifting

The x.sight device weighs between 30 kg and 35 kg (depending on model). Please always

use two persons to lift and carry the device. Grab the device underneath the housing, close

to the feet frame. Keep the device in a stable and even position. Do not carry it over head.

Do not place it on another side, just on its feet.

8.2 Space requirements

The x.sight device dimensions are 600 x 400 x 240 mm (WxDxH) as shown below in the

left figure. To work with the device in a comfortable manner, it is recommended to include

additional free space around the device. Recommended space requirements are 670 x

450 x 500 mm (WxDxH) as shown below in the right figure.

As the x.sight comes with monitor, keyboard and computer mouse, a free table is required

next to the instrument. Recommended space requirements to set up monitor and controls

are 600 x 400 x 500 mm (WxDxH).


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8.3 Airflow and cooling

The x.sight devices contain active air cooling with environmental air. The embedded fans

produce an air flow of 1 m²/min. Air inflow is through bottom grooves, air outflow through

rear grooves.

Air cooling is used to cool electronics, power supplies and the embedded PC. There is no

airflow around the substrate and dispensing unit. The processing part (plate, cartridge,

etc.) is separated from the cooling airflow by a vertical sheet inside the device.

Airflow is designed in a way that standard biosafety cabinet airflow is not disturbed.

8.4 Installation requirements

The device should be installed on an even, flat surface that can carry the load. This can

be a laboratory table, desk or similar. Installation inside a biosafety cabinet is also possible.

Hint: When installing the device inside a biosafety cabinet, make sure that it does

not cover the cabinets air flow grooves. Otherwise, the cabinets air flow might be

disturbed and biosafety class might no longer be guaranteed.

Also make sure, that the following requirements are met:

• Do not place the device next to strong heat sources like lamps, heaters, etc. High temperature may deform the protective lid and may compromise the device functionality.

• Keep the device separated from sources of stronger vibrations like e.g. centrifuges or shakers. Vibrations may negatively influence cell detection during operation (camera vibrates).

• When bringing in the device from outside, let it adapt to room temperature and conditions, before switching it on and bringing it into operation. Axes, dispenser and electronics may work differently when the device is cold.

• Make sure that no condensed water is on the device before starting it. This may happen when bringing the device from a cold environment to a warm environment. Condensed water may lead to electrical failure or similar errors.


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8.5 Connections

Connect all required peripherals according to the following instructions. The rear

connectors for the embedded PC provide the following interfaces:

A. DisplayPort (PD++) B. GigE LAN C. HDMI D. USB-3.0 (SS) E. Main power cord F. Main power switch

Connect the peripherals to the following interfaces (A-F):

• Monitor to (A) via DP++ or (C) via HDMI

• Computer mouse and keyboard to (D)

• Power cord to (E)

• Optional: LAN cable to (B)

As soon as all connectors are properly installed switch on the screen. Turn on the main

switch (F) and start the device by pressing the power button at the front side. The

instrument is operable after appearance of the Windows 7 desktop.


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9 Calibration

The models b.sight and f.sight require optical calibration before operation. Calibration has

to be done for each new cartridge which is loaded. The c.sight is set up during installation

and does not require regular re-calibration.

For both models it is essential to calibrate the position of the cartridge nozzle in the image

plane. Three fine-tune screws are provided: (A) focus, (B) lateral position and (C) vertical

position.

The influence on the image respective to which screw is used is the following:


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Besides correct location of the nozzle, camera exposure time, background subtraction

threshold and – for f.sight only – laser intensity are more parameters to tune. The following

chapters deal with correct calibration of the models b.sight and f.sight prior to operation.

For both models, calibration has to be performed every time a new cartridge is taken or

the device has been moved.

9.1 b.sight calibration

Due to its high-resolution optics, the field of view of the b.sight camera is considerably

smaller than on the c.sight or f.sight. To enable detection, the cartridge nozzle has to be

centered inside the field of view.

Follow the instructions after instrument and software start:

1. In software “print” tab, choose “toggle enlarged view” button to display an enlarged view of the camera window. Bring the window to the front.

2. Open b.sight lid

3. Mount a cartridge and lock the dispenser in operation position

4. Use the lateral (B) and vertical (C) fine-tune screws to move the nozzle in the image center. Check in enlarged view camera window

5. Use the focus (A) fine-tune screw to bring the image in focus. Make sure it looks like the reference image below:

Fig.: b.sight reference image with bacterial cell (marked by arrow)

6. Close the lid and return to software

7. Go to “Options” and set the value for threshold.


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8. Return to “Print” tab and run “Collect sample” until you have a suitable amount of data points collected (e.g. 20-50).

9. Check if sample cells are correctly detected by clicking on sample points in the graphics plot. You will see enlarged thumbnails of the target cell below that data point. Make sure the cells outline (shape) is fully detected.

10. If the contour is too large compared to the object, increase threshold value

11. If the contour is too small compared to the object or if it is incomplete, readjust the focus by using fine-tune screw (A).

12. If the camera appears dark, go to “Options” tab and increase the “Exposure” value until you see the image. Make sure it is comparable to the reference image.

Hint: Please note, that focus adjustment is critical for correct detection of bacteria.

When the system is defocused, the software will not be able to detect bacteria

cells. In order to find the optimal focus, try to bring the alignment marks – circle

and cross – besides the nozzle in good focus.

9.2 f.sight calibration

The f.sight features a spot laser in addition to bright field detection. The laser spot is

centered on a specific area in the camera field of view. To enable fluorescence detection,

the cartridge nozzle has to be centered over the laser spot. Only such, the fluorescent

cells can be excited for sorting. Follow the instructions after instrument and software start:

1. Open the lid, a bigger screen showing the nozzle will appear.

2. Use the three fine-tune screws: (A) focus, (B) lateral position and (C) vertical position


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to center the nozzle in the camera window using the target contour:

3. First find the right focus by using screw (A). If you see the ring and the plus in focus you are close to a good focus. Make sure you can see the cells clearly in the cartridge.

4. In “Settings” tab set “Threshold” to 15

Hint: Please note, that focus adjustment is critical for correct detection of cells.

When the system is defocused, the software will not be able to detect cells and

their fluorescent signals. In order to find the optimal focus, try to bring the

alignment marks – circle and cross – besides the nozzle in good focus.

Hint: Please note, that laser adjustment is critical for correct fluorescence

readout. The software can only excite and read fluorescent signals, when laser

spot and cartridge nozzle are inline.


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9.3 f.sight channel mapping

The two independent cameras have to be aligned by software before the system is used

to sort cells. This ensures that both channels (bright field and fluorescence) match with

each other. The alignment mode is automatically started with a dialog when either [Collect

Sample] or [PLAY] buttons are pressed.

It is recommended to run this alignment once for each new cartridge. Although the dialog

is always displayed, you can skip this step by pressing [No] every time. The algorithm will

then use the settings from the run before. Choose [Yes] to start the alignment process.

The algorithm will run collect samples in the background and perform the matching on the

acquired cell images from both channels. You will get the results displayed in the dialog:

On the left side, the result image of the cell cam is shown (bright field and fluorescence

channel combined). On the right side the matching result will be displayed (background

isolated fluorescence signal plus contour of the cell from the bright field channel).


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First, check the black picture with blue contour and the white object. Make sure that

contour and object superpose each other like shown in (A) below. Cases where both do

not superpose (B+C), where only the blue contour is present but no object (D) or where it

is unclear if the blue contour belongs to a specific object (E+F) should be discarded.

Second, check the left bright field + fluorescence picture. Make sure that object (cell) in

bright field is superposing the fluorescence signal (A). Discard cases where object and

fluorescence signal only partly overlap (B) or do not overlap all at (C).

A real mismatch case (example B from above) would look like:

Confirm whether the algorithm has performed the matching correctly or not. In case of a

correct matching, confirm by clicking [Ok] in the dialog. If you are not sure about the result

and/or want to perform the matching again, press [Retry]. You can also exit the dialog and

return to the main window by pressing [Cancel].


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The compensated image (as overlay) should like the figure below:

Fig.: Overlay image after successfully completed channel mapping

Hint: Please note, that focus adjustment is critical for correct detection of cells.

When the system is defocused, the software will not be able to detect the cells

outer contours correctly. In order to find the optimal focus, try to bring the

alignment marks – circle and cross – besides the nozzle in good focus.


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10 Use

The following sections explain the use of the device and its components in detail. The

single steps described hereafter briefly explain the order in which the device is prepared

for an experiment. Please note that it is mandatory to previously install and calibrate the

device. It is further assumed that you have prepared your cell sample.

Preparation work flow:

1. Start the device by pressing the main switch (front left)

2. Login to Windows

3. Use desktop icon to start the software

4. Choose blank substrate or preconfigured experiment from start screen

5. Open the lid

6. Swing out the P9 dispenser

7. Unpack a cartridge and load your sample to it

8. Mount the cartridge to the P9 dispenser

9. Swing back the dispenser until it locks magnetically

10. Attach a tip to the agitation adapter

11. Place the tip inside the cartridge reservoir and lock the adapter magnetically

12. Place your plate on the substrate carrier

13. Check that plate is correctly placed and P9 is locked

14. Close the lid

15. Configure your experiment in software

16. Press [Start] button in software

Work flow after final experiment:

1. Close the software

2. Shut down the PC (via Windows “shut down”)

3. Open the lid

4. Unload the plate from the carrier

5. Detach agitation tip and dispose it

6. Dismount agitation adapter and clean it

7. Swing out the P9 dispenser

8. Unload the cartridge and dispose it

9. Dismount piston guide and clean it

10. Clean substrate carrier and DQC waste plate

11. Clean device and lid


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10.1 Unpacking a cartridge

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

top side and remove cartridge by hand (wear gloves!).

Caution: Do not touch the chip face side respectively the nozzle area. Mechanical

impact on the face side may damage or destroy the exterior coating!

10.2 Sample properties

Your sample has to meet the following conditions to be compatible to the x.sight

technology

characteristic single-cell suspension

Cell types Eukaryotic (c.sight, f.sight)

Prokaryotic (b.sight)

Min. cell size 5 µm (c.sight, f.sight)

0.5 µm (b.sight)

Max. cell size 35 µm (c.sight, f.sight)

5 µm (b.sight)

Max. concentration 5e6 cells/ml

Ideal concentration 1e6 cells/ml

Min. sample volume to be prepared 10 µl

Ideal sample volume to be prepared 1 ml

Sample volume used per 100 single cells approx. 1 µl


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10.3 Load sample into cartridge

Use a standard hand pipette to load the prepared suspension sample into the cartridge.

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

self-fill via capillary forces. Please note that at least 10 µl sample is required to ensure

proper capillary filling. Add 0.5 µl sample per 100 single cells you want to deposit (e.g. for

each 96-well plate to be filled with single cells, calculate 0.5 µl additional sample).

The following schematic shows you how to dispense the sample into the cartridge reservoir

using a hand pipette. Dip the loaded pipette tip inside the reservoir until the bottom is

reached. Dispense slowly while moving the tip out of the reservoir.


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There are several mistakes that should be avoided when filling a cartridge.

• 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. This likely when using filter tips.


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10.4 Lid

The loading area for substrate and cartridge can only be accessed by opening the lid (A)

on the right front side of the x.sight. The lid it hold by two friction lock hinges (B) that keep

it in every position the user puts it. To open and close the lid, use its handle (C).

The lid has two major functions:

• Protection of sample and substrate from the environment and the user

• Protection of the user from moving parts (axes) and laser radiation (f.sight only)

To ensure that the user is protected from moving parts, the lid contains a security switch.

It is located in the top right corner inside the lid. Opening the lid interrupts the power supply

for moving parts, stopping them immediately. When closing the lid, make sure that it is

fully closed to power up the moving parts and continue operation.


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10.5 Loading a cartridge

The dispenser is mounted on a hinge. This allows it to fold outwards and so to load or

change the cartridge easily. The easiest way to access it is to fold the dispenser completely

outwards, as shown in the figure below. When folding back, make sure that the dispenser

snaps in the magnetic lock. This ensures the correct alignment with the camera so that the

system is ready for use.

To load or change the cartridge follow the steps:

A. Open the lid B. Swing out the P9 dispenser C. Load the cartridge (see mounting instructions for details) D. Swing back the P9 dispenser E. Until it snaps in the magnetic lock F. Close the lid

10.6 Mounting a cartridge

In order to mount a new cartridge, the lid needs to be opened and the dispenser must be

folded outwards. 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. Use the torsion hex tool provided by

cytena.

Caution: Do not use tools other than those provided with the device!

Inappropriate use of screws or screwing tools may damage the dispenser!


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The following schematic shows correct orientation of the cartridge. Turn the cartridge

such that the microfluidic 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.

Insert the M2x13 hex screws into the front top and rear bottom hole in the cartridge as

shown below (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.

A correct mounted cartridge with tightened screws (A):


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10.7 Applying agitation

The agitation system prevents sedimentation of cells inside the cartridge reservoir over

time. This ensures a continuously homogeneous distribution of cells inside the chip.

Please follow the steps to ensure correct function of the agitation system:

A. Detach the adapter from its magnetic mount B. Pick up a pipette tip from the rack or box. Make sure it tight fits to the adapter. C. Place the tip inside the cartridge reservoir D. Reattach the adapter to its magnetic mount

After these steps have been completed, the system can be activated. Refer to the x.sight

software manual to learn how to start the agitation in the software.

The agitation adapter is supporting the following tip models:

Eppendorf epT.I.P.S 2-200 µl


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10.8 Substrate carrier

The x.sight standard substrate carrier (A) can carry and process skirted SLAS plate

formats with 96-well and 384-well. For deep-well or low-profile substrates an additional

carrier may be required. For partly skirted or non-skirted plates (e.g. PCR plates, low

volume plates) an additional adapter insert (C) is required.

To load a substrate, chose the respective carrier and load the plate manually. The standard

carrier comes with four spring loaded clips (two on the rear and two on the right side). Clip

the skirted plate into the carrier, following the schematic description below.


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10.9 Loading a substrate

The following section deals with substrates and substrate loading.

Warning: Please note the maximum clearance between substrate holder and

dispenser. The inserted well-plate must not be higher than this clearance,

otherwise it will collide with the dispenser and / or the housing.

Such collision will likely damage the device and must be avoided.

Please ask the support for adjustment of the clearance when planning to use

higher well-plates.

All supported SLAS skirted substrates can be mounted by simply clipping them in the

foreseen carriers on the baseplate. The following schematic demonstrates the easy way

of clipping with a plate.

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

1. Orientate the well plate with well A1 located to the front 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:


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• Well A1 does not face front left side of the carrier

• 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 as guideline to what can go wrong:

Caution: Avoid incorrect placement of the substrate (typical situations shown in

the schematic above). Incorrect placement will very likely lead to physical collision

between dispenser and substrate and/or housing!

Only use supported plate types and supported carriers and adapters!

Before starting your experiment, assert that the plate and its carrier are placed

correctly. The device has not sensors and will not detect incorrect mounted plates!


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10.10 Cleaning

Please follow the cleaning instructions carefully.

Warning: Use only specified cleaning agents and follow the instructions in this

manual. The use of unspecified cleaning agents and cleaning procedures can lead

to damage to the device or its failure. Material compatibilities are listed in section

4 (technical information).

When being in regular use, cleaning of the device on a regular basis is recommended to

avoid contamination. Cytena recommends to use 70%-Ethanol as cleaning solution. You

can spray or wipe the surfaces.

The follow table provides information on cleaning of specific parts:

Housing Spray, wipe, UV

Lid Spray, wipe, UV

Optics / lenses Wipe with special optics tissue only, UV

Substrate carrier Wipe, UV

Axis system Wipe, UV

DQC waste plate Wipe, UV

Caution: On the loading dock, there are slits that are required to move the plate

into the device. Do not spray or spill liquid inside the device through these slits. In

case larger amounts of liquid have entered the device, please shut it down, detach

the power cord and contact the technical support.

In addition agitation adapter (A) and piston guide (B) can be disconnected from the device

for cleaning.


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To clean the DQC waste container (red circle) pull the slider towards you (red arrow) and

clean the container. Push it back to the top position after cleaning.

Please follow the protocol listed below to clean the x.sight device after having completed

your single-cell printing experiment runs for the day. The protocol refers to 70%-Ethanol

as the standard cleaning and disinfection agent.

1. Complete your printing experiment 2. Unload your substrate (well-plate) 3. Detach agitation adapter, unplug and dispose tip 4. Unload cartridge and dispose it 5. Unplug piston guide from P9 dispenser 6. Unplug vacuum tubing (LuerLock) from x.sight device 7. Place piston guide incl. tubing in 70%-Ethanol for 5 min 8. Clean wipe agitation adapter with 70%-Ethanol


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9. Clean the DQC waster container 10. Spray and/or clean wipe the device 11. Dry all parts on a clean wipe before reassemble the system

Hint: You may also use your preferred solution for cleaning and disinfection. For

a list of material compatibilities please refer to section on technical information in

this manual.

10.11 Disposal

The pipette tip used for agitation and the cartridge used for single-cell printing are one-

way disposables. They have to be disposed according to your laboratory rules.


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11 Maintenance

The x.sight devices are installed by a qualified field service engineer or field application

specialist. As part of the installation procedure, a calibration is performed.

It is recommended to perform a preventive maintenance (PM) on a yearly basis. The PM

includes a comprehensive review of all system components and features. Defective or

worn parts can be replaced. Refer to the service options available from cytena or your

local distributor.

(Re-) calibration of P9 dispenser, optical components, LEDs, lasers and axis system must

only be performed by a qualified field service engineer. Do not open the device yourself.

Do not perform calibration yourself. Refer to the technical support available from cytena or

your local distributor.


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Brands and Trademarks

The cytena logo, the name “cytena”, the product series name x.sight™ and the product names

c.sight™, f.sight™ and b.sight™ are registered trademarks of cytena GmbH in Europe and/or

other regions. Without written permission from cytena GmbH it is not allowed to use our

registered trademarks and/or logos.

cytena GmbH

Neuer Messplatz 3

D-79108 Freiburg

Germany

Email: [email protected]

Website: www.cytena.com

Tel: +49 (0) 761 70 88 900

Amtsgericht Freiburg

HRB 711600

VAT: DE295807841

Managing Director / CEO:

Dr. Jonas Schoendube

http://www.cytena.com/

x.sight user manual - molecular devices

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