Nanolive 3D Cell Explorer-fluo User manual

3D Cell Explorer-uo
User Manual

1. Safety Notes 3
• 3D Cell Explorer-uo 3
• CoolLED illumination system 5
2. Maintenance & Care 6
• 3D Cell Explorer-uo 6
• CoolLED’s illumination system 7
3. Introduction 8
• Instrument Description & Main Features 8
• Typical Field of Applications 9
• 3D Cell Explorer-uo terminology 10
• CoolLED illumination system terminology 11
4. Technical Specifications Sheet 12
5. Computer Technical Specifications 13
6. Start-Up 14
7. Handling 29
8. Sample Preparation 34
9. Cleaning procedure for optical elements 35
10. Software STEVE 37
11. Troubleshooting 58
12. Compliance 60
Contents

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1. Safety Notes for the 3D Cell Explorer-uo
• Verify that the computer is plugged and grounded earthed
with its own power supply during usage with your 3D Cell
Explorer-uo.
• Switch the 3D Cell Explorer-uo OFF before connecting it to
a power supply.
• Disconnect and lockout the power supplies of the 3D Cell
Explorer-uo before completing any maintenance work tasks
or making adjustments.
• Do not use electrical equipment in wet conditions or damp
locations.
• Do not clean tools with ammable or toxic solvents.
2. Supply the 3D Cell Explorer-uo only with the provided power
supplies.
3. No special laser safety required when operating the 3D Cell
Explorer-uo (Class I laser).
For measurements, the 3D Cell Explorer-uo uses a Class I laser.
A Class I laser is harmless for your eyes under any circumstances and
thus does not require laser safety goggles when properly operated.
By no means should the user open the covers and thus have access
to the laser path within the microscope frame without a clear
authorization from Nanolive.
Operating the microscope in non-appropriate conditions may then
be a risk. It is thus forbidden for a non-authorized user to open the
covers and manipulate the laser.
Thank you for purchasing the Nanolive 3D Cell Explorer-uo. Before using your 3D Cell Explorer-uo and the CoolLED illumination system, carefully
read the safety notes to ensure safe handling and usage of the device at all times. Failure to do so may result in personal injury or damage to other
items.
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1. Safety Notes
3D Cell Explorer-uo

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4. Contaminations
The user has the responsibility to avoid disastrous contaminations
by biological samples. Nevertheless, contamination might occur
when working with biological samples. Please ensure appropriate
cleaning of the 3D Cell Explorer-uo. Please refer to our Main-
tenance and Care notes for proper cleaning instructions.
5. Cleaning
Please note that alcohol is highly ammable, do keep it away from re or
potential sources of electrical sparks, and use it in a well- ventilated room
when using it for cleaning the 3D Cell Explorer-uo or other measures.
6. The 3D Cell Explorer-uo conforms to the requirements of
the Safety Standards as follows:
• EN/IEC 61010-1:2010 Safety Requirements for Electrical
Equipment for Measurement, Control and Laboratory use.
• EN62471:2008 Photo-biological Safety of Lamps and Lamp
Systems/Guidance on manufacturing requirements relating
to non-laser optical radiation safety. No risk
1. Safety Notes
3D Cell Explorer-uo

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7. Safety Notes for the CoolLED illumination system
• UV light may be emitted from this product depending on the
conguration selected. Avoid eye and skin exposure. Never look
directly into the light output beam from the Light Source or
accessories. The emissions could damage the cornea and retina
of the eye if the light is observed directly.
• Always ensure that the Light Source is securely attached to the
microscope with its light guide, prior to turning on the power.
This will minimize the risk of injury and damage.
• If for any reason the Light Source is to be operated when
not attached to a microscope, all personnel should wear eye
shielding and clothing to protect the exposed skin.
• Disconnecting the main supply is achieved by unplugging the
power cord from the power supply block or the Light Source.
Only plug in the power cable, once the Light Source is attached
to the microscope.
• There are no serviceable parts within the Light Source.
Removing any of the screws and covers will result in the safety
of the Light Source being impaired. The DC power supply unit
should be inspected periodically throughout the lifetime of the
system.
• To clean the exterior of the Light Source, use a slightly dampened
cloth with a simple water/detergent solution only. Avoid the
optical surfaces and lenses. Cleaning of optics should only be
carried out using the provided optical swabs with grade isopro-
panol or ethanol. Please note that the DC power supply unit
should be isolated prior to cleaning.
• Safety precautions must always be exercised when using
electrical equipment to prevent operator shock, re hazard or
equipment damage.
• FIRE HAZARD: Do not drape or cover the light source or the light
guide while it is in use. Do not place the light guide on a drape
while it is in use.
8. Compliance:
The light source conforms to the requirements of the Safety
Standards as follows:
• EN/IEC 61010-1:2010 Safety Requirements for Electrical
Equipment for Measurement, Control and Laboratory use.
• EN62471:2008 Photo-biological Safety of Lamps and Lamp
Systems/Guidance on manufacturing requirements relating
to non-laser optical radiation safety. Risk Group 3.
1. Safety Notes
CoolLED illumination system
All warnings may not be applicable depending on the version/wavelength being used.

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Maintenance and Care for the 3D Cell Explorer-uo
1. Carefully, open the shipment box with the top face up to avoid damaging the 3D Cell Explorer-uo.
2. Handle the 3D Cell Explorer-uo from the side or the bottom plate; unplug all cables before moving it.
3. When moving the microscope, carefully carry it without lifting it from the camera and avoid any shocks.
4. Keep the instrument out of direct sunlight, high temperature or humidity, dusty and easy shaking environments.
Make sure the table surface is at, horizontal and rm.
5. All elements (e.g. microscope objective, mirrors, lenses) have been specially and carefully adjusted; please do
not dismount, reposition, or modify them. In such case the warranty becomes automatically void and rework
shall be charged to the customer.
6. Do not block the rotating arm or prevent it to rotate freely.
7. Do not disassemble any parts of the microscope, as this affects the function or reduces the performance of the
microscope. Please be aware that the warranty expires if you remove the covers.
8. Keep the instrument clean, and do not contaminate the optical elements when wiping away the dust on the
instrument. It is highly recommended to leave the red cap in place on the microscope objective when the
microscope is not in use in order to prevent contaminations to come on the optical surfaces.
9. Contaminations on the objective and the mirrors, like ngerprints and oil smudges, could be gently wiped with
the provided cleaning swab and a bit of alcohol (Ethanol or Isopropanol). More information on the cleaning
procedure to be found in section “Cleaning Procedure for the optical elements”.
10. Do not attempt to use organic solvents to clean the microscope. To clean it, use a lint-free, soft cloth slightly
moistened with clear water.
11. During use, if the microscope is splashed by liquid, cut off the power at once, and wipe away the splash.
12. Place the instrument in a cool, dry position. When not using the microscope or carrying it, keep it covered with
the provided dust cover. Furthermore, protect the microscope objective with the red vinyl cover.
2. Maintenance & Care
3D Cell Explorer-uo

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Maintenance & Care for the CoolLED illumination system
1. Carefully unpack the components from the shipping cartons.
2. The CoolLED illumination system requires little or no maintenance throughout its life. There are no eld service-
able parts so there is no need to remove the covers.
3. Cleaning of the external surfaces can be carried out with a mild soap and water solution used to lightly dampen a
lint-free cloth. Ensure that no liquid is allowed to enter the product through vents and panel edges.
Avoid optical surfaces.
4. Cleaning of optical surfaces maybe necessary if debris or nger prints accidentally come into contact with the
excitation lters during installation. In the rst instance remove any loose debris with an air duster (aerosol or
rubber blower).
5. Finger prints or other liquid type contaminants should be removed using standard lens cleaning procedures.
Do not ood the optical surfaces with uid as liquid could enter the product and cause damage.
Care of the light guide
The light guide is a ne, high quality optical instrument. The useful life can be prolonged by following these
simple guidelines.
1. Avoid stretching the light guide, forming congurations, involving sharp angels or kinks, or contact with sharp or
pointed objects. The internal light bers are made of glass, a material that breaks under stress. Fiber breakage
will result in diminished light output.
2. Any inadvertent cut or puncture to the outer wall will render the light guide unsafe for use and must be taken
out of service immediately.
2. Maintenance & Care
CoolLED illumination system

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3. Introduction
Instrument description and main features
Plug & Play systems
• 2 imaging modalities:
- Physical structural imaging (based on refractive index, RI)
- Chemical uorescence imaging
• 3 Fluorescence Channels: 2 congurations available:
- DAPI (Excitation 392 nm / Emission 432 nm), FitC (Excitation 474 nm /
Emission 515 nm), TritC (Excitation 554 nm / Emission 595 nm)
- FitC (Excitation 474 nm / Emission 515 nm), TritC (Excitation 554 nm /
Emission 595 nm), Cy5 (Excitation 635 nm / Emission 730nm)
• 4 Fluorescence Channels:
- DAPI (Excitation 392 nm / Emission 432 nm), FitC (Excitation 474 nm /
Emission 515 nm), TritC (Excitation 554 nm / Emission 595 nm), Cy5
( Excitation 635 nm / Emission 730nm)
Note: TritC and Cy5 excitation lters are exchangeable on channel 3
Please refer to the following link for details on the Filter conguration:
https://www.semrock.com/SetDetails.aspx?id=3297
Likewise, refer to the following link for further data on the LED source:
http://www.coolled.com/product-detail/led-wavelengths/
• Operation modes:
- 2D epiuorescence snap-shot (every second) & time-lapse (up to 96 hours)
- 3D RI snap shot & time-lapse 3D RI & 2D epiuorescence snap-shot (every
3 seconds) & time-lapse (up to 96 hours)
• Co-localisation of Fluorescence (3 channels) and Digital Stains (7 channels at
single acquisition)
• Time-lapse possibility up to 96 hours
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The typical fields of applications for the 3D Cell Explorer-uo
• Merging of uorescence microscopy and 3D cell tomography
• Calibration of the refractive index map with markers (Chemical dyes, uorescent antibodies, protein
labeling with GFP, etc.)
• Combination of structural RI information with chemical information for specic protein/drug tracking,
protein localization and mapping
• long term (over a week) live cell imaging in 3D at high temporal resolution (1 image every 2 sec.) thank
to our technology that generates no phototoxicity
• Powerful partner for advanced image analysis strategies: high resolution and no phototoxicity allows
to obtain more reliable cell measurements in subsequent image analysis
• Possibility to use 10 markers in parallel
3. Introduction
Typical Fields of Applications

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3. Introduction
3D Cell Explorer-uo terminology
Rotating arm
Scanning head
Peripheral mirror
Injection Head USB 2.0 slot Power switch
Power supply
plug port
BNC cable
plug port
Hi-grade stage for
sample screening
Z knob for
optical focusing
Cell container
interface
XY knob for
sample screening
Camera
with USB
3.0 slot
Camera
with USB
3.0 slot
3D Cell Explorer-uo – front view 3D Cell Explorer-uo – side view 3D Cell Explorer-uo – rear view

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3. Introduction
CoolLED illumination system terminology
CoolLED illumination system – front view
CoolLED illumination system – rear view
CoolLED illumination system – side view 1 Liquid Light Guide (LLG)
CoolLED illumination system – Top/Side View
Not needed BNC plugs
Excitation lters slots
Global Trigger

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4. TechnicalSpecicationSheet(TSS)
3D Cell Explorer-uo & CoolLED illumination system
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Specification 3D Cell Explorer-uo /
Holotomography
Specifications CoolLED illumination system /
Epifluorescence
Dimensions (width x depth x height in mm) 380 x 170 x 445 77 x 186 x 162
Weight (in kg) 8 4
Environmental Operating Conditions Permissible ambient temperature, and can be
operated between 20° C and 30° C
5-35°C
Voltage ranges 100-240 VAC / 50-60 Hz / 1.0-0.5 A 110-240 VAC / 50-60 Hz / 2 A
Camera USB3.0 CMOS Sony IMX174 sensor
Quantum Eciency (typical) 70 % (at 545 nm)
Dark Noise (typical) 6,6 e¯
Dynamic Range (typical) 73,7 dB
Microscope Objective (MO) Dry objective / 60x magnication / NA 0.8
Illumination Source Class I laser low power (λ=520 nm,
sample exposure 0.2 mW/mm2)
(Class I. No eye protection needed)
High speed switchable <100 μs
Lifetime >20’000 hours each channel
Field-of-View (FoV) 90 x 90 x 30 μm390 x 90 μm
Lateral resolution 200 nm 400 nm
Axial resolution 400 nm -
Tomography frame rate (acquisition) 0.5 fps 3 fps each channel
Self-adjusting time < 90 seconds (Depending on sample mounting
medium thickness)
Channels Up to 7 simultaneously 3: DAPI (blue), FitC (green), TritC (orange)
3: FitC (green), TritC (orange), Cy5 (red)
4: DAPI (blue), FitC (green), TritC (orange) and
Cy5 (red) (Note: TritC and Cy5 excitation lters
are exchangeable on channel 3)

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5. ComputerTechnicalSpecications
3D Cell Explorer-uo
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The 3D Cell Explorer-uo is provided without a computer. Therefore, before you acquire a computer, please be aware of the minimum
requirements listed below which are absolutely necessary for correct operation of the instrument.*
CPU: 1.5 GHz or faster multi-core CPU
CPU Memory: minimum 8 GB DDR3 RAM
Hard disk: 450 MB free space for STEVE and 1 TB for acquisition data
GPU**: NVIDIA GeForce GTX 780 or later NVIDIA GTX or Quadro GPU of the Kepler or newer architectures
GPU RAM: minimum 4 GB dedicated
USB ports: 3 ports, minimum one USB 3.0 port
Operating System: 64-bit versions of Windows Vista, 7, 8 or 10
(Updated drivers: you need to have the latest Nvidia Graphics Card drivers, USB 3.0 drivers and fully updated Windows Operating System)
In addition: Internet access for STEVE installation and for the newest updates.
*We are happy to provide you with a list of computers and laptops that we have tested and validated. Please contact us.
**If you are unsure about the minimal requirements, please contact us for an updated list of tested GPU cards.
Please pay particular attention to the graphics card requirements. The full version of STEVE performs heavy calculations using a Nvidia GPU while
the microscope is acquiring data. For this reason, please refer to the minimum required specications of the Nvidia GPU above. In addition, you
need at least one USB 3.0 port for camera image acquisition.

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6. Start-up
Components
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The 3D Cell Explorer-uo package contains the following:
(1) the 3D Cell Explorer-uo
(2) 1 USB 2.0 cable
(3) 1 USB 3.0 cable
(4) 1 power supply
(5) 1 power cable
(6) 1 cleaning set
(7) WEEE certicate
(8) protective cap for the MO and
(9) protective cap for the LLG
connection to the microscope
(10) 1 BNC cable.
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2
3
4
5
6
WEEE
Certicate
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1

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The CoolLED Illumination System packaging contains the following:
(11) LED Light Source
(12) DC Power Supply
(13) IEC Power Cable
(14) 1 USB 2.0 cable
(15) liquid light guide (LLG) protected with red vinyl caps (use the red vinyl caps to protect both end of the LLG when not in use)
(16) 3 holders equipped with excitation lters.
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6. Start-up
Components
11 16
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Important Information: It is critical to follow the installation and setup steps as described in the following pages to ensure the safety standards
of the CoolLED illumination system. Please refer to chapter 1: Safety Notes.
1. Install the 3D Cell Explorer on a stable, at, horizontal and rm workbench.
• Lift the 3D Cell Explorer-uo carefully out of the box by holding it at the indicated spots in the image below.
• Never lift the 3D Cell Explorer-uo by holding the camera.
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WARNING: Nanolive does not bear the damage produced by wrong handling of the
3D Cell Explorer-uo while it is removed out of its box.
In case of damages, Nanolive reserves the rights to examine the 3D Cell Explorer-uo
and verify how the damage occurred. All warranty claims will be cancelled in case of
mishandling the 3D Cell Explorer-uo by ignoring the instructions.
The warranty does not apply even if the 3D Cell Explorer-uo has been subject to
accidental damage.
6. Start-up
Installation and setup

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2. Carefully remove the protective accessories
• Properly dispose of the original packaging, or keep it for storage or return of the instrument to the manufacturer.
• Remove the disposable plastic foil and red vinyl protective cap.
6. Start-up
Installation and setup
Disposable plastic foil Red vinyl cap
For safety reasons, please proceed to install the CoolLED illumination system rst.

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3. Install the CoolLED Illumination System on a stable, at, horizontal and rm workbench
• Lift the CoolLED Illumination System carefully out of the box together with its accessories.
6. Start-up
Installation and setup
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4. Install the excitation lters on the CoolLED Illumination System
• Place the little drawers containing the excitation lters in the dedicated slots.
• Mind the order and the direction.
6. Start-up
Installation and setup
Channel 1
Channel 2
Channel 3

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6. Start-up
Installation and setup
5. Install the LLG on the CoolLED Illumination System
• Remove the red cap from the LLG from one end (Figure 1)
• Insert the LLG as far as it can go into the LED Light Source as shown below (Figure 2).
• Use an Allen key n°1.5 to secure the LLG in place. Once the screw is in mechanical contact with the LLG header, gently apply an extra force
to make sure the LLG is maintained in place. There is a risk of damaging the LLG header and reducing the coupled power from the LED Light
Source to the sample if too much force is applied by the screw.
LLG headerProtective cap
Pressure screw
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Figure 1
Figure 2