3D Cell Explorer-fluo

User Manual

November 2019
Reference: SS-DM-IN-005
Revision: 2
Contents
1. Safety Notes 3
• 3D Cell Explorer-fluo 3
• CoolLED illumination system 5
2. Maintenance & Care 6
• 3D Cell Explorer-fluo 6
• CoolLED’s illumination system 7
3. Introduction 8
• Instrument Description & Main Features 8
• Typical Field of Applications 9
• 3D Cell Explorer-fluo 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 38
12. Compliance 40
13. Technical Issues 41
1. Safety Notes
3D Cell Explorer-fluo

Thank you for purchasing the Nanolive 3D Cell Explorer-fluo. Before using your 3D Cell Explorer-fluo 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 and void your guarantee.

1. Safety Notes for the 3D Cell Explorer-fluo
• Verify that the computer is plugged and grounded earthed
with its own power supply during usage with your 3D Cell
­Explorer-fluo.
• Switch the 3D Cell Explorer-fluo OFF before connecting it to
a power supply.
• Disconnect and lockout the power supplies of the 3D Cell
­Explorer-fluo 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 flammable or toxic solvents.
2. Supply the 3D Cell Explorer-fluo only with the provided power
supplies.
3. No special laser safety required when operating the 3D Cell
­Explorer-fluo (Class I laser).
For measurements, the 3D Cell Explorer-fluo uses a Class I laser.
A Class I laser is harmless for your eyes and thus does not require
laser safety goggles when properly operated.
By no means should the user open the covers and thus have a ­ ccess
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.
✖ ✖

✔ ✖

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1. Safety Notes
3D Cell Explorer-fluo

4. Contaminations 6. The 3D Cell Explorer-fluo conforms to the requirements of

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-fluo. Please refer to our Main-
tenance and Care notes for proper cleaning instructions.
5. Cleaning
Please note that alcohol is highly flammable, do keep it away from fire or
potential sources of electrical sparks, and use it in a well-­ventilated room
when using it for cleaning the 3D Cell Explorer-fluo or other measures.
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.

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1. Safety Notes
CoolLED illumination system

7. Safety Notes for the CoolLED illumination system • Safety precautions must always be exercised when using
electrical equipment to prevent operator shock, fire hazard or
• UV light may be emitted from this product depending on the
equipment damage.
configuration selected. Avoid eye and skin exposure. Never look
directly into the light output beam from the Light Source or • FIRE HAZARD: Do not drape or cover the light source or the light
accessories. The emissions could damage the cornea and retina guide while it is in use. Do not place the light guide on a drape
of the eye if the light is observed directly. while it is in use.
• Always ensure that the Light Source is securely attached to the
microscope with its light guide, prior to turning on the power. 8. Compliance:
This will minimize the risk of injury and damage.
The light source conforms to the requirements of the Safety
• If for any reason the Light Source is to be operated when ­S­tandards as follows:
not ­attached to a microscope, all personnel should wear eye
­shielding and clothing to protect the exposed skin. • EN/IEC 61010-1:2010 Safety Requirements for Electrical
­Equipment for Measurement, Control and Laboratory use.
• Disconnecting the main supply is achieved by unplugging the
power cord from the power supply block or the Light Source. • EN62471:2008 Photo-biological Safety of Lamps and Lamp
Only plug in the power cable, once the Light Source is attached ­Systems/Guidance on manufacturing requirements relating
to the microscope. to non-laser optical radiation safety. Risk Group 3.
• 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 All warnings may not be applicable depending on the version/wavelength being used.

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.

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2. Maintenance & Care
3D Cell Explorer-fluo

Maintenance and Care for the 3D Cell Explorer-fluo

1. Carefully, open the shipment box with the top face up to avoid damaging the 3D Cell Explorer-fluo.
2. Handle the 3D Cell Explorer-fluo 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 flat, horizontal and firm.
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 objective protective 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 fingerprints 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
a dust cover. Furthermore, protect the microscope objective with the red or black vinyl cover.

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2. Maintenance & Care
CoolLED illumination system

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 field 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 finger prints accidentally come into contact with the
excitation filters during installation. In the first 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 flood the optical surfaces with fluid as liquid could enter the product and cause damage.

Care of the light guide

The light guide is a fine, high quality optical instrument. The useful life can be prolonged by following these
simple guidelines.
1. Avoid stretching the light guide, forming configurations, involving sharp angels or kinks, or contact with sharp or
pointed objects. The internal light fibers 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.

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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 fluorescence imaging
• 3 Fluorescence Channels: 2 configurations 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 filters are exchangeable on channel 3

Please refer to the following link for details on the Filter configuration:
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 epifluorescence snap-shot (every second) & time-lapse (up to 96 hours)
- 3D RI snap shot & time-lapse 3D RI & 2D epifluorescence 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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3. Introduction
Typical Fields of Applications

The typical fields of applications for the 3D Cell Explorer-fluo

• Merging of fluorescence microscopy and 3D cell tomography

• Calibration of the refractive index map with markers (Chemical dyes, fluorescent antibodies, protein
labeling with GFP, etc.)
• Combination of structural RI information with chemical information for specific 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

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3. Introduction
3D Cell Explorer-fluo terminology

3D Cell Explorer-fluo – front view 3D Cell Explorer-fluo – side view 3D Cell Explorer-fluo – rear view

Scanning head

Peripheral mirror

Rotating arm

Cell container
interface
Hi-grade stage for
XY knob for sample screening
sample screening
Z knob for
Camera optical focusing
with USB
3.0 slot

Camera Injection Head USB 2.0 slot Power switch

with USB
3.0 slot
BNC cable Power supply
plug port plug port

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3. Introduction
CoolLED illumination system terminology

CoolLED illumination system – front view CoolLED illumination system – side view 1 Liquid Light Guide (LLG)

Not needed BNC plugs

Global Trigger

CoolLED illumination system – rear view CoolLED illumination system – Top/Side View

Excitation filters slots

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4. Technical Specification Sheet (TSS)
3D Cell Explorer-fluo & CoolLED illumination system

Specification 3D Cell Explorer-fluo / Specifications CoolLED illumination ­system /

Holotomography 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 5-35°C
operated between 20° C and 30° 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 Efficiency (typical) 70 % (at 545 nm)
Dark Noise (typical) 6,6 e¯
Dynamic Range (typical) 73,7 dB
Microscope Objective (MO) Dry objective / 60x magnification / NA 0.8
Illumination Source Class I laser low power (λ=520 nm, High speed switchable <100 μs
sample exposure 0.2 mW/mm2) Lifetime >20’000 hours each channel
(Class I. No eye protection needed)
Field-of-View (FoV) 90 x 90 x 30 μm3 90 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 m ­ ounting
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 filters
are exchangeable on channel 3)

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5. Computer Technical Specifications
for operating the 3D Cell Explorer-fluo

The 3D Cell Explorer-fluo is provided without a computer. Therefore, before you acquire a computer, please be aware of the recommended require-
ments listed below which are necessary for correct operation of the instrument.*

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 recommended specifications of the Nvidia GPU above. In addi-
tion, you need at least one USB 3.0 port for camera image acquisition.

CPU: 2.00 GHz or faster quad-core CPU

CPU Memory: minimum 16GB DDR3 RAM
Hard disk: 2 GB free space for STEVE and 1 TB for acquisition data
GPU**: NVIDIA GPU with 8GB dedicated memory
USB ports: 4 ports, minimum one USB 3.0 port
Operating System: 64-bit versions of Windows 10
(Updated drivers: you need to have the latest Nvidia Graphics Card drivers,
USB 3.0 drivers and fully updated Windows)
In addition: Internet access for STEVE installation and for the newest updates.

*We are happy to provide you with a reference list of computers and laptops. Please contact us.
**If you are unsure about the minimal requirements, please contact us.

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6. Start-up
Components

The 3D Cell Explorer-fluo package contains the following:

(1) the 3D Cell Explorer-fluo (7) WEEE certificate

(2) 1 USB 2.0 cable
(3) 1 USB 3.0 cable
(4) 1 power supply
(5) 1 power cable
(6) 1 cleaning set
(8) protective red or black cap for the MO and
(9) protective cap for the LLG
connection to the microscope
(10) 1 BNC cable.

6 8
1 2

3 9

4
10

5
7
WEEE
Certificate

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6. Start-up
Components

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 filters.

11 13 14 15 16

12

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6. Start-up
Installation and setup

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, flat, horizontal and firm workbench.
• Lift the 3D Cell Explorer-fluo carefully out of the box by holding it at the indicated spots in the image below.
• Never lift the 3D Cell Explorer-fluo by holding the camera.

WARNING: Nanolive does not bear the damage produced by wrong handling of the
3D Cell Explorer-fluo while it is removed out of its box.
In case of damages, Nanolive reserves the rights to examine the 3D Cell Explorer-fluo
and verify how the damage occurred. All warranty claims will be cancelled in case of
­mishandling the 3D Cell Explorer-fluo by ignoring the instructions.
The warranty does not apply even if the 3D Cell Explorer-fluo has been subject to
­accidental damage.

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6. Start-up
Installation and setup

2.   Carefully remove the protective accessories

• Please keep the original packaging for the period of your warranty in case you need to return the device to the manufacturer.
• Remove the disposable plastic foil and red or black vinyl protective cap.

Disposable plastic foil Objective protective cap

For safety reasons, please proceed to install the CoolLED illumination system first.

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6. Start-up
Installation and setup

3. Install the CoolLED Illumination System on a stable, flat, horizontal and firm workbench
• Lift the CoolLED Illumination System carefully out of the box together with its accessories.

13 14 15 16

12

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6. Start-up
Installation and setup

4. Install the excitation filters on the CoolLED Illumination System

• Place the little drawers containing the excitation filters in the dedicated slots.
• Mind the order and the direction.

Channel 2

Channel 1

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)

Protective cap LLG header

Figure 1

• Insert the LLG as far as it can go into the LED Light Source as shown below (Figure 2).

✖ ✔ Pressure screw

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.

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6. Start-up
Installation and setup

6. The CoolLED illumination system with the inserted LLG should look like the image below.
Place the light source close to the 3D Cell Explorer-fluo in order to avoid any strain on the LLG or too short radius turns.

Figure 3
200 mm 200 mm

Ensure that the Light Source sits upright on a flat surface and keep Clear space Clear space
a clearance of 200mm on each side to ensure adequate airflow for the
­cooling system.

Figure 4

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6. Start-up
Installation and setup

7. Connect the LLG to the 3D Cell Explorer-fluo

• Remove the red cap from the LLG from the other end (Figure 5)

Protective cap LLG header

Figure 5

• Insert the LLG as far as it can go into the injection head on the 3D Cell Explorer-fluo as shown below (Figure 6).

✖ ✔

Figure 6 Injection Head

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6. Start-up
Installation and setup

• Use an Allen key n°1.5 to secure the LLG in place.

• Bring the 3 screws first in mechanical contact without effort one after the other. Gently apply some force to keep the LLG in place one after
the other. Please be aware that any damage made to the LLG might lead to irreversible loss of coupled power brought to the sample (Figure 7).

Figure 7

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6. Start-up
Installation and setup

8. Handling note on the LLG (Figure 8)

• The LLG is carrying the light from the CoolLed illumination system to the 3D Cell Explorer-fluo. It is fragile and should be handled with care.
• Avoid scratches and damages to the LLG ends (especially the silica surfaces) when connecting them to the CoolLed illumination system and
the 3D Cell Explorer-fluo.

Metal

Teflon

Fused Silica
Adequate bending radius: about
Figure 8 90 mm in the picture below

• Do not stretch, squeeze or bend the LLG excessively.

✔
Important Note

Short-term bending radius Rshort > 40 mm /

Long-term bending radius Rpermanent > 75 mm (Figure 9)
To avoid permanent damage, it is highly r­ ecommended to
leave a clear and safe path for the LLG from the CoolLed
R
­illumination system to the microscope without any strain
(Figure 10).

Figure 9

Figure 10

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6. Start-up
Installation and setup

9. Diverse connections of your CoolLed illumination system

• Plug the power connector from the DC power supply to the CoolLED illumination system as shown below. Ensure that the DC power s­ upply
is the one supplied with the product. Using a non-CoolLED power supply may damage the illumination system and will invalidate the
­warranty. At this stage do not connect the main power lead to the DC power supply (Figure 11).
• Plug the CoolLED illumination system USB 2.0 cable into the USB 2.0 port on the top of the CoolLED illumination system and connect it to
one of the USB slots on your computer (Figure 11).
• Then plug the BNC cable to the rear panel of the CoolLED illumination system and to the rear panel of the 3D Cell Explorer-fluo (Figure 12).
USB 2.0 cable between Not Used Power supply
module and computer

Not Used

BNC cable

Figure 11 Figure 12

Important Note

Orientation of the power plug: Plug the power cable with the flat surface facing inward (Figure 13)

CoolLED module Power plug Flat surface

Figure 13
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6. Start-up
Installation and setup

• With the CoolLED illumination system now attached to the microscope it is safe to connect the power cable. Connect the power cable
­supplied to a convenient socket, plug in the IEC connector into the DC power supply and switch the power on at the socket.

Camera / data transfer USB 3.0

Computer

USB 2.0

3D Cell Explorer
Nanolive SA
SN: YY MM DD 00 00 X X XX XX

Power supply &

power cable
BNC cable

LLG

Power supply &

power cable
USB 2.0

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6. Start-up
Installation and setup

10. Diverse connections of your 3D Cell Explorer-fluo (Figure 14)

• Make sure the power switch of your 3D Cell Explorer-fluo is off.
• Connect the Nanolive power cable to the Nanolive power supply. Then, plug the Nanolive power supply into the power port.
• Plug the Nanolive USB 3.0 cable into the USB 3.0 port on the side of the 3D Cell Explorer (the camera), tightly screw the locks and connect
it to the USB 3.0 slot on your computer.
• Plug the Nanolive USB 2.0 cable into the USB 2.0 port on the back of the 3D Cell Explorer-fluo and connect it to one of the USB slots on
your computer.

LLG

BNC cable

Power supply
cable

USB 2.0 cable

Figure 14

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6. Start-up
Installation and setup

11. Download and install STEVE

• Switch on your PC and make sure it is connected to a power source.
• USB 3.0 drivers and fully updated Windows. We highly recommend to update your Nvidia Graphics Card drivers directly from Nvidia's
­website: http://www.nvidia.com/Download/index.aspx
• Go to www.nanolive.ch/register, fill out the form to register the 3D Cell Explorer-fluo and download the software.
• When requested, insert the serial number (you can find your serial number on the bottom of your 3D Cell Explorer-fluo, the delivery note
or on the back of the quick start guide).
• Download and install STEVE.

Please note that you must run Steve with Nvidia graphics processor
• right click on the Steve exe file
• then click on "run with graphics processor"
• select "Nvidia high-performance"

• Start STEVE while still connected to the internet.

• The user’s manual and important information are downloaded during installation.
• Switch on your 3D Cell Explorer-fluo and make sure it is connected to a power source.
• Connect the power supply of the CoolLED source to the grid.

Before to start exploring your cells, check our Sample Preparation Manual on how to properly prepare your samples.

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7. Handling
Correct handling of the 3D Cell Explorer-fluo

The following section’s purpose is to explain how to avoid any damages to the 3D Cell Explorer-fluo during moving, carrying, transportation and
repacking.

If you want to move, carry, transport or repack the 3D Cell Explorer-fluo, please make sure to:

1. Unplug all cables from both devices (3D Cell Explorer-fluo and CoolLED illumination system): (a) power, (b) USB2.0, (c) USB3.0 (d) BNC and (e)
light guide.
2. Secure the sample stage of the 3D Cell Explorer-fluo in top position.
3. Protect the Microscope Objective with its dedicated objective protective cap.
4. Protect the injection head with its dedicated black vinyl cap
5. Respect the holding precautions of the 3D Cell Explorer-fluo.
6. Protect both ends of the LLG with the red vinyl cap.
7. Avoid stretching the LLG, forming configurations, involving sharp angels or kinks, or contact with sharp or pointed objects.
8. Transport the 3D Cell Explorer-fluo and the CoolLED illumination system in their original packaging.

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7. Handling
Correct handling of the 3D Cell Explorer-fluo

When planning to move the device short distances, for example from worktop to worktop follow the steps below to secure the stage
correctly and avoid potential damage.

If planning to move longer distances or using transport to move, refer to our Nanolive guidelines for travelling with your microscope.
Please contact Nanolive for further information and Nanolive approved packaging.

✔ ✖

Secure top position for
transport with z-screw in top
position
High-risk of damages during
transport
Further secure the high- Underside view with stage
grade stage using the red removed, be sure the screw
locking screw. Also uninstall is fully secured.
red locking screw if ever
removing the high-grade
stage.

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7. Handling
Correct handling of the 3D Cell Explorer-fluo

Instructions on how to protect the 3D Cell Explorer-fluo Objective (MO) with the black vinyl protective cap

To be used when the 3D Cell Explorer-fluo is not in use and during transportation.

✔ ✔ ✔ ✖

Place the protective cap on
the MO through the sample
stage aperture.
Push lightly until fully Correctly installed, levelled Incorrectly installed, not
covering black rubber on the device. levelled on the device, not
soufflet and/or touching the providing protection.
objective heater which may
be installed on your device
if using a Nanolive-OKO
incubator.

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7. Handling
Correct handling of the 3D Cell Explorer-fluo

Instructions on how to protect the LLG header and how to properly take care of the LLG
• Use the protective cap for both ends when the LLG is not connected to the devices (Figure 15 & 16)
• The light guide is a fine, high quality optical instrument. The useful life can be prolonged by following these simple guidelines
• Avoid stretching the light guide, forming configurations, involving sharp angels or kinks, or contact with sharp or pointed
objects. Light guide breakage will result in diminished light output (Figure 16).
• 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.

Protective cap LLG header

Metal

Teflon

Fused Silica

Figure 17 Figure 15

• Avoid scratch or damage to the LLG ends (especially the silica ­surfaces) Storage of LLG: minimum
when connecting them to the CoolLed illumination s­ ystem and the 3D bending radius of 75 mm
Cell Explorer-fluo (Figure 17).
• Clean ends with optical tissue and grade alcohol

Figure 16

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7. Handling
Correct handling of the 3D Cell Explorer-fluo

Instructions on how to carry the 3D Cell Explorer-fluo correctly

Once all cables are unplugged and the sample stage is secured and in top position, hold/carry your device as shown below.

✔ ✖

Secure hand positions at the places provided for Do not lift the device by its sample
lifting, carrying, and holding stage or by the rotating arm

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8. Sample Preparation

Sample Preparation Manual:

The 3D Cell Explorer-fluo allows measuring the inside of a living cell offering the researcher the possibility to acquire high resolution 3D
images of a cell within seconds:
• cells fixed on glass coverslips or FluoroDish™
• cells grown on FluoroDish™
Thanks to extremely low light exposure and compatibility with cell culture accessories, the 3D Cell Explorer-fluo is suitable for long-term
live cell imaging. We have designed a Sample Preparation Manual to guide researchers through the sample preparation methods for
­observation.

Please download here our Sample Preparation Manual.

Note: If you are using fluorescent markers to label your cells, please follow the known concepts of Sample Preparation for Fluorescence
Microscopy!

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9. Cleaning Procedure for optical elements

First, inspect the optical elements to determine the location of the con-
taminants. This allows you to anticipate the cleaning (typically by a swiping
movement) so that the contaminant is removed from the surface of the
optical element as soon as possible (avoid dragging it around).

Optical surfaces are sensitive to scratches and must thus be cleaned

carefully with the appropriate equipment. We advise to use only new clean
swabs (Texwipe™ Microdenier or Alpha series) soaked with ethanol or iso-
propanol to clean the optics.

Take a fresh and clean swab soaked in ethanol or isopropanol, and with-
out pressure swipe the contaminant away from the center of the optical
element. Use a fresh side of the swab after each contact with the optical
surface in order to avoid re-deposition of removed contaminants.

Please clean first the most exposed optical surfaces, with thus higher
probability of contamination:

• The MO is quite exposed to contamination and it may thus be required

to clean it regularly. (Figure 18)
• The scanning mirror of the rotating arm is not much exposed, and its
cleaning should not be required too often. (Figure 19)
• Finally, the central mirror is not exposed and its cleaning should be
exceptional. (Figure 20)

High concentrations of contaminants may require repeated cleaning.

Figure 18

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 9. Cleaning Procedure for optical elements

Figure 19 Figure 20

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10. Software STEVE
Important Note: Fluorescent filter configuration

Please refer to our tutorial videos on our "Supporting Material" page for further assistance with STEVE.

Fluorescence filter configuration

Before you start your acquisitions, set up STEVE with the correct fluorescence filter configuration as shown below.
The 3D Cell Explorer-fluo is delivered with the options FITC (green), TRITC (orange) and DAPI (blue) or Cy5 (red).
The configuration may be changed from the Microscope —> Options menu. (Figure 21 & 22)

Figure 21 Microscope Options

Figure 22 Fluo channels configuration window

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11. Troubleshooting

Calibration
Calibration failed err.1.1
If applicable, reduce the quantity of liquid above your sample (-> Check our Sample Preparation Protocol in Section 5). Check that no opaque object
can intersect the laser beam while the arm is rotating. If using a dish, check that you are imaging close to its center such that the laser beam cannot
intersect the wall of the dish instead of the top.
Calibration failed err.1.4
Sample may be too absorbent. Try to move in a different area and re-do the acquisition. If the error persists, please get in touch with our customer
service.

Acquisition
Acquisition failure err.2.1
If applicable, try to move to another position within your sample. If the error persists try to clean the microscope objective and check that you com-
ply with the sample preparation protocol ( -> Check our Sample Preparation Protocol).

Connection
Connection failure err.3.1
If STEVE cannot establish communication with the 3D Cell Explorer, please verify that the backside USB 2.0 is correctly plugged in to the 3D Cell
­Explorer and to your computer, that the microscope has electrical power and that it is switched on. If the error persists, unplug all cables, reboot
your computer and plug the cables back (check Start-up - Section 4).
Connection failure err.3.2
If STEVE cannot establish communication with the 3D Cell Explorer, please verify that the USB 3.0 cable coming from the camera is correctly
screwed in and that it is properly connected to your computer’s USB 3.0 port.

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11. Troubleshooting

Connection failure err.3.3

STEVE cannot establish communication with the 3D Cell Explorer. The port to which the USB 3.0 cable (coming from the camera) was connected to
was recognized as USB 2.0. This cable must be connected to a USB 3.0 port. If you have correctly connected the USB 2.0 and USB 3.0 cables and the
error persists, please verify that your USB 3.0 adapter drivers are properly installed or contact your system administrator.
Connection failure err.3.4
STEVE cannot establish communication with the 3D Cell Explorer and the microscope cannot initialize itself. Please make sure the rotating arm is
not obstructed in any way. Try to switch it off and on again. If the error persists observe whether the arm rotates when you switch the microscope on.
If this is not the case, please contact our service support.
Connection failure err.3.6
The microscope needs maintenance. Please contact your distributor if applicable or our service support.
Connection failure err.3.7
The connection to the fluorescence module failed. Please make sure that the fluorescence module is powered and that the USB cable from the
module is connected to a USB port on your computer.
Connection failure err.3.8
Microscope firmware update error. Your microscope likely needs maintenance. Please contact our service support.

Processing
Processing error err.5.1
The STEVE processing tool chain encountered a fatal error. Please make sure your computer meets the STEVE hardware requirements. In particular,
make sure your graphical card is from the NVIDIA brand with at least 4GB of dedicated GPU memory. Please upgrade your graphical drivers using
the NVIDIA website at www.nvidia.com/Download/index.aspx. If the error persists, please contact our customer service including error detailed
information displayed in the error popup.

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12. Compliance

WEEE
All qualifying products that are subject to the WEEE Directive and
supplied by Nanolive are compliant with the WEEE marking requirements.
Such products are marked with the "crossed out wheelie bin" WEEE
symbol and in accordance with European Standard EN 50419.

RoHS
Based on information obtained from our component suppliers, this
statement certifies that ALL products manufactured and supplied by
CoolLED Ltd are in compliance with Directive 2011/65/EU of the
European Parliament and of the Council of 8 June 2011 on the restriction
of the use of certain hazardous substances in electrical and electronic
equipment (also known as “RoHS”).
This declaration is correct to the best of CoolLED Ltd knowledge,
information and belief at the date of its release.

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13. Technical Issues

If you encounter any technical issues, please contact our service support at support@nanolive.ch.

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