Malvern NANOSIGHT NS300 User manual

NANOSIGHT NS300
OPERATING MANUAL
MAN0516-08-EN-00 January 2016

NanoSight NS300 Operating Manual
(MAN0516-08-EN-00) 2
Copyright © 2016 Malvern Instruments Ltd.
Malvern Instruments pursues a policy of continual improvement due to technical development. We therefore reserve the
right to deviate from information, descriptions, and specifications in this publication without notice. Malvern Instruments
shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing,
performance or use of this material. No reproduction or transmission of any part of this publication is allowed without the
express written permission of Malvern Instruments Ltd.
Head office:
Malvern Instruments Ltd.
Enigma Business Park,
Grovewood Road,
Malvern,
Worcestershire WR14 1XZ
United Kingdom.
Tel + [44] (0)1684-892456
Fax + [44] (0)1684-892789
www.malvern.com
NanoSight, Malvern and the 'hills' logo are registered trademarks in the UK and/or other countries, and are owned by
Malvern Instruments Ltd.

NanoSight NS300 Operating Manual
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NANOSIGHT NS300
OPERATING MANUAL
Contents
1.1 INTRODUCTION ......................................................................................................................................... 5
1.2 EXPLANATION OF WARNING SYMBOLS ................................................................................................. 5
1.3 IMPORTANT NOTES .................................................................................................................................. 5
1.3.1 SAFETY ................................................................................................................................................... 5
1.3.1.1 Laser Safety ................................................................................................................................... 5
1.3.1.2 Electrical Safety.............................................................................................................................. 6
1.3.1.3 General Safety ............................................................................................................................... 6
1.3.2 SERVICING ............................................................................................................................................... 7
1.3.3 MAINTENANCE ......................................................................................................................................... 7
1.3.4 HANDLING............................................................................................................................................... 7
1.3.5 UNPACKING AND INITIAL INSPECTION............................................................................................................. 8
1.3.6 INSTALLATION AND RELOCATION .................................................................................................................. 8
1.3.7 RETURNING EQUIPMENT.............................................................................................................................. 8 
1.3.8 WARRANTY ............................................................................................................................................. 9
2. NANOSIGHT NS300 HARDWARE.............................................................................................................. 10
2.1 NS300 TECHNICAL SPECIFICATIONS ...................................................................................................... 10
2.1.1 NS300 INSTRUMENT HOUSING.................................................................................................................. 10
2.1.2 NS300 LASER MODULE ........................................................................................................................... 10
2.1.3 LASER CLASSIFICATION ............................................................................................................................. 10
2.2 NS300 SETUP ........................................................................................................................................... 11
2.2.1 INSTRUMENT COMMUNICATIONS ................................................................................................................ 11
2.2.2 LASER MODULE CONFIGURATION ............................................................................................................... 11
2.2.3 TOP-PLATE SELECTION.............................................................................................................................. 11
2.2.4 EXTERNAL FLUIDIC SETUP .......................................................................................................................... 12
3. LOW VOLUME FLOW CELL ....................................................................................................................... 13
3.1 SYSTEM SETUP ........................................................................................................................................ 13
3.1.1 FLOW CELL ASSEMBLY ............................................................................................................................. 13
3.1.2 FLOW CELL TUBING CONNECTION .............................................................................................................. 14
3.2 FLOW CELL USAGE.................................................................................................................................. 15
3.2.1 PRIMING TUBING..................................................................................................................................... 15
3.2.2 CHANGING INLET TUBING SYRINGE ............................................................................................................. 15
3.2.3 LOADING AN INITIAL SAMPLE ..................................................................................................................... 15
3.2.4 CHANGING SAMPLES ............................................................................................................................... 16
3.2.5 MANUAL CLEANING PROCEDURES .............................................................................................................. 17
3.2.5 DRYING THE LOW-VOLUME FLOW CELL ....................................................................................................... 18

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3.3 CARE AND MAINTENANCE ..................................................................................................................... 19
3.3.1 GASKET COMPONENT REPLACEMENT .......................................................................................................... 19
4. O-RING TOP PLATE.................................................................................................................................... 20
4.1 SYSTEM SET-UP....................................................................................................................................... 20
4.1.1 O-RING TOP-PLATE ASSEMBLY FOR MANUAL INJECTION ................................................................................. 20
4.1.2. O-RING TOP-PLATE ASSEMBLY FOR SYRINGE PUMP USE................................................................................. 20
4.1.3 O-RING SYRINGE PUMP TUBING CONNECTION ............................................................................................. 21
4.2 O-RING TOP-PLATE USAGE..................................................................................................................... 21
4.2.1 MANUAL INJECTION OF A SAMPLE .............................................................................................................. 21
4.2.2 LOADING A SAMPLE FOR USE WITH A SYRINGE PUMP...................................................................................... 22
4.2.2.1 Priming Tubing ............................................................................................................................ 22
4.2.2.2 Changing Inlet Tubing Syringe ..................................................................................................... 22
4.2.2.3 Loading the Sample through the Syringe Pump Tubing................................................................23
4.2.3 CHANGING SAMPLES ............................................................................................................................... 24
4.2.4 MANUAL CLEANING PROCEDURES .............................................................................................................. 24
4.2.5 DRYING THE O-RING TOP-PLATE ................................................................................................................ 25
4.3 CARE AND MAINTENANCE ..................................................................................................................... 25
5. MOUNTING THE LASER MODULE ............................................................................................................ 26
6. DAILY USE.................................................................................................................................................. 26
7. NANOSIGHT NS300 CONTROL SOFTWARE ............................................................................................. 27
7.1 NTA SOFTWARE STARTUP............................................................................................................................ 27
7.2 CAMERA SETUP ......................................................................................................................................... 28
7.3 ADJUSTING THE NS300 TO OBTAIN A VIDEO .................................................................................................... 28
7.4 THRESHOLDING PIXELS (HIGH-SENSITIVITY SYSTEMS ONLY) .................................................................................. 29
7.5 FLUORESCENCE MODE ................................................................................................................................ 29
7.5.1 Viewing fluorescently labelled particles ........................................................................................... 29
7.6 CAPTURE VIDEOS ....................................................................................................................................... 30
7.7 TEMPERATURE CONTROL ............................................................................................................................. 30
7.7.1 Operation ....................................................................................................................................... 30
7.7.2 Initialising ....................................................................................................................................... 31
7.7.3 Temperature Setting ....................................................................................................................... 31
8. SPARE PARTS LIST .................................................................................................................................... 32

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1.1 Introduction
The NanoSight NS300 instrument is a patented, laser-based, light scattering system which provides an easy-to-use,
reproducible platform for specific and general nanoparticle characterisation.
Particles suspended in a liquid are loaded into the laser module sample chamber and viewed in close proximity to the optical
element. The NS300 device illuminates the particles using a specially aligned and focussed laser beam. This allows extremely
small particles (down to 10 nm, dependent on refractive index) to be seen directly and individually by conventional
microscopy.
Particles in the liquid sample which pass through the beam path are seen as small points of light moving rapidly under
Brownian motion, allowing information on particle properties to be obtained. With the NS300 you can analyse the
presence, size distribution, concentration and fluorescence of all types of nanoparticles from 10 nm to 2000 nm, depending
on the instrument configuration and sample type.
The laser module contains thermoelectric Peltier elements, allowing the sample temperature to be controlled. This is fully
programmable using the NTA Software Suite.
The installed tubing and the viewing chamber surfaces allow the use of all non-flammable, water-based solvents with
neutral pH. Other solvents may not be compatible with wetted surfaces and should not be used without confirming
suitability. If in doubt about the choice of solvent and its compatibility with any part of the device, please contact Malvern
Instruments ([email protected]om) for further information.
This manual is designed to provide an introduction to help familiarise the user with the NanoSight NS300 operation. Further
advice and help to get the best out of your instrument can be found in the NanoSight Application Notes, Technical Notes,
NTA Software Operation Manual, Quick-start Guides and User Training Videos. These can be found on our website
(www.malvern.com) or by contacting Malvern Instruments.
For technical assistance, please contact Malvern Instruments.
Malvern Instruments Technical Support:Tel: + [44] (0)1684-892456 or Email: [email protected]
1.2 Explanation of Warning Symbols
Warning: read instructions to understand possible hazard prior to use.
Electric shock hazard
1.3 Important Notes
1.3.1 Safety
1.3.1.1 Laser Safety
•The NanoSight NS300 device is classified (to BS EN 60825-1 (2001)) as a Class 1 laser device. The instrument
contains a Class 3B laser which must never be removed from the laser module housing.
•The laser module should not be plugged into the main body of the NS300 without the flow cell or top plate being
secured in position with the screws supplied.
•The laser module should always be removed from the main body of the NS300 (cutting power to the laser) prior to
removing the flow cell or top-plate screws.

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1.3.1.2 Electrical Safety
•
Fuses must be only replaced with a fuse of the type and rating - F5AH250V Ø5mm x 20mm long.
• Use only the power adapter and other accessories supplied with the instrument.
• If the mains cordset needs to be replaced, please select another cordset which conforms to the following
specifications:
Voltage Rating
125 V AC if being used with a 100-120 V supply.
250 V AC if being used with a 220-240 V supply.
Current Rating
6 A minimum.
Temperature Rating
60 °C minimum.
Length
3 m Maximum.
Fittings
Grounded plug for attachment to power outlet.
IEC appliance coupling.
In addition to the above specifications, the mains cordset should be certified by one of the following institutions.
UK – BSI USA – UL Europe – VDE Japan – JET, JQA, TUV
• If the appliance is being used outside of the above areas the local regulations for mains power cords must be
checked and a cordset which complies with the relevant standards should be sourced for use with the equipment.
• The safety of the equipment can no longer be warranted if a non-approved cordset is used.
1.3.1.3 General Safety
•
The instrument must not be used in hazardous areas.
• The instrument is for use in moderate climates only. Never use the equipment in damp or wet conditions.
• Avoid excessive heat, humidity, dust and vibration.
• Do not place liquid filled containers on the equipment.
• Do not use where the equipment may be subjected to dripping or splashing liquids.
• If the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment
may be impaired.
• The instrument can be used with all non-flammable water-based solvents of neutral pH. Other solvents may not be
compatible and must not be used without first contacting Malvern Instruments to confirm suitability.
• When using biologically or chemically hazardous sample material, it is the responsibility of the operator to
determine the requisite protection for each application.

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•The NS300 system contains no user serviceable part. It should not be modified in any way. Any modification will
void the warranty and could make the device unsafe.
•Use of controls, adjustments or performance of procedures other than those specified herein may result in
hazardous laser radiation exposure.
•The laser module may become hot when operating the temperature control. Caution should be taken when
handling the device.
•The temperature control should always be turned off when the instrument is unattended.
1.3.2 Servicing
•The NS300 must only be serviced by qualified Malvern Instruments personnel, or Malvern Instruments approved
agents.
•The NS300 contains no user-serviceable parts. The instrument casing should not be opened by any user.
•Removing the instrument casing or opening the housing of the laser module voids all warranties and could expose
users to hazardous voltages or Class 3B laser radiation.

1.3.3 Maintenance
•The NS300 system contains no user serviceable parts. It should not be modified in any way. Any modification will
void the warranty and could make the device unsafe.
•The casing of the NS300 body and the laser module should be kept clean with the use of a damp cloth. Do not
wet or allow excess moisture to penetrate any part of the system. Do not use solvents.
•To maintain best functionality and to protect against cross contamination during and following use, the sample
flow cell and tubing should be cleaned as described in this manual.
•Regularly inspect fluidic tubing and replace any that show signs of wear. Please contact Malvern Instruments for
additional sets of tubing (P/N NTA4161 NS300 Complete Tubing Kit).
1.3.4 Handling
The NanoSight NS300 incorporates a rugged housing designed to protect the integrity of the instrument. However, it is a
sensitive scientific instrument and should be treated as such. The laser module contains a static-sensitive laser diode and
should never be used in circumstances when a static discharge may damage the diode.
During use and when cleaning it is important to ensure that liquid does not enter the inside of the laser module. Use only a
damp tissue for cleaning, never expose the laser module to excess fluids. During sample loading and instrument operation,
ensure that the sample chamber is properly sealed and that there is no evidence of any leaking fluids.
Between uses of the NanoSight NS300, or for longer term storage, the unit must be cleaned and dried as described in this
manual.

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1.3.5 Unpacking and Initial Inspection
The standard instrument is supplied with the following accessories:
•Mains power lead
•USB cable
•CMOS Firewire cable
•Spare NS300 Tubing Kit
•Spare low volume flow cell gasket component
•Instruction manuals
•Allen/Hex-key set
•Particle size standards
Inspect the shipping container when the NS300 is received. Carefully check the contents for completeness and condition.
The NS300 is supplied in a specially designed packing case which should be returned to Malvern Instruments after delivery.
The packing case can be kept if required, although an additional charge will be made.
Notify Malvern Instruments ([email protected]om) if the contents are incomplete, or if the instrument or accessories
appear to be damaged in any way. Keep all damaged packaging, materials and goods for inspection by the carrier.
1.3.6 Installation and Relocation
Initial installation will be carried out by qualified Malvern Instruments personnel.
In the event that the instrument needs to be relocated the following instructions must be adhered to:
Always disconnect the equipment from the mains and ancillary units before moving.
The instrument should be located in an area of good ventilation and with sufficient space for safe and efficient
operation and maintenance.
The supplied PC should be sited to avoid dangers from spillage and splashing.
The equipment must be connected to an earthed power supply with a voltage corresponding to that on the power
adapter.
Ensure that the mains plug is easily accessible to allow the unit to be disconnected from the mains supply.
Always use the mains lead supplied. Your sales representative can provide a lead suitable for your country.
1.3.7 Returning Equipment
If, for any reason, you experience problems with your instrument, contact Malvern Instruments (helpde[email protected]).
In the unlikely event you experience a problem with the NS300 system that requires returning the unit for repair, please
contact Malvern Instruments for instructions and documentation:
The following information will need to be supplied:
•Sender’s name and address;
•Sender’s contact telephone number and email address;
•Complete list of equipment being returned including serial numbers;
•A detailed description of the problem or reason why the equipment is being returned;
•Declaration that, if the instrument has been used with biologically or chemically hazardous sample
material, all equipment has been fully decontaminated before return.

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On receipt of this information Malvern Instruments will provide a Return Merchandise Authorization (RMA) number. An
RMA number must be obtained from Malvern Instruments before returning any equipment, and should be clearly displayed
on the return shipment and included on all subsequent correspondence.
If the original shipping case is not available, shipping cases will be supplied by Malvern Instruments as required to ensure
safe return of the system.
1.3.8 Warranty
Malvern Instruments warrants that the NS300 system as supplied with its accessories is free from defects in materials and
workmanship for a period of one year from shipping to the customer. During this warranty period, Malvern Instruments
will, at its discretion, repair or replace defective products.
Any liability under this warranty extends only to the replacement value of the equipment.
This warranty is void if:
•The NS300 or its accessories have been partly or completely disassembled, modified or repaired by
persons not authorised by Malvern Instruments
•The instrument or instrument system is installed or operated other than in accordance with these
operating instructions.
No other warranty is expressed or implied. Malvern Instruments is not liable for consequential damages except as limited by
English law.

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2. NanoSight NS300 Hardware
2.1 NS300 Technical Specifications
2.1.1 NS300 Instrument Housing
Size: 400 mm x 400 mm x 250 mm
Weight: ~12 kg
Operating temperature: 10 °C to 40 °C
Humidity: Up to 80% rH at 31 °C then decreasing linearly to 50% at 40 °C
Casing material: Plastic, aluminium
Voltage: 100-240 V rms +/- 10%
Frequency range: 50 – 60 Hz
Max power: 60 VA
2.1.2 NS300 Laser Module
Size: 140 mm x 74 mm x 68 mm
Weight: 650 g
Temperature control range: from 5 °C below ambient to 50 °C
Temperature control accuracy: +/-1 °C
Time to temperature: 3 minutes (to indication of within 1 °C)
Humidity: 5-95% non-condensing
Casing material: Anodised aluminium alloy
Top plate materials: Low Volume Flow-cell: PMMA, silicone, glass, PDMS,
SU8 epoxy, Norland Optical Adhesive 61
O-ring Top-Plate: Anodised aluminium alloy, glass and viton rubber
Wetted Parts: PEEK connectors, Delrin connectors, PTFE tubing
Low Volume Flow-cell: PMMA, silicone, glass, PDMS,
SU8 epoxy, Norland Optical Adhesive 61
O-ring Top-Plate: Anodised aluminium alloy, glass, viton rubber
2.1.3 Laser Classification
Embedded laser: Red 642 nm CW, max power < 50 mW
Green 532 nm CW, max power < 60 mW
Blue 488 nm CW, max power < 55 mW
Violet 405 nm CW, max power < 70 mW

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2.2 NS300 Setup
2.2.1 Instrument Communications
1) The NS300 instrument communicates with the PC via a USB connection. Plug
the USB cable into the USB port on the right side of the NS300 instrument casing
(labelled USB-1) and connect it to one of the USB ports on the supplied PC.
2) Plug the firewire camera cable into the firewire port on the side of the NS300
(labelled Firewire B) and connect it to the PC firewire port.
3) Set up the monitor, keyboard and mouse and turn on the PC.
4) Connect the mains lead to the NS300 instrument and turn the instrument on
at the power switch, located on the left side of the case.
5) Start the NTA software, and confirm that no hardware detection error
messages are displayed in the status panel (see also Section 7 of this manual and
NanoSight NS300 NTA Software guide)
2.2.2 Laser Module Configuration
The laser module contains a specially configured and focussed laser source,
mounted within a sealed housing. An optical flat element is fixed in place on the top cover of the housing. The electrical
contacts on the end of the housing connect to the contacts within the NS300 to provide the laser module with power.
Optical flat
Power contact
2.2.3 Top-Plate Selection
The NS300 is supplied with two different top-plates that can be mounted on the laser
module. The assembly, fluidic connections and the instructions for cleaning will vary
according to which design of top-plate is being used. Please refer to the appropriate
section of this manual for the low volume flow-cell top-plate or the O-ring top-plate.
The low volume flow-cell (LVFC) top-plate (Fig. 4) is suitable for use with chemically
compatible aqueous solutions and can be cleaned by flushing wash fluid through the
chamber, avoiding the need to manually disassemble and clean the chamber after
every sample.
The LVFC is suitable for use with non-flammable, water-based solvents with
neutral pH. Ethanol and other solvents may not be compatible with wetted
surfaces and should not be used without confirming suitability with the listed
materials.
Figure 4: Low Volume Flow Cell
Figure 1: USB and camera connection
Figure 2: Power connection
Figure 3: NS300 laser module

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The O-ring top-plate (ORTP) provides greater chemical compatibility for non-
aqueous solvents, or samples which are more viscous or contain larger particles,
which may block the flow-cell.
The ORTP (Fig. 5) must be disassembled from the laser module and manually cleaned
after each sample to prevent particle carryover.
2.2.4 External Fluidic Setup
Both top-plates can be used with fluidic tubing which attaches to a tubing port holder on
the right-hand side of the NS300 casing (Fig 6). The holder can be slid in and out of the
case for easy access to the fluidic connectors. The tubing port holder is held in place with
a black nylon screw on the inside of the casing, accessed by opening the front hatch.
Tubing is supplied for use with the low volume flow cell or when the O-ring top-plate is
being used with the syringe pump accessory.
In both cases, the left-hand port on the tubing holder is the inlet port, used to introduce
sample or wash fluid into the system. Connect the inlet tubing TUB0281 to the left port
with a Luer port fitting on the end to load sample into the system via a disposable
syringe.
The right-hand tubing port is the waste outlet. Connect the outlet tubing TUB0288 to the
right port and to one of the ports on the waste bottle cap using the fittings supplied. This
is important as raising the waste prevents syphoning of liquid when changing sample
syringes.
The internal tubing should be attached to the top-plate inside the casing as described in
the following sections for the low volume flow cell or the O-ring top-plate.
Figure 5: O-Ring Top Plate
Figure 6: External NS300
Fluidic Connections

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3. Low Volume Flow Cell
3.1 System Setup
3.1.1 Flow Cell Assembly
The low volume flow cell top-plate consists of two separate parts – the manifold (NTA0065), with ports to attach the tubing
fittings (Fig 7A), and a gasket component (NTA0066) which contains an embedded microchannel and chamber seal (Fig.
7B). The LVFC is supplied with NS300 systems with the two components already assembled. The complete flow cell
assembly should be mounted on the optical glass flat in the NS300 laser module using the four sprung fastening bolts
supplied.
Check that there are no fibers or dust particles on either sealing surface before attaching the flow cell to the
laser module. Contamination of the sealing area can cause fluid leaks. If such contamination is present do not
attempt to remove physically but follow instructions for cleaning, described later.
A B
Figure 7; A: Low volume flow cell manifold
(NTA0065 )
B: Low volume flow cell gasket component
(NTA0066)
C D
C: LVFC assembly with PDMS chamber seal on
the underside of the gasket component D: LVFC assembly mounted on laser module with sprung fastening bolts

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The supplied sprung bolts should be used to connect the flow cell to the laser module. The bolts should be gently finger
tightened until an increased resistance is felt when the bolts reach the end of the screw thread. This attaches the gasket
component with PDMS chamber seal to the optical glass flat, forming a chamber in which the sample can be measured
with the NS300 (Fig 7C).
Do not overrtighten the fastening screws as this can cause damage to the screw threads
When using the LVFC, liquid is loaded into the system through fluidic tubing connected to ports on the flow cell manifold,
set up as described in Section 3.1.2.
Important!
The LVFC is suitable for use with non-flammable, water-based solvents with neutral pH. Ethanol and other solvents may not
be compatible with wetted surfaces and should not be used without confirming suitability with the listed materials. If in
doubt about the choice of solvent and its compatibility with any part of the Malvern device, please contact
[email protected] for further information.
3.1.2 Flow Cell Tubing Connection
When using the low volume flow-cell, liquid is always loaded into the system using a disposable 1ml syringe. The syringe
connects via a Luer fitting to the inlet fluidic tubing on the left port of the tubing holder on the outside of the NS300
casing.
Inside the casing, inlet and outlet tubing is connected from the corresponding ports on the inside of the tubing holder to
ports on the flow cell using the fittings supplied. The inlet and outlet tubing connections are as shown in Fig. 8.
Figure 8: Low Volume Flow Cell Tubing Configuration
The inlet tubing TUB0281 connects to the tubing holder port nearest the front of the NS300 and the left port of the low
volume flow cell manifold (looking from the tubing connection end of the manifold). Note that before using the system to
load samples, it is recommended to rinse out the inlet tubing with sample or buffer to remove any trapped air before
connecting the inlet fitting to the flow cell (see Section 3.2.1 – Priming Tubing)
The outlet tubing TUB0288 connects to the tubing holder port nearest the back of the NS300 and the right port of the low
volume flow cell manifold.
Ensure there are no kinks or blockages in the outlet tubing. This will cause an increase in pressure inside the flow
cell, and could cause the seal to leak.

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3.2 Flow Cell Usage
3.2.1 Priming Tubing
Important: For the low volume flow cell, the inlet fluidic tubing should be rinsed out with buffer or sample, before the
tubing is connected to the top-plate and the top-plate primed for use. This improves bubble clearance from the tubing on
initial priming, reducing the likelihood of air bubbles entering the sample chamber and causing problems in subsequent
measurements.
To rinse through the inlet tubing with buffer or sample:
Make sure the inlet tubing fitted to the inside of the NS300 casing is not connected to the top-plate
Place the end of the inlet tubing into a suitable waste container
Insert a 1ml syringe of liquid into the Luer port and push ~900ul of the liquid through the inlet tubing as fast as the
back pressure will allow (should take approximately 5-10 seconds).
Leave the syringe with the remaining liquid attached to the Luer port to prevent any air being introduced
Initially rinsing the inlet tubing at higher speeds allows you to better remove any air initially trapped in the tubing
or connectors. Take care that the pressure generated does not force the syringe out of the Luer port.
3.2.2 Changing Inlet Tubing Syringe
It is important to ensure that liquid to liquid contact is always maintained at the syringe port when changing over the
syringe fitted to the inlet tubing e.g. changing between buffer and sample, or when replacing an empty syringe.
Before changing syringes, have the next syringe prepared, ensuring there are no air pockets present at the tip and that
there is a small positive meniscus (bead of liquid) protruding from the syringe. Keep the Luer port as low as possible (at
bench level) when changing syringes to prevent liquid draining from the Luer port. Remove the old syringe from the Luer
port and insert the new syringe into the Luer port (keeping syringes and Luer port horizontal) such that the two meniscuses
combine without trapping an air bubble.
3.2.3 Loading an Initial Sample
We recommend that before proceeding with analysis, a sample of any buffer or diluent is checked to confirm that it doesn’t
contain any contaminating nanoparticles.
The sample should be loaded with the laser module outside of the instrument. As the sample is loaded, the presence of any
air pockets or bubbles can be detected and removed.
Sample is loaded into the flow cell chamber with a 1ml disposable syringe connected to the Luer fitting on the inlet tubing.
Important: The low volume flow cell must only be used with disposable 1ml syringes with Luer port fitting.
Using syringes with larger volume than 1ml, or exceeding the maximum rated flow speed of 0.05ml per second
(1ml total in 20 seconds) may result in leaking or damage to components.
Once the flow cell is mounted onto the laser module and the inlet tubing has been pre-rinsed with buffer or sample (See
Section 3.2.1), the inlet tubing can then be connected to the flow cell manifold to load liquid into the sample chamber.

NanoSight NS300 Operating Manual
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i. Connect the end of the inlet tubing inside the NS300 casing
to the LEFT port of the low volume flow cell manifold
ii. Fill a 1ml disposable syringe with the appropriate buffer or
sample.
iii. Remove any air bubbles from the syringe
iv. Insert the new syringe into the Luer port, ensuring liquid-to-
liquid contact is maintained (See Section 3.2.2 for additional
guidance on changing the syringe)
v. Introduce the buffer or sample slowly into the chamber. The
flow cell should not be loaded at speeds exceeding 0.05ml
per second (1ml total in 20 seconds).
vi. The low volume flow cell is now loaded ready for use. If
using a syringe pump, place the syringe into the syringe
pump holder and operate as described in the NanoSight
Syringe Pump Operating Manual.
Important:
Occasionally a bubble may be present in the chamber and in the path of the laser beam. This might cause some degree of
specular reflection of the laser beam off the bubble surface. This will degrade image quality and should be removed before
analysis. If bubbles are routinely observed when a sample is firsty loaded, the flow cell should be cleaned manually to
remove any contamination which might be preventing good wetting of the surface. Certain samples may generate bubbles
through out-gassing of dissolved gases over extended periods. In both cases, the image collected by the camera supplied
will show a high intensity region (or may be completely saturated) indicative of the presence of such bubbles. If samples
repeatedly show evidence of bubble formation, it is advisable to de-gas the sample before analysis,
3.2.4 Changing Samples
The flow-cell top-plate has been designed so that the system can be flushed clean between samples (dependent on sample
type) with particle carryover of less than 1%. It is not necessary to remove the flow cell or disconnect the tubing for flush
cleaning.
i. Flush the system by loading a 1ml syringe of clean water or buffer solution. The flow cell should not be flushed at
speeds exceeding 0.05ml per second (1ml total in 20 seconds).
Note: Ensure that liquid to liquid contact is maintained at the Luer port when changing over the syringe
ii. Confirm the cleanliness of the chamber by checking for any particles present in the NTA software image
iii. Repeat the flush if necessary to remove remaining sample particles
iv. Load the next sample syringe into the system. Make sure to load at least 800ul of the new sample through the
system to prevent significant dilution of the sample with wash fluid remaining in the outlet waste tubing.
Important: Using syringes with larger volume than 1ml, or exceeding the maximum rated flow speed of 0.05ml
per second (1ml total in 20 seconds) may result in leaking or damage to components.
If sample particles persist in the image after rinse through cleaning, or if many particles stuck to the optical flat are visible,
increasing the background noise on the images, the flow-cell should be cleaned manually as described in Section 3.2.5.
When using samples which commonly adhere to the optical glass surfaces, it is more appropriate to use the O-ring top-
plate, which is dissassembled and thoroughly cleaned between each measurement.
Figure 9: Loading a sample with the low volume flow cell

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3.2.5 Manual Cleaning Procedures
The LVFC has been designed not to require manual cleaning between samples (dependent on sample type). Between
samples, push at least 1 ml of water or diluent through the sample chamber (following the advice in Section 3.2.4), to
remove any particles present. This ensures carry over of less than 1 %.
A manual clean is only necessary if there is visible cloudiness or sample residue stuck to the optical flat or the glass window
in the gasket component. The LVFC must be removed from the laser module but does not need any further disassembly for
manual cleaning.
Whilst the unit is splash-proof, under no circumstances must excess liquid be allowed to enter the
housing of the laser module at any time. This will cause irreparable damage to the laser mounted
within the unit.
It is important to take care not to abrade or scratch the optical flat surface, or introduce particulates or contaminants onto
the surface. Treat all optical surfaces with the same care as would be employed with equivalent surfaces on your
microscope. If the optical flat in the laser module becomes damaged, it can be replaced by Malvern Instruments (pricing on
request).
The recommended practice for manually cleaning the optical flat on the laser
module and the low volume flow cell top-plate is given below:
Before performing a manual clean, the system should be flushed to clean
sample from the tubing and fittings.
Once all tubing assemblies are flushed clean, empty the fluidics by loading a
1ml syringe full of air through the system (do not exceed maximum rated
flow speed of 0.05ml per second).
Open the door on the NS300 instrument and rotate the red lever to the left
to release the laser module. Slide out the laser module from inside the
NS300 so that the LVFC can be easily accessed
Disconnect the tubing fittings from the low volume flow cell top-plate,
remove the sprung fastening bolts, and lift the flow cell gently off the laser
module
Do not wipe or touch the chamber seal on the underside of the
gasket component
Wet a tissue with water, (or a solution of up to 10% ethanol if needed),
and use this to wipe the optical flat on the laser module.
Do not pour any liquid over the laser module, as this could
penetrate the casing and damage the laser inside.
Wipe the flat gently with a soft dry tissue (e.g. Mediwipes) to remove any
streaks from the optical surface and then dry with compressed air.
Figure 10: Unmounting the laser
module from the NS300
Figure 11: Cleaning the optical flat on the
laser module

NanoSight NS300 Operating Manual
(MAN0516-08-EN-00) 18
Using a dripping or low-pressure source, e.g. a water bottle, rinse the manifold ports and the area inside the chamber
seal on the underside of the gasket with water, (or a solution of up to 10% ethanol if needed), to remove any
remaining sample particles.
If, and only if, any sample residue is visible on the glass window surface,
the underside of the glass can be gently cleaned using a small cotton bud
dampened with water or a solution of up to 10% ethanol (Fig. 12).
When cleaning the glass window inside the chamber seal area, take care to
limit any rubbing of the surrounding soft PDMS seal.
The glass window in the top-plate is fragile, and should be
treated with care during manual cleaning. Do not apply any
pressure to the top surface of the glass as this may cause the
viewing window to become separated from the gasket
component.
Do not use a more concentrated than 10 % ethanol solution
as this may damage LVFC components.
Important: If the gasket component repeatedly leaks after a manual
clean, it should be replaced (See Section 3.3.1)
After manual cleaning, the low volume flow cell must be dried as described in Section 3.2.5 before it can be
remounted on the laser module.
3.2.5 Drying the Low-Volume Flow Cell
Direct compressed air through the tubing ports on the manifold to thoroughly dry the embedded microchannel inside
the gasket component (Fig. 13A).
Once the channel is dry, turn the flow cell assembly over and, keeping the nozzle of the compressed air at least 15 cm
from the seal, lightly dry the underside of the component and the area inside the chamber seal (Fig. 13B).
Repeat as necessary until the gasket component is completely dry.
Figure 13, A: Drying gasket component through
manifold tubing ports
B: Drying the chamber seal on the
underside of the gasket component
Avoid touching or rubbing the seal when drying the underside of the gasket component. Keep the
compressed air nozzle at least 15cm away, to avoid the seal being damaged by high pressures
Once the low volume flow cell is dry, check that there are no fibers or dust particles on the gasket component seal. The
flow cell is then ready for reattaching to the NS300 laser module
Figure 12: Cleaning the underside of the
glass window in the gasket component

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3.3 Care and Maintenance
3.3.1 Gasket Component Replacement
When supplied with new NS300 systems, the LVFC is supplied fully assembled and
does not need to be disassembled for normal usage and cleaning.
If the flow cell needs to be disassembled, i.e. if the gasket component seal
becomes worn or damaged and needs replacing, use a 2 mm Allen / Hex key
(part contained in NTA4111) to undo the fixing bolt on the underside of the
flow cell and disconnect the gasket component from the manifold (Fig. 14).
Before reassembling with a new gasket component,
ensure that the circular seals are bedded fully down in the
ports on the manifold (Fig. 15).
If the circular seals have lifted out during disassembly, wet
the seals with deionised water and push them fully into the
sockets on the manifold, ensuring that they are fully seated
and level. If the circular seals need replacing, 2 spare seals
are provided with each gasket component supplied.
Lay the gasket component into the recess on the manifold ensuring the
holes in the gasket line up with those in the manifold and then hold in
place by inserting the fixing bolt.
Tighten the bolt until the gasket component is held securely – the circular
seal contact area can be viewed from the underside of the manifold and
should be checked to confirm an visibly unbroken seal contact (Fig. 16)
Check that there are no fibers or dust particles on the PDMS chamber seal
on the underside of the gasket component (Fig.7C). The flow cell is then
ready for reattaching to the NS300 laser module.
Figure 14: Disassembly of the
flow cell
Figure 15: Circular seals pressed fully down into the
manifold (only just protruding from the surface)
Figure 16: Visual of the circular seal contact
areas from underneath, confirming a good
connection of the gasket component to the
manifold

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4. O-Ring Top Plate
4.1 System Set-Up
The O-ring top-plate contains an embedded O-ring seal. The supplied sprung bolts should be used to connect the top-plate
to the laser module, forming the sample chamber. The bolts should be gently finger tightened until an increased resistance
is felt when the bolts reach the end of the screw thread.
Do not overrtighten the fastening screws as this can cause damage to the screw threads
The O-ring top-plate can be setup in two different ways, according to how samples are to be loaded into the system.
4.1.1 O-Ring Top-Plate Assembly for Manual Injection
Samples can be loaded into the chamber using a disposable 1ml syringe, directly connected to a luer port on the top-plate.
A push-fit elbow luer connector is provided so that the laser module fits into the NS300 with the syringe remaining in place.
4.1.2. O-Ring Top-Plate Assembly for Syringe Pump Use
A sample can also be loaded into the O-ring top-plate through the NS300 fluidic
tubing for use with a syringe pump accessory.
In this case, the Luer ports should be removed and the tubing connected using the
fittings supplied, as described in Section 4.1.3.
Sprung
fastening
screws
(spring held
by circlip
attached to
the bolt)
O-ring seal
Figure 17; A Sprung fastening bolts and
O-Ring Top-plate (ORTP)
B: ORTP with Luer ports and push fit
elbow Luer connector
C: ORTP mounted on the laser module
inside the NS300
Figure 18: O-ring Top-Plate with tubing
fittings for syringe pump use