pyroscience Piccolo2 User manual

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Piccolo2
FIBER-OPTIC OXYGEN METER
USER MANUAL

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Document Version 2.04
Refers to Pyro Oxygen Logger Software version >3.2
The Piccolo2 is manufactured by
Pyro Science GmbH
Hubertusstr. 35
52064 Aachen
Germany
Phone [phone]
Fax [phone]
Email [e-mail]
Internet www.pyro-science.com
Registered: Aachen HRB 17329, Germany

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TABLE OF CONTENT
1OVERVIEW ..............................................................................5
2SAFETY GUIDELINES.............................................................. 6
3INTRODUCTION TO THE PICCOLO2......................................... 8
4SOFTWARE INSTALLATION.................................................... 9
5OXYGEN SENSOR TYPES.......................................................10
5.1 ROBUST PROBE ....................................................................... 10
5.2 DIPPING PROBE ....................................................................... 11
5.3 SENSOR SPOTS........................................................................ 11
5.4 FLOW-THROUGH CELLS ............................................................ 13
5.5 RESPIRATION VIALS.................................................................. 14
5.6 NANOPROBES ......................................................................... 16
5.7 CONNECTING THE SENSORS AND OPTICAL FIBERS ......................... 18
5.8 CLEANING AND MAINTENANCE OF THE SENSORS........................... 20
6THE SOFTWARE "PYRO OXYGEN LOGGER" ...........................22
6.1 MAIN WINDOW........................................................................ 22
6.2 SETTINGS................................................................................ 30
6.2.1 Basic Settings.................................................................... 31
6.2.2 Advanced Settings............................................................. 32
6.2.3 Conditions in the Sample ................................................... 33
6.2.4 Options ............................................................................. 34
6.3 RAW DATA WINDOW ................................................................ 36
7CALIBRATION OF OXYGEN SENSORS ....................................38
7.1 CALIBRATION .......................................................................... 38
7.1.1 Calibration Mode: Factory.................................................. 39
7.1.2 Calibration Mode: 1-Point .................................................. 40
7.1.3 Calibration Mode: 2-Point .................................................. 42
7.1.4 Calibration Mode: Custom.................................................. 44
7.2 ADVANCED ADJUSTMENTS......................................................... 45
8CALIBRATION STANDARDS...................................................47
8.1 THE AIR CALIBRATION STANDARD .............................................. 47

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8.1.1 Ambient Air ....................................................................... 49
8.1.2 Water-Vapor Saturated Air ................................................ 49
8.1.3 Air Saturated Water........................................................... 50
8.2 THE 0% STANDARD.................................................................. 51
8.2.1 Water Mixed with a Strong Reductant ................................ 51
8.2.2 Water Flushed with Nitrogen Gas....................................... 51
8.2.3 Nitrogen Gas ..................................................................... 52
9CALIBRATION OF CONTACTLESS SENSORS .......................... 53
9.1 CALIBRATION PROCEDURE......................................................... 53
9.2 MANUAL BACKGROUND COMPENSATION .................................... 54
10 APPENDIX.............................................................................56
10.1 SPECIFICATIONS OF THE PICCOLO2.............................................. 56
10.2 TROUBLESHOOTING ................................................................. 57
10.3 MEASURING PRINCIPLE ............................................................. 58
10.4 OPERATING SEVERAL PICCOLO2IN PARALLEL................................60
10.5 DEFINITION OF OXYGEN UNITS................................................... 61
10.6 TABLE OF OXYGEN SOLUBILITY .................................................. 63
10.7 EXPLANATION OF THE SENSOR CODE .......................................... 65

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1Overview
The ultra-compact Piccolo2 is a fiber-optic oxygen meter
integrated in a small USB stick housing. Despite its small size, it
features the proven REDFLASH technology from Pyro Science (see
Appendix 10.3 for more details). The Piccolo2 can be used in
combination with a variety of optical oxygen sensors, like robust
probes, dipping probes or contactless sensors (sensor spots,
respiration vials, flow-through cells, nanoprobes). The optimized
optics of the Piccolo2 enable contactless oxygen measurements up
to a window thickness of 20 mm. The sensors can be used in water
samples (dissolved oxygen, DO), as well as in the gas phase
(gaseous oxygen, O2). Simply plug the Piccolo2 into a USB port of
your Windows PC or Windows tablet, connect the oxygen sensor of
your choice and start measuring with the comfortable logging
software "Pyro Oxygen Logger".
More information concerning our products can be found at
www.pyro-science.com
or contact us under [e-mail]
Your Pyro Science Team

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2Safety Guidelines
The Piccolo2 is a laboratory instrument to be used at constant
experimental temperatures with optical oxygen sensors (optodes)
from Pyro Science. In order to guarantee an optimal performance
of the Piccolo2, please follow these operation instructions and
safety guidelines.
Please note that opening the housing will void the warranty. There
are no serviceable parts inside the device.
The Piccolo2 and the sensors should be used in the laboratory by
qualified personnel only, following the operation instructions and
safety guidelines of this manual. They should be kept and stored
out of reach of children in a secure place under dry and clean
conditions at room temperature, avoiding moisture, dust,
corrosive conditions and heating of the instrument.
The Piccolo2 and the sensors are not intended for medical, military
or other safety-relevant areas. They must not be used for
applications in humans; not for in vivo examination on humans, not
for human-diagnostic or therapeutic purposes. The sensors must
not be brought in direct contact with foods intended for
consumption by humans.
Please follow the appropriate laws and guidelines for safety like
EEC directives for protective labor legislation, national protective
labor legislation, safety regulations for accident prevention and
safety data-sheets from manufacturers of chemicals used during
the measurements.
When used in the field, extreme environmental conditions (like
high humidity, dust, and exposure to (salt) water or intense solar
radiation) can cause damage to the Piccolo2.

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Before using the Piccolo2 and the sensors, carefully read the
instructions and user manuals.
In case of problems or damage, disconnect the instrument and
mark it to prevent any further use! Consult Pyro Science for
advice! There are no serviceable parts inside the device. Please
note that opening the housing will void the warranty!
The Piccolo2 is not watertight, is sensitive to corrosive
conditions and to changes in temperature causing
condensation. Avoid temperatures above 50°C (122°F) or below
0°C (32°F). Avoid any elevated humidity causing condensing
conditions.
Handle the sensors with care, especially after removal of the
protective cap! Prevent mechanical stress to the fragile sensing
tip! Avoid strong bending of the fiber cable!
Calibration and application of the sensors are on the user’s
authority, as well as data acquisition, treatment and
publication!
The sensors and the oxygen meter Piccolo2 are not intended for
medical, diagnostic, therapeutic, or military purposes or any
other safety-critical applications. The sensors must not be used
for applications in humans and must not be brought in direct
contact with foods intended for consumption by humans.
The sensors should be used in the laboratory by qualified
personnel only, following the user instructions and the safety
guidelines of the manual, as well as the appropriate laws and
guidelines for safety in the laboratory!
Keep the sensors and the oxygen meter Piccolo2 out of reach of
children!

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3Introduction to the Piccolo2
The Piccolo2 is a miniature USB-driven fiber-optic oxygen meter
for usage in the laboratory. It is compatible to a variety of oxygen
sensors from Pyro Science, like robust probes, dipping probes and
contactless sensors, including sensor spots, respiration vials, flow-
through cells and nanoprobes.
These sensors are available in versions for the full range (0-
50% O2, max. range 0-100% O2), and selected sensor types are
additionally available for the trace range (0-10% O2). The Piccolo2
utilizes a measuring principle based on red light excitation and
lifetime detection in the near infrared using the proven REDFLASH
indicators (REDFLASH technology, see Appendix 10.3 for more
details).
The Piccolo2 can be connected directly to a Windows PC with the
USB plug, providing energy supply and data exchange with the PC.
If a greater distance between the PC and the Piccolo2 is needed,
the delivered USB extension cable can be used.
Comfortable calibration and logging functions are provided by the
logging software Pyro Oxygen Logger, available as free download.
IMPORTANT: The Piccolo2 is intended for oxygen measurements
at constant and defined temperatures! Do not submerse the
housing of the Piccolo2 into liquid samples! This destroys the
device. Protect the Piccolo2 against water spray!

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4Software Installation
IMPORTANT: Do not connect the Piccolo2 to your PC before the
Pyro Oxygen Logger software has been installed. The software will
install the appropriate USB-drivers automatically.
System requirements:
PC or tablet with Windows XP / Vista / 7 / 8 / 10 (but not
Windows RT) and min. 200 MB free disk space
Installation steps:
•download the installer package for the newest version of the Pyro
Oxygen Logger software from the Pyro Science homepage:
www.pyro-science.com/downloads.html
•unzip, then start the installer and follow the instructions
•connect the Piccolo2 to a free USB port of the computer
After successful installation, a new program group "Pyro Oxygen
Logger" is added to the start menu, and a shortcut named "Oxygen
Logger" can be found on the desktop.

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5Oxygen Sensor Types
For the Piccolo2 meter, Pyro Science offers a range of optical
oxygen probes and contactless oxygen sensors, comprising sensor
spots, flow-through cells, respiration vials and nanoprobes. For an
overview of all available oxygen sensor types, please visit the Pyro
Science website.
5.1 Robust Probe
Characteristics: robust probe for Piccolo2 (item no. OPROB3) with
stainless steel tubing 3 mm in diameter and
30 mm in length
oxygen sensor with optical isolation located
in the 3 mm disc at the tip of the tubing
(sensor end, see chapter 5.7)
fiber cable 3 mm in diameter and 1 m in
length
Note: The fiber cable needs to be handled with care, as strong
bending can lead to breakage and damage of the robust probe.
Applications:
standard laboratory applications in gases and liquids
for aqueous liquids: permanent stirring is recommended, as
diffusion limitation of oxygen towards the 3 mm tip has a
measurable effect on the response time in liquid samples (this
diffusion limitation is negligible in gas samples)

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5.2 Dipping Probe
Characteristics: dipping probe for Piccolo2 (item no. OPDIP20)
with
stainless steel tubing 3 mm in diameter and
200 mm in length
oxygen sensor with optical isolation located
in the 3 mm disc at the tip of the tubing
(sensor end, see chapter 5.7)
Applications:
standard laboratory applications in gases and liquids
full probe length can be dipped into the sample
in aqueous liquids: permanent stirring is recommended, as
diffusion limitation of oxygen towards the 3 mm tip has a
measurable effect on the response time in liquid samples (this
diffusion limitation is negligible in gas samples)
Important: No part of the Piccolo2 device itself must be
submersed into liquid samples!
5.3 Sensor Spots
Measuring principle: oxygen sensor spots (item no. OXSP5)
oxygen measurements with contactless read-out
through transparent container walls

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sensor spot with REDFLASH indicator needs to
be glued to the inner container wall
excitation and detection of oxygen indicator
luminescence emission through the
transparent container wall with an optical fiber
fixed at the outer container wall
Characteristics:
PET foil coated with REDFLASH indicator and optical isolation
standard diameter of 5 mm
to be glued with the green back side on transparent, clean and dry
inner container walls (e.g. glass or acrylic glass)
black sensing surface has to be covered completely by the gaseous
or aqueous sample
can be autoclaved few cycles at 121°C for 15 min (requires
calibration afterwards)
Accessories for readout: 0-10 mm thick transparent windows
basic spot adapter SPADBAS: 12 mm in diameter, needs to be
fixed tightly or glued to the outer container wall at the spot
position
optical fiber PICFIB2: black fiber cable, 3 mm in
diameter, 1 m in length, connecting the basic
spot adapter SPADBAS with the Piccolo2 meter
(see also chapter 5.7) or, alternatively,
optical fiber rod PICROD2:stainless steel tubing, 3 mm diameter,
40 mm length, connecting the basic spot adapter SPADBAS with
the Piccolo2 meter (see also chapter 5.7)
transparent silicone glue SPGLUE: based on acetic acid, for gluing
sensor spots and basic spot adapters to the container wall
The PICFIB2 and PICROD2 are also compatible with the flow-
through cells and respiration vials from Pyro Science.

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Note: A change of position of the spot adapter after calibration of
the sensor spot might require a new calibration.
Accessories for readout: 10-20 mm thick transparent windows
optical fiber rod PICROD3: ca. 50 mm in length, needs a
customized fixation on the outer container wall (not compatible
with the spot adapter SPADBAS)
The optical fiber rod PICROD3 is not compatible with the basic
spot adapter SPADBAS. The user has to build a custom fixation at
the wall of the sample container.
Applications:
multi-sampling measurements
measurements of oxygen at greater scales
measurements in closed sample containers,
e.g. in respiration chambers, bioreactors, cell
biological approaches and industrial process water-monitoring
CAUTION: Oxygen measurements in air-tight containers or setups
require special precautions, like constant conditions during
measurements. More details on request.
Options:
trace range version (item no. TROXSP5): for measurements close
to 0% O2 (range: 0-10% O2)
5.4 Flow-Through Cells
Characteristics: flow-through cells (item no. OXFTC, OXFTC2)
flow-through cells with integrated oxygen sensor
available as small version OXFTC and as large version OXFTC2

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equipped with Luer lock connectors on both tubing ends
included Luer lock adapters allow connection of gas-
tight tubings (e.g. stainless steel, ISO-VERSINIC®) with
about 1.0-2.0 mm inner diameter to the OXFTC and
about 3.0-4.0 mm to the OXFTC2
Accessories for readout:
optical fiber PICFIB2: black fiber cable, 3 mm in diameter, 1 m in
length, connecting the flow-through cell with the Piccolo2 meter
(see also chapter 5.7)
Applications:
contactless measurement of oxygen in a
gaseous or liquid sample pumped through
the cell
CAUTION: For liquids, a flow rate of ca. 10-100 mL/min for the
OXFTC and of 20-500 mL/min for the OXFTC2 is recommended!
Lower flow rates are in principle possible; however the user has to
ensure sufficient gas tightness of the connecting tubing.
Options:
trace range version (item no. TROXFTC): for measurements close
to 0% O2 (range: 0-10% O2)
5.5 Respiration Vials
Characteristics: respiration vials (item no. OXVIAL4, OXVIAL20)
ready assembled respiration vials with integrated stripe of the
oxygen sensitive REDFLASH indicator with optical isolation
available with a (uncalibrated) volume of ca. 4 mL (OXVIAL4)
or ca. 20 mL (OXVIAL20)

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elongated shape of the integrated sensor allows
oxygen measurements at different heights within the vial
Note: A change of position of the adapter ring after sensor
calibration might require new calibration.
Accessories for readout:
adapter rings ADVIAL4 / ADVIAL20: for easy fixation of the optical
fiber PICFIB2 to OXVIAL4 / OXVIAL20
optical fiber PICFIB2: black fiber cable, 3 mm in diameter, 1 m in
length, can be fixed in the adapter ring with the clamping screw,
connecting the respiration vial with the Piccolo2 meter (see also
chapter 5.7)
Applications:
for oxygen measurements in e.g. stirred aqueous liquids or
gaseous sample
small-scale respirometry and metabolic rate measurements of e.g.
cell cultures, eggs, larvae, small crustaceans, small fish, water-,
plant-, algal-samples etc.
CAUTION: Oxygen measurements in air-tight vials require special
precautions, like e.g. constant temperature. More details on
request.

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5.6 Nanoprobes
Measuring principle: oxygen nanoprobes (item no. OXNANO)
nanoparticles coated with oxygen-
sensitive REDFLASH indicator
dispersible in aqueous solutions, like e.g.
water, culture media
contactless readout from outside through
transparent windows
Characteristics:
oxygen nanoprobes OXNANO in 5 x 10 mg units
dispersible in aqueous solutions
ultra-fast response times
simple batch calibration
measurements at different positions or in
different vials containing the same dispersion
Accessories for readout: vials,microwell plates
basic spot adapter SPADBAS: 12 mm in diameter, needs to be
fixed tightly or glued to the outer container wall
optical fiber PICFIB2: black fiber cable, 3 mm in diameter, 1 m in
length, connecting the basic spot adapter with the Piccolo2 meter
(see also chapter 5.7) or, alternatively,
optical fiber rod PICROD2:stainless steel tubing, 3 mm in
diameter, 40 mm in length, connecting the basic spot adapter with
the Piccolo2 meter (see also chapter 5.7)
Accessories for readout: microfluidic applications
optical fiber with integrated focusing lens PICFIB2-LNS: black fiber
cable, 1 m in length, stainless steel tip 3 mm in diameter,
connecting the basic spot adapter or custom fixation with the
Piccolo2 meter (see also chapter 5.7) or, alternatively,

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optical fiber rod with integrated focusing lens PICROD2-LNS:
stainless steel tubing, 3 mm in diameter, 40 mm in length,
connecting the basic spot adapter or custom fixation with the
Piccolo2 meter (see also chapter 5.7)
Applications:
real-time monitoring
high through-put screening in e.g. microwell
plates
contactless measurements of fast processes,
like enzymatic reactions
in microfluidic devices (microfluidic chip,
lab-on-a-chip device)
more details on request
CAUTION: Oxygen measurements in closed vials require special
precautions, like constant conditions during measurements. More
details on request.

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5.7 Connecting the Sensors and Optical Fibers
The robust probe OPROB3, the dipping probe OPDIP20, and the
optical fibers PICFIB2 / PICFIB2-LNS or the optical fiber rods
PICROD2 / PICROD3 / PICROD2-LNS must be connected to the
sensor port of the Piccolo2 with their instrument end. At the
instrument end IE, the core of the optical fiber protrudes ca. 1 mm
from the fiber jacket. For OPROB3, OPDIP20 and PICFIB2 the
instrument end IE is marked with "Instrument" on the attached
label.
First, remove the protective caps from both ends of the optical
probe / fiber / rod. Then slightly unscrew and loosen the nut at the
sensor port of the Piccolo2. The nut must not be removed from the
sensor port. Typically, unscrewing the nut with a single half turn
(180°) is sufficient. Insert now the instrument end IE carefully into
the sensor port of the Piccolo2, the insertion length is about 24
mm (see pictures with insertion of a) a dipping probe and b) an
optical fiber rod).

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Insert the optical probe / fiber / rod carefully as deep as possible
(ca. 24 mm) into the sensor port of the Piccolo2!
Fix the optical probe / fiber / rod by screwing down the nut tightly
onto the sensor port of the Piccolo2.
a)
b)
Then, insert the adapter end (AE) of the optical fibers PICFIB2 /
PICFIB2-LNS or optical fiber rods PICROD2 / PICROD2-LNS into
the basic spot adapter, adapter ring or flow-through cell, and fix it
with the clamping screw.
For OPROB3 and OPDIP20, insert the sensor end (SE) into the gas
or (stirred) aqueous liquid sample.

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5.8 Cleaning and Maintenance of the Sensors
Oxygen sensors for the Piccolo2 can be sterilized with ethylene
oxide (EtO) and can be cleaned with ethanol, peroxide (3% H2O2)
or soap solution. They can be applied in gas phases, in aqueous
solutions, as well as in ethanol, methanol or isopropanol (robust /
dipping probe: only short-term application in diluted ethanol,
methanol or isopropanol). Other organic solvents and gaseous
chlorine (Cl2) interfere with the oxygen sensor reading. No cross-
sensitivity is found for pH 1-14, CO2, CH4, H2S and any ionic
species.
A signal drift of the sensor can indicate photo-bleaching of the
oxygen-sensitive REDFLASH indicator, depending on the ambient
light intensity, as well as the intensity of the excitation light and
the sample interval. This can necessitate new calibration of the
sensor and eventually also readjustment of the Sensor Settings
(LED intensity; see also chapter 6.2.2). In case of sensor spots, re-
positioning of the spot fiber on the sensor spot and subsequent
new calibration might be necessary. If the signal intensity is getting
too low (typically after several millions of data points), as indicated
by the horizontal indicator bar in the main window of the Pyro
Oxygen Logger software and by the respective warning (see
chapter 6), the sensor needs to be replaced.
After finalization of the measurements, the sensor tip of the robust
and dipping probe should be rinsed carefully with demineralized
water. Especially after application in seawater, it is recommended
to clean the sensor thoroughly with demineralized water. After
drying, put on carefully the protective caps onto both ends of the
probe.
For sensor spots and respiration vials, wet cotton swabs can be
used for carefully wiping the sensing surface. Rinse the sensing