BMG Labtech PHERAstar FS User manual

Operating Manual
Revision A

PHERAstar FS Operating Manual BMG LABTECH
2/26 0471B0001A 2009-09-22
This manual was designed to guide PHERAstar FS users through the basic hardware features of the
microplate reader instrument.
Although these instructions were carefully written and checked, we cannot accept responsibility for problems
encountered when using this manual. Suggestions for improving this manual will be gratefully accepted.
BMG LABTECH reserves the right to change or update this manual at any time. The revision number is
stated at the bottom of every page.
For contact information please visit www.bmglabtech.com or send an email to [email protected]
Copyright 2009 BMG LABTECH. All rights reserved. All BMG LABTECH brand and product names are trademarks of BMG
LABTECH. Other brand and product names are trademarks or registered trademarks of their respective holders.

BMG LABTECH PHERAstar FS Operating Manual
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The PHERAstar FS is a multifunctional microplate reader that can perform a wide variety of applications for
fluorescence intensity, fluorescence polarization, time-resolved fluorescence, HTRF®, luminescence,
AlphaScreen®and full spectrum absorbance. The PHERAstar FS has high-end performance in all plate
formats and in all detection modes. By using proprietary technology in a variety of important parts, the
PHERAstar FS will deliver high-end results.
PHERAstar FS

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TABLE OF CONTENTS
1TECHNICAL SPECIFICATIONS 5
2OVERVIEW 7
2.1 FRONT AND RIGHT SIDE OF PHERASTAR FS 7
2.2 BACK OF PHERASTAR FS 8
3INSTALLATION 9
3.1 TRANSPORT LOCKS 9
3.1.1 UNLOCKING THE PLATE CARRIER 9
3.1.2 LOCKING THE PLATE CARRIER 10
3.1.3 UNLOCKING AND REMOVING THE OPTIC MODULE TRANSPORT SYSTEM PROTECTION PLATE 10
3.1.4 INSERTING AND LOCKING THE OPTIC MODULE TRANSPORT SYSTEM PROTECTION PLATE 11
3.2 POWER AND COMMUNICATION CONNECTIONS 11
4DESCRIPTION OF COMPONENTS AND PERIPHERALS 12
4.1 PHERASTAR FS OPERATING LAMP 12
4.2 PLATE CARRIER IN/OUT BUTTON 12
4.3 PHERASTAR FS SHIPMENT BOX 12
4.4 INSTALLING AND CHANGING OPTIC MODULES 13
4.4.1 BUILT-IN BARCODE READER FOR OPTIC MODULES 14
4.5 DESCRIPTION OF OPTIC MODULES 15
4.5.1 FLUORESCENCE INTENSITY AND TRF OPTIC MODULE 15
4.5.2 FLUORESCENCE POLARIZATION OPTIC MODULE 15
4.5.3 TIME-RESOLVED FLUORESCENCE OPTIC MODULE,FRET AND TR-FRET, FOR EXAMPLE HTRF®16
4.5.4 LUMINESCENCE OPTIC MODULES 16
4.5.5 ALPHASCREEN®MODULE 17
4.6 REAGENT INJECTION SYSTEM 18
4.6.1 REAGENT INJECTORS 18
4.6.2 SLIDING DOOR 19
4.6.3 NEEDLE HOLDER 19
4.6.4 HOLDER FOR COMPOUND VESSELS 20
4.6.5 PUMP PRIMING 20
4.7 PHERASTAR FS -STACKER CONNECTION 22
4.8 AUTOMATIC HEIGHT SENSOR 23
5TROUBLESHOOTING 23
5.1 LOCKING THE PLATE CARRIER MANUALLY 23
5.2 TRANSPORT SYSTEM OFFSET 24
6INSTRUMENT DISINFECTION 25

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1 Technical Specifications
Detection Modes - Absorbance Spectra UV/Vis
- Fluorescence Intensity - including FRET
- Fluorescence Polarization - FarRed™ and Transcreener®
- Time-Resolved Fluorescence - including DELFIA®
- TR-FRET - including HTRF®, LanthaScreen®, LANCE®
and other TR-FRET assays
- Luminescence (flash & glow) - including BRET
- High-End AlphaScreen®
Measurement Modes - Endpoint and kinetic measurements
- Sequential dual excitation measurements
- Simultaneous dual emission measurements
- Ratiometric measurements with real-time ratio
- Top and Bottom reading
- High-Resolution cell layer scanning
Light Source - High-Energy Xenon flash lamp
- Solid state laser for AlphaScreen®
- UV Nitrogen 337 nm laser for TR-FRET
Detectors - Four photomultiplier tubes optimized for reading modes, automatic gain selection,
manual gain available
Optic modules - Up to six application specific optic modules built-in
Additional optic modules available
Spectral Range - 230 nm – 750 nm for FI, FP, LUM
- 230 nm – 900 nm for TRF
- 220 nm – 1000 nm for Absorbance
Absorbance
Spectrometer - Spectral range: 220 to 1000 nm
Full spectrum captured in < 1 s/well
Selectable spectral resolution: 1 nm, 2 nm, 5 nm, 10 nm
OD range: 0 to 4 OD
Accuracy: < 1% at 2 OD
Precision: < 0.5% at 1 OD and < 0.8% at 2 OD
Sensitivity - FI (top): 0.4 pM Fluorescein (10 amol/well) (384)
- FI (top): 2.0 pM Fluorescein (16 amol/well) (1536)
- FI (bottom): 5.0 pM Fluorescein (0.2 fmol/well) (384)
- FP: 1.0 mP SD at 1 nM Fluorescein (384)
- FP: 3.0 mP SD at 1 nM Fluorescein (1536)
- TRF: 25 fM Europium (0.5 amol/well) (384)
- TRF: 100 fM Europium (0.8 amol/well) (1536)
- HTRF®:
Reader Control Kit (Eu) after 18h (384)
Delta F > 1100 % (High Calibrator)
Delta F > 25 % (Low Calibrator)
- LUM: 10 amol/well ATP (384)
- AlphaScreen®: < 100 amol * (384)
Microplate Format - 6 to 1536, user definable
Reading Speed 1 flash: 14 s (384)
1 flash: 27 s (1536)

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10 flashes: 38 s (384)
10 flashes: 1 min 52 s (1536)
50 flashes: 1 min 29 s (384)
50 flashes: 5 min 18 s (1536)
Reagent Injectors Up to two built-in reagent injectors
Injection at measurement position (6 to 384-well)
Individual injection volumes for each well (3 to 350 µL)
Variable injection speed (100 to 420 µL/s with a 500 µL syringe)
Up to four injection events per well
Reagent back flushing
Gain control - Software selectable gain for F, FP, L, and AS.
Height adjustment - Automatic Z-adjustment with curve monitoring
Temperature Control - Incubation range from 5°C above ambient to 45°C
- Temperature monitoring (with and without incubation)
Plate Carrier - Auto lock microplate carrier
All microplate formats up to 1536-well in all detection modes
Microplates should adhere to the SBS specification and non-SBS formats
should fit: (lxwxh) (mm) max: 128x86x20; min: length 124
Shaking - Double orbital, orbital and linear shaking
- Programmable shake time and diameter
Computer interface - USB 2.0, compatible to USB 1.1
Power requirements - 100 or 115 or 230 V, 50/60 Hz, auto ranging
- Consumption: max. 300 VA
Fuses - T 5A/250V for main power 100 - 240 V
(use original type Wickmann only)
Dimensions and weight - Height: 46 cm, width: 46 cm and depth 51 cm. Weight 50 kg max
Ambient conditions - Operating temperature: 15°C to 35°C
- Storage temperature: -10°C to 50°C
- Humidity of atmosphere: 20 % to 80 %
- Non-condensing environment.
Instrument conformity - Over voltage category II; contamination class II; protection class I;
Laser class I and IIIB
Robotic capabilities - Easy to integrate into all existing robotic systems
For medium level throughput, an optional BMG LABTECH
Stacker with magazines for 50 microplates is available
The fluorescence ratio associated with the HTRF®readout is a correction method developed by Cisbio international and
covered by the US patent 5,527,684 and its foreign equivalents, for which Cisbio international has granted a license to
BMG LABTECH. Its application is strictly limited to the use of HTRF® reagents and technology, excluding any other TR-
FRET technologies.
* Limit of detection < 100 amol of biotinylated and phosphorylated polypeptide (P-Tyr-100 assay kit, PerkinElmer, #6760620C),
measured in white 384 small volume microplates (17 µL/well).
AlphaScreen, DELFIA, and LANCE are registered trademarks of Perkin Elmer, Inc.
HTRF is a registered trademark of Cisbio international.
LanthaScreen is a registered trademark of Invitrogen Corp.
Transcreener is a registered trademark of BellBrook Labs
Specifications are subject to change without notice.

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2 Overview
2.1 Front and Right Side of PHERAstar FS
Figure 1: PHERAstar FS
Figure 2: Optic module door
Figure 3: Right side of the PHERAstar FS, showing the reagent injection system
Press the button on the
grip and the optic module
door can be slid aside.
Service lid for laser cartridge
Plate carrier in/out button
Optic module door
Operating lamp
Plate carrier door
Sliding Door
Magnetic
Clamp Reagent
Injectors
Priming
Buttons
Needle
Holder
Waste
Container
Holder for
Compound
Vessels

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2.2 Back of PHERAstar FS
Figure 4: Back of PHERAstar FS
Figure 5: Back of PHERAstar FS
Main power on/off switch
Fan
Air inlet
Miscellaneous labeling such as serial
Number, CE-marking, and voltage
information
Main power on/off switch
USB connection to PC
RS-232 connection to Stacker
USB connection to PC

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3 Installation
This chapter includes the initial important points that must be followed before a measurement can be
performed. Please follow these steps.
When unpacking the instrument, please check to ensure that all parts are included.
The shipping box should contain:
PHERAstar FS instrument
•Control and evaluation software (CD ROM in a cover inside the manual)
•Manual (also on CD ROM)
•Power cord
•USB cable
•Shipment box containing:
Optic Modules
2 extra fuses-Wickmann original:
T 5A/250V for main power 100 - 240 V
•Compound vessel holder type 1 and 2
•Waste container for pump priming
Call BMG LABTECH immediately if any of these items are missing.
The area designated for the instrument should be free of dust, liquids and acidic vapours. The table's surface
should be flat and even. Avoid areas subject to vibrations and direct sunlight. Behind the instrument there
should be a minimum distance of 10 cm.
The operator of the PHERAstar FS microplate reader is assumed to be trained in the
correct operation of the instrument and the safety issues. Throughout this manual the
word "you" refers to this trained operator.
Upon unpacking and positioning the reader, make sure to unlock the transport locks
(section 3.1.1 and 3.1.3 below) before any power connection (section 3.2 Power and
Communication Connections).
3.1 Transport Locks
When the PHERAstar FS microplate reader is shipped, the transport lock for the plate carrier must be
unlocked and the Optic Module transport system protection plate must be unlocked and removed.
If the PHERAstar FS has to be moved or shipped to another location, it is very important to
lock and secure the two transport systems in order to avoid damage (see chapter 3.1.1
and 3.1.4 below).
3.1.1 Unlocking the Plate Carrier
The transport pin that locks the plate carrier can be accessed inside the instrument. Open the Optic
Module door and you will see a silver screw for locking/unlocking the plate carrier. By hand, turn the
screw counter-clockwise to unlock the pin of the plate carrier (figure 6).
If the screw moves freely up and down then the plate carrier is unlocked and ready to be used. Once
the PHERAstar FS has been turned on, press the In/Out button (figure 10) briefly and the plate carrier
should move out.

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Figure 6: Unlocking the plate carrier
3.1.2 Locking the Plate Carrier
Upon closing the software or pushing the plate carrier in/out button for more than three seconds (figure 10),
the plate carrier will move into the respective lock position. Push the screw down and turn it clockwise to lock
the plate carrier (figure 7). The screw has to be tight and must not be moving as it was in the unlocked
position.
Figure 7: Locking the plate carrier
Never open the plate carrier door during measuring.
3.1.3 Unlocking and Removing the Optic Module Transport System Protection Plate
Open the optic module door and unscrew the three screws and remove the (red) protection plate
(figure 8). Because of a spring loading mechanism, the screws will stay connected to the protection
plate. This plate is also used for offset determination of the optic module transport system. Store the
protection plate in the shipment box (figure 11).
Figure 8: The optic module transport lock

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3.1.4 Inserting and Locking the Optic Module Transport System Protection Plate
Upon closing the software or pushing the plate carrier in/out button for more than three seconds (see figure
10), the Optic Module transport system will move into its respective reference and lock position. Hereafter,
turn off the PHERAstar FS and you can position the red protection plate (with the spring loaded screws
connected to it) in the position shown in figure 9. First position and fix (finger tight) the middle positioned
screw; after that position and fix the two other screws.
Figure 9: Carefully position the red protection plate and start fixing
the screw in the middle position, thereafter the other two screws
Both the plate carrier transport and the optic module transport needs to be locked
before moving or shipping the reader.
3.2 Power and Communication Connections
Check that the power switch on the back of the instrument is in the "OFF" position.
Inspect the voltage information on the label next to the power switch (see figure 4) to ensure that it
corresponds to the local main power specifications. Also make sure the power cable is grounded. Hereafter,
connect only the power cable to the instrument.
In order to make a ‘Connection check’, the Control Software needs to be installed before
connecting the USB cable to the instrument. Please refer to the software manual part of
this binder to install the software and how to perform a connection check.
Make sure the plate carrier and the optic module transport systems are unlocked (see
chapter 3.1.1 and 3.1.3 above). Put the power switch in the “ON” position.
Connection check
Connect the USB cable to the USB port labelled “PC” located on the back of the PHERAstar FS and to the
USB port on the PC. If a Stacker is included in the package, please see section 4.7 below. Once the USB
cable is installed open the PHERAstar FS Control Software and perform the connection check.
Only connect a computer that corresponds to EN 60950 and UL 1950 for data processing
instruments.

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4 Description of Components and Peripherals
4.1 PHERAstar FS Operating Lamp
The operating lamp (see figure 10) delivers a constant blue light when the instrument is turned on.
A flashing blue lamp (once per second) means the instrument is busy (e.g. performing a measurement, plate
in/out, etc.).
A fast flashing lamp (five flashes per second) indicates that the reader has stopped for uncommon reasons.
The function of the plate carrier in/out button will be disabled during measurement and
during Stacker operation!
When the plate carrier is out, the internal fan will reduce its speed, because the amount of air needed to
maintain an acceptable inner environment can be acquired more easily through an open plate carrier door.
4.2 Plate Carrier In/Out Button
The plate carrier in/out button on the front (figure 10) is an easy way to get the plate carrier in and out. The
plate carrier in/out button works independently from the PHERAstar FS Control Software.
Figure 10: Plate in/out button and operating lamp
Pressing the plate carrier in/out button for more than three seconds moves the transport systems of the plate
carrier and the optic module to the reference positions in which they can be locked, as described in section
3.1.1 and 3.1.4 above.
4.3 PHERAstar FS Shipment Box
Figure 11: Shipment box
Optic Modules
Optic module transport lock
and alignment tool
Extra bags and
Allen keys
Fuses
Operating lamp
Plate in/out button

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Figure 13: Hold the optic
module by the handle
Figure 14: The groove of
the optic module transport
system
Figure 15: When installing or
removing the optic module, hold
the handle and move it up and out
4.4 Installing and Changing Optic Modules
All PHERAstar FS readers can be equipped with six optic modules. Five optic modules can be exchanged
and one optic module (Luminescence) is preinstalled.
In case of shipment or moving the PHERAstar FS, it is important to remove the optic
modules from the optic module transport.
Figure 12: Optic module, front view and side view
When the PHERAstar FS is in its permanent position and powered ON, the optic modules can be installed.
The PHERAstar FS has five positions for optic modules. Once the optic module door has been opened the
optic module transport will automatically move to a position where it can be accessed. Install the optic
modules by holding it by the handle (figure 13) and then position the optic module positioning pin in the
groove (figure 14 and figure 15).
Be careful not to touch any optical surfaces while handling the optic modules.
The various types of optic modules are described in more details in sections 4.5.1 to 4.5.4.
Optic module
positioning pin
Excitation
Emission
channel B
Handle to hold
optic module
Emission
channel A
Label with bar-code,
measurement
principle,
wavelengths and
batch-nr. code

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4.4.1 Built-in Barcode Reader for Optic Modules
With the built-in optic module barcode reader the PHERAstar FS will automatically detect the optic modules
installed and automatically transfer the information into the optic module list. If changes have been made,
they will automatically be recognized.
When no optic modules are in position, the barcode reader will detect an empty position and this information
is then transferred to the optic module list.
If the optic module is a customer inter-changeable type, a message will appear informing you manually to
type in wavelengths in the optic module table.
The barcode detection will be triggered by a) opening and closing the optic module door or b) performing a
connection check. After turning on reader and control software this will always be done automatically.
The barcode label on the optic module (figure 16) holds the information about optic module name, filter
wavelengths and bandwidths.
Figure 16: Barcode labeled Optic Module
The automatic barcode detection takes place in
the far right position of the optic module
chamber (figure 17).
If there is a position without optic module the
barcode label in the back of the compartment
will be detected.
Figure 17: Barcode detection of Optic Modules

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4.5 Description of Optic Modules
There are single channel optic modules as well as dual channel optic modules for various detection
technologies.
Depending on the type of optic module, it can consist of filters, dichroic mirrors, beam splitters and
polarization film. Emission channel A is always active (whatever measurement principle is in use) and
Emission channel B is active when dual emission detection is used (for instance as in fluorescence
polarization, HTRF®and dual luminescence assays).
The optic module is an important part of the PHERAstar FS and all parts used in the optic module are
carefully selected and accurately aligned and tested for optimal performance.
4.5.1 Fluorescence Intensity and TRF Optic Module
Standard fluorescence intensity optic modules are available in an almost unlimited variety of different
configurations (contact sales rep with your wishes). All standard fluorescence intensity optics are configured
as can be seen in figure 18.
Figure 18: Optic module for single
channel fluorescence intensity
The configuration of the filters can be freely chosen from a long list of optimized filters available.
4.5.2 Fluorescence Polarization Optic Module
The fluorescence polarization optic module consists of many parts, all specially selected and aligned for the
best performance. Fluorescence polarization optic modules (figure 19) can be customized to fit any
polarization assay, also at wavelengths other than the standard excitation 485 nm and emission at 520 nm
and 520 nm.
Figure 19: Optic module for fluorescence polarization

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4.5.3 Time-Resolved Fluorescence Optic Module, FRET and TR-FRET, for example HTRF®
The time-resolved fluorescence optic module (figure 20) is optimized for HTRF®. The beam splitters, dichoic
mirror, as well as the filters, are all specially selected to give the very best performance.
Figure 20: Optic module for time-resolved
fluorescence, for example HTRF®
4.5.4 Luminescence Optic Modules
Luminescence assays are mostly towards light of unspecific wavelength and therefore the default optic
module is an empty module. The empty module (figure 21) still needs to be there because it limits reflection
and scatter.
Figure 21: Optic module for luminescence, wavelength not specified.
The LUM+ Module is intended for microplate formats up to 384 wells.

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If the assay demands a specific wavelength to be measured, this can be carried out by having a filter in front
of the measurement channel. And if the demand is to measure two different wavelengths, this can be carried
out by including a dichroic mirror and a second filter in front of the emission channel B, for example BRET
assays. (figure 22).
Figure 22: Left: luminescence optic module to measure light of a specific wavelength
Right: luminescence optic module to measure light of two specific wavelengths
4.5.5 AlphaScreen®Module
AlphaScreen®is a unique assay technology that measures the interaction of two molecules bioconjugated to
donor and acceptor beads. When interactions occur between these molecules, the beads are brought close
enough to cause a chemical reaction. Upon laser excitation at 680 nm, a singlet oxygen transfer occurs that
leads to a strong light emission between 520 to 620 nm, which is directly proportional to the amount of
binding (figure 23).
Figure 23: Optic Module for single channel
AlphaScreen® measurement

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4.6 Reagent Injection System
The PHERAstar FS is equipped with an internal on board reagent injection system located on the right side
of the instrument (figure 24). Use the sliding door to open, close, and to gain access to the reagent injection
system:
Figure 24: Overview of the PHERAstar FS Reagent Injection System
The reagent needles are made of stainless steel, the tubing and valve housing are made of Teflon and Kel-
F, and the syringe barrel is made of glass. All reagent injector materials are among the most chemical
resistant materials that are available.
Note: Before using a pump in a test run, you need to prime the pump. This can be done using the Control
software (menu command ‘Measure | Prime’). You can also prime the pumps without using the
software. When you open the reagent door, you will see two buttons for priming. Refer to chapter
4.6.5 for a detailed description of the priming procedure.
4.6.1 Reagent Injectors
In order to retain optimal performance, it is recommended to follow these guidelines:
Do not use the syringes more than two cycles without liquid.
After each use flush the syringes thoroughly with distilled water.
If the plunger is removed from the syringe barrel, it should be wiped with ethanol before replacing.
Syringes should be cleaned each week using one of the following procedures:
To remove cellular debris and viscous solutions from the syringe barrel:
Take off the syringe barrel and rinse it with distilled water. It may be useful to use the wire syringe cleaners
(can be found in the service box) to scrape particles off the walls.
Cleaning with weak detergent or 10% bleach
1. Fill the syringe with a weak detergent or 10% bleach solution
2. Leave the solution in the syringe for 30 minutes
3. Flush the syringe a minimum of 10 times with distilled water
Sliding Door
Magnetic
Clamp
Reagent
Injectors
Priming Buttons
Needle Holder
Waste
Container
Holder for
Compound
Vessels

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Cleaning with acid / base (best procedure if cells are used in the syringe)
1. Fill the syringe with 0.1M NaOH and leave it in the syringe for 10 minutes.
2. Flush the syringe a minimum of 10 times with distilled water.
3. Fill the syringe with 0.1M HCl, and leave the solution in the syringe for 10 minutes.
4. Flush the syringe a minimum of 10 times with distilled water.
4.6.2 Sliding Door
The Sliding Door can be opened by pressing onto the two ends of the handle and sliding it upwards. The
door will stay at the position it is released. The PHERAstar FS can not start a test run if the sliding door is in
the open position. Thus always close the sliding door before starting a test run.
4.6.3 Needle Holder
The needle holder is an S-shaped device and houses one or two needles connected to the respective
reagent injectors (figure 25). The arrangement of the needles and the architecture of the needle holder itself
allow simultaneous injection and measurement for all detection modes except absorbance. The tubing
coming from the syringe barrels are assembled together in a magnetic clamp to minimize dead volume and
to guide them to the needle holder. The magnetic clamp can easily be removed by gently pulling it away from
the instrument. In order to execute external priming, to exchange the needle holder or to clean the needles it
is necessary to first pull away the magnetic clamp from the reader. The magnetic clamp is also equipped
with a sensor which prevents the PHERAstar FS from executing a test run when the magnetic clamp and the
needle holder are not properly installed.
Figure 25: Needle holder assembly
Figure 26: Waste container
The needle holder slides easily in and out off its reference position. Note that the tubing assembly, the
magnetic tube clamp, and the needle holder always come as one unit. The waste container for internal
priming comes as a unit but can be taken apart for cleaning as shown in figure 26.
Two versions of the needle holder assembly are available. Needle holder A1 incorporates 2 needles and is
used for the 96/384 square well plate format. Needle holder A2 has 1 needle and is used for the 96/384
round well plate format. Each version is recognized in the Control software by a sensor placed on the inside
of the magnetic tube clamp.
Connection to the valves
of the syringes
Magnetic Clamp
Needle holder with
needles

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4.6.4 Holder for Compound Vessels
BMG LABTECH provides the PHERAstar FS user with two different compound holders (figure 27). They
accommodate the most commonly used compound storage tubes, flasks, and even custom-made probe
holders for small volumes.
Figure 27: Compound vessel holders type 1 and 2 for different storage vessels
4.6.5 Pump Priming
The reason for priming is to fill the tubing and needles of the reagent system with solution prior of executing
a test run. This is necessary to avoid bubbles which could lead to false results. The priming procedure can
be performed in two ways: internal or external priming:
Internal priming requires that the waste container and the needle holder are installed (figure 28). The waste
container must be placed underneath the needle holder and holds a total volume of max 15 ml. Once 15 ml
solution volume has been reached an alert message will inform the user to take out the waste container and
discard the solution. If further priming is required install the waste container again.
To start priming, place the waste container into its normal operating position, a compound solution into the
compound vessel, and insert the tubing into the solution. Priming is then initiated either via the Control
Software or by a double click of the respective priming buttons.
Figure 28: Internal priming conditions
Internal Priming will only start when the magnetic clamp, needle holder, and waste
container are securely placed in the operating position. They are equipped with a
sensor and hence priming will not start until operating position is in place.