Molecular Devices SpectraMax Paradigm User Manual
SpectraMax®Paradigm®
Multi-Mode Detection Platform
User Guide
5014038 E
May 2015
SpectraMax Paradigm Multi-Mode Detection Platform User Guide
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2 5014038 E
Contents
Safety Information 7
Warnings, Cautions, Notes, and Tips 7
Symbols on Instrument Labels 8
Before Operating the Instrument 9
Electrical Safety 9
Laser Safety 10
Chemical and Biological Safety 11
Moving Parts Safety 12
Cleaning and Maintenance Safety 13
Chapter 1: Introduction 15
Applications 16
Dual Photomultiplier Tubes 16
Microplate Controls 16
Environmental Controls 18
Chapter 2: Read Modes and Read Types 19
Supported Read Types 19
Absorbance Read Mode 23
Fluorescence Intensity Read Mode 28
Luminescence Read Mode 33
Time-Resolved Fluorescence Read Mode 38
FRET Read Mode 44
HTRF Read Mode 45
Fluorescence Polarization Read Mode 49
AlphaScreen Read Mode 53
Western Blot TRF Read Mode 57
Chapter 3: Unpacking and Setting Up the Instrument 61
Contents of the Package 62
Unpacking the Instrument 63
Removing the Transport Locks 67
Connecting the Instrument Cables 72
Unlocking the SpectraMaxParadigm Instrument 75
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SpectraMax Paradigm Multi-Mode Detection Platform User Guide
Connecting and Disconnecting a Gas Supply 76
Chapter 4: Using the Instrument 79
Front Panel Controls and Indicators 80
Turning the Instrument On and Off 82
Using Detection Cartridges 84
Loading and Unloading Microplates 90
Chapter 5: Available Detection Cartridges 93
Absorbance Detection Cartridge 94
Tunable Wavelength (TUNE) Detection Cartridge 97
Multi-Mode (MULTI) Detection Cartridge 102
AlphaScreen Detection Cartridges 107
Cisbio HTRF Detection Cartridge 110
Time Resolved Fluorescence (TRF) Detection Cartridge 113
Fluorescence Intensity (FI) Detection Cartridges 116
Fluorescence Intensity (FI) GeneBLAzer Detection Cartridge 119
Fluorescence Intensity Dual Label (FI-DL) (MultiTox-Fluor) Detection Cartridge 122
Fluorescence Polarization (FP) Detection Cartridges 125
Glow Luminescence (LUM) Detection Cartridges 128
Dual Color Luminescence (LUM) (BRET2) Detection Cartridge 131
Dual Color Luminescence (LUM) (Chroma-Glo) Detection Cartridge 136
ScanLater Western Blot (WB) Detection Cartridge 142
Chapter 6: Maintenance and Troubleshooting 147
Doing Preventive Maintenance 148
Cleaning the Instrument 149
Replacing Fuses 149
Moving the Instrument 152
Packing the Instrument for Storage or Service 153
Troubleshooting 164
Obtaining Support 165
Appendix A: Instrument Specifications 167
Computer System Specifications 167
Physical Specifications 168
4 5014038 E
Appendix B: System Diagrams and Dimensions 171
Appendix C: Electromagnetic Compatibility 173
Glossary 175
Index 183
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SpectraMax Paradigm Multi-Mode Detection Platform User Guide
6 5014038 E
Safety Information
The safety information section provides information on the safe use of the instrument,
including the use of user-attention statements in this guide, a key to understanding the
safety labels on the instrument, precautions to follow before operating the instrument, and
precautions to follow while operating the instrument.
Please read and observe all warnings, cautions, and instructions. Remember, the most
important key to safety is to operate the instrument with care.
WARNING! If the instrument is used in a manner not specified by Molecular
Devices, the protection provided by the equipment might be impaired.
Warnings, Cautions, Notes, and Tips
All warning symbols in the user guide are framed within a yellow triangle. An exclamation
mark is used for most warnings. Other symbols can warn of other types of hazards such as
biohazard, electrical, or laser safety warnings as are described in the text of the warning.
When warnings and cautions are displayed in this guide, be careful to follow the specific
safety information related to them.
The following user-attention statements can be displayed in the text of Molecular Devices
user documentation. Each statement implies a particular level of observation or
recommended procedure as described:
WARNING! A warning indicates a situation or operation that could cause
personal injury if precautions are not followed.
CAUTION! A caution indicates a situation or operation that could cause damage to
the instrument or loss of data if correct procedures are not followed.
Note: A note calls attention to significant information.
Tip: A tip provides useful information or a shortcut, but is not essential to the
completion of a procedure.
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SpectraMax Paradigm Multi-Mode Detection Platform User Guide
Symbols on Instrument Labels
Each safety label located on the instrument contains an alert symbol that indicates the type
of potential safety hazard related to the label. The following table lists the alert symbols that
can be found on Molecular Devices instruments.
Table S-1: Instrument Label Alert Symbols
Symbol Indication
This symbol indicates that the product documentation needs to be consulted.
This symbol indicates a potential laser hazard. The instrument is rated a Class 1 Laser
Product because it can house one or more laser modules, and the laser light cannot be
accessed. See Laser Safety on page 10.
This symbol indicates a potential lifting hazard. To prevent injury, use a minimum of
two people to lift the instrument. For information about the weight of the instrument,
see Physical Specifications on page 168.
This symbol indicates a potential pinch hazard.
This symbol on the power switch indicates power on. See Turning the Instrument On
and Off on page 82.
This symbol on the power switch indicates power off. See Turning the Instrument On
and Off on page 82.
This symbol on the product is required in accordance with the Waste Electrical and
Electronic Equipment (WEEE) Directive of the European Union. It indicates that you
must not discard this electrical or electronic product or its components in domestic
household waste or in the municipal waste collection system.
For products under the requirement of the WEEE directive, please contact your
dealer or local Molecular Devices office for the procedures to facilitate the proper
collection, treatment, recovery, recycling, and safe disposal of the device.
8 5014038 E
Before Operating the Instrument
Make sure that everyone involved with the operation of the instrument has:
Received instruction in general safety practices for laboratories.
Received instruction in specific safety practices for the instrument.
Read and understood all Safety Data Sheets (SDS) for all materials being used.
Electrical Safety
To prevent electrically related injuries and property damage, properly inspect all electrical
equipment before use and immediately report all electrical deficiencies. Contact Molecular
Devices technical support for servicing of equipment that requires the removal of covers or
panels.
WARNING! HIGH VOLTAGE. Within the instrument is the potential of an
electrical shock hazard existing from a high voltage source. All safety instructions
should be read and understood before proceeding with the installation,
maintenance, and servicing of all modules.
Safety Information
Do not remove the instrument covers. To prevent electrical shock, use the supplied power
cords only and connect to a properly grounded wall outlet. Use only multi-plug power strips
that are provided by the manufacturer.
To protect against fire hazard, replace the fuses only with the same type and rating as the
original factory-installed fuses. See Replacing Fuses on page 149.
To ensure sufficient ventilation and provide access for disconnecting power from the
instrument, maintain a 20cm to 30cm (7.9in. to 11.8in.) gap between the rear of the
instrument and the wall.
Molecular Devices recommends turning the power off when the instrument is not in use.
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SpectraMax Paradigm Multi-Mode Detection Platform User Guide
Laser Safety
WARNING! LASER LIGHT. This symbol indicates that a potential hazard to
personal safety exists from a laser source. When this symbol is displayed in this
guide, be careful to follow to the specific safety information related to the
symbol.
The SpectraMaxParadigm Multi-Mode Detection Platform is rated a Class 1 Laser Product
because it houses one or more laser modules, and the laser light cannot be accessed.
The embedded laser module inside the SpectraMaxParadigm Multi-Mode Detection
Platform basic instrument is used for the plate height detection and has the following
specifications.
Table S-2: Embedded Laser Module Specifications
Item Description
Lasertype Diode laser
Wavelength 650nm
Maximumoutputpower 0.9mW, cw
Laser class Class2 (IEC60825-1, ed. 2.0:2007)
Fan angle 58°
The SpectraMaxParadigm Multi-Mode Detection Platform is equipped with a redundant
laser safety system. A hardware interlock prevents the laser module from turning on, unless
the microplate chamber flap is closed and the front cover of the detection cartridge drawers
are in place. The user or the service engineer is not exposed to radiation from the laser
module during operation, maintenance, or service. The closed microplate chamber provides
the protective housing.
WARNING! LASER LIGHT. The instrument must be operated only when all the
doors and panels of the instrument are in place and closed.
Laser or Laser Diodes in Detection Cartridges
Some detection cartridges can have a laser or laser diode up to Laser Class 4 inside the
detection cartridge. The lasers are non-operational until after the detection cartridges are
properly installed in the SpectraMaxParadigm Multi-Mode Detection Platform.
10 5014038 E
Chemical and Biological Safety
Normal operation of the instrument can involve the use of materials that are toxic,
flammable, or otherwise biologically harmful. When using such materials, observe the
following precautions:
Handle infectious samples based on good laboratory procedures and methods to
prevent the spread of disease.
Observe all cautionary information printed on the original containers of solutions before
their use.
Dispose of all waste solutions based on the waste disposal procedures of your facility.
Operate the instrument in accordance with the instructions outlined in this guide, and
take all the necessary precautions when using pathological, toxic, or radioactive
materials.
Splashing of liquids can occur. Therefore, take applicable safety precautions, such as
using safety glasses and wearing protective clothing, when working with potentially
hazardous liquids.
Use a correctly contained environment when using hazardous materials.
Use a compressed gas supply in a well-ventilated area. The instrument is not air-tight,
and so gas can escape into the atmosphere surrounding the instrument. When using
potentially toxic gas, always observe the applicable cautionary procedures as defined by
your safety officer to maintain a safe working environment.
Observe the applicable cautionary procedures as defined by your safety officer when
using flammable solvents in or near a powered-up instrument.
Observe the applicable cautionary procedures as defined by your safety officer when
using toxic, pathological, or radioactive materials.
Safety Information
WARNING! Never use the instrument in an environment where potentially
damaging liquids or gases are present.
CAUTION! Use of organic solvents (such as dichloromethane) can cause harm to the
optics in the instrument. Extreme caution is recommended when using organic
solvents. Always use a plate lid and do not place a plate containing these materials in
the microplate chamber for prolonged periods of time. Damage caused by the use of
incompatible or aggressive solvents is NOT covered by the instrument warranty.
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SpectraMax Paradigm Multi-Mode Detection Platform User Guide
Moving Parts Safety
To prevent injury due to moving parts, observe the following:
Never try to exchange labware, reagents, or tools while the instrument is operating.
Never try to physically restrict the moving components of the instrument.
Keep the instrument work area clear to prevent obstruction of the movement. Provide
clearance in the front of the instrument of 18cm (7.1in.) for the microplate drawer and
15cm (5.9in.) for the detection cartridge drawer.
The instrument has adjustable optics to define the read height, or z-height. In a top
read, the read height is the gap between the lens and the top of the microplate, or the
top of the lid if the microplate is lidded.
CAUTION!To prevent damage to the instrument, the height of the microplate must
not exceed 25mm, including the lid if the microplate is lidded.
Transport locks are placed on the detection cartridge drawers and the microplate drawer to
protect the instrument from damage during shipping. The transport locks must be removed
before powering on the instrument.
To move the microplate drawer or the detection cartridge drawers into or out of the
instrument, always use the buttons on the keypad or the controls in the software. See Using
Detection Cartridges on page 84 or Loading and Unloading Microplates on page 90.
CAUTION! To prevent damage to the installed detection cartridges and the
instrument, do not manually slide the detection cartridge drawer in or out when the
instrument is powered on or when one or more detection cartridges are installed in
the drawer.
Note: Observe all warnings and cautions listed for all external devices attached to or in
use during the operation of the instrument. See the applicable user guide for the
operating and safety procedures of that device.
12 5014038 E
Cleaning and Maintenance Safety
Observe the cleaning procedures outlined in this guide for the instrument.
Do the following before cleaning equipment that has been exposed to hazardous material:
Contact the applicable Chemical and Biological Safety personnel.
Review the Chemical and Biological Safety information contained in this guide.
Do only the maintenance described in this guide. Maintenance procedures other than those
specified in this guide can be done only by Molecular Devices qualified personnel. See
Obtaining Support on page 165.
WARNING! BIOHAZARD. It is your responsibility to decontaminate
components of the instrument before requesting service by a service engineer or
returning parts to Molecular Devices for repair. Molecular Devices will not accept
items that have not been decontaminated where it is applicable to do so. If parts
are returned, they must be enclosed in a sealed plastic bag stating that the
contents are safe to handle and are not contaminated.
Safety Information
For approved cleaning and maintenance procedures, see Maintenance and Troubleshooting
on page 147.
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14 5014038 E
Chapter 1: Introduction
The SpectraMax®Paradigm® Multi-Mode Detection Platform from Molecular Devices® is a
user-upgradeable, multi-mode microplate reader capable of performing absorbance,
fluorescence, time-resolved fluorescence (including HTRF), fluorescence polarization,
AlphaScreen®, AlphaLISA®, and luminescence measurements. An external computer running
the SoftMax® Pro provides integrated instrument control, data display, and statistical data
analysis.
Detection cartridge modularity lets you configure the system to meet your current needs,
while providing flexibility to address future applications. Up to six detection cartridges can be
installed in each of the two detection cartridge drawers. The software detects the installed
cartridge configuration and does all measurement types supported by the detection
cartridges. For information about detection cartridges, see Available Detection Cartridges on
page 93.
Depending on the application, the instrument can read 6, 12, 24, 48, 96, and 384-well
microplates. For micro-volume measurements, the instrument supports SpectraDrop 24-well
Micro-Volume Microplates and SpectraDrop 64-well Micro-Volume Microplates. The
instrument is capable of reading 1536-well microplates when used with specific detection
cartridges. See Selecting Suitable Microplate Types on page 91.
1
CAUTION! To prevent damage to the instrument, the height of the microplate must
not exceed 25mm, including the lid if the microplate is lidded.
The SoftMax Pro Software can collect data from one or more microplates and store it in a
single data file, using the same or different instrument settings for different microplates.
Assays requiring a read in two or more read modes or read types can be combined in a single
experiment and run with a single command in the software, by defining separate microplate
reads and enabling Auto Read. For information on the acquisition and analysis capabilities of
the software, see the SoftMax Pro Software application help or user guide.
The SpectraMaxParadigm Instrument can be integrated with an automated laboratory
system. When integrated, the detection protocols are accessed by the robotic controller
software.
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Applications
The high sensitivity and flexibility of the SpectraMaxParadigm Instrument make it useful for
applications in the fields of biochemistry, cell biology, immunology, molecular biology, and
microbiology.
Typical applications include ELISA, nucleic acid, protein, enzymatic type homogeneous and
heterogeneous assays, microbial growth, endotoxin testing, and pipettor calibration.
Application notes with specific application protocol suggestions can be found in the
Information Center and the Knowledge Base on the Molecular Devices web site at
www.moleculardevices.com.
Dual Photomultiplier Tubes
The SpectraMaxParadigm Instrument is equipped with two photo multiplier tubes (PMTs).
The dual PMTs let the instrument measure two separate emissions successively or
simultaneously, resulting in faster read times and increased throughput.
Microplate Controls
Microplate controls include Shake and On-the-Fly Detection. The instrument can also detect
the height and position of a microplate in the microplate drawer.
Shake
The Shake feature of the instrument permits the contents of the wells in a microplate to be
mixed automatically inside the microplate chamber before each read cycle, making it
possible to do kinetic analysis of solid-phase, enzyme-mediated reactions.
Shake must be selected before you start a read. The process related to the Shake setting
depends on the selected read mode:
In endpoint reads, Shake shakes the plate for a definable number of seconds and then
reads at all selected wavelengths.
In kinetic reads, Shake can shake the plate for a definable number of seconds before the
initial reading, and for a definable number of seconds before each subsequent reading.
The following Shake settings are available for the SpectraMaxi3x Instrument:
Intensity: Low, Medium, or High. The actual shake speed is based on the microplate
format.
Direction: Linear or Orbital patterns.
Duration: Length of time in seconds (1 to 60).
Molecular Devices strongly recommends the use of Shake for ELISAs and other solid-phase,
enzyme-mediated reactions to enhance accuracy.
16 5014038 E
Chapter 1: Introduction
On-the-Fly Detection
With some detection cartridges, the instrument can read microplates as the microplate
drawer is moving within the chamber instead of pausing the microplate drawer to read each
well. This results in shorter read times.
There are two On-the-Fly Detection modes:
Selecting Performance results in a faster read time than not using On-the-Fly Detection,
but not as fast as the Speed mode. Performance provides considerably better results
than Speed for demanding assays.
Selecting Speed results in the fastest possible read time per microplate. However, there
is a trade-off between the data quality and read speed because each well is sampled for
shorter integration times.
The following table compares the read time for different plate types in each of the on-the-fly
detection modes. These read times do not include the time needed for the microplate
drawer to move the plate into the instrument and start the read, and then move the plate
out of the instrument, which can add approximately 25 seconds to the overall read time.
Table 1-1: Plate Read Times for On-the-Fly Detection (±5seconds)
Mode 96-well 384-well 1536-well
Optimized for speed 12seconds 25seconds 50seconds
Optimized for performance 20seconds 40seconds 80seconds
Microplate Height Sensing
Microplates up to a height of 25mm can be placed in the microplate drawer of the
instrument. A sensor detects the height of a microplate positioned in the microplate drawer
and confirms that the height is consistent with the selected microplate type and that it is
positioned properly on the microplate drawer.
CAUTION! To prevent damage to the instrument, the height of the microplate must
not exceed 25mm, including the lid if the microplate is lidded.
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Environmental Controls
The environmental controls of the instrument include temperature regulation and a gas
inlet.
Temperature Regulation
The temperature inside the microplate chamber can be maintained at ambient plus 4°C ± 1°C
up to 45°C. When using a detection cartridge that has a flash lamp, temperature can be
maintained at ambient plus 5°C ± 1°C up to 45°C. You can set and control the temperature by
using the software. See the SoftMax Pro Software application help or user guide.
Note:The temperature sensors detect the temperature of the air inside the chamber,
not the temperature of the samples in the microplate. If you use the instrument to
warm the samples, Molecular Devices recommends that you use a seal or lid on the
microplate to prevent evaporation of the sample. Using a seal or lid also helps to
maintain uniform temperature. Letting the prepared sample equilibrate inside the
microplate chamber can take an hour or more. You can speed up equilibration by prewarming the sample and the assay reagents to the desired temperature before
placing the microplate in the chamber. Validate the data quality to determine whether
the seal or lid can stay on the microplate for the read.
Gas Inlet
The gas inlet permits the partial pressure of CO2, nitrogen, or other gas inside the microplate
chamber to be applied. This is useful when reading a cell-based assay in which the CO
environment needs to be controlled to keep cell cultures alive. The gas supply is not
controlled or monitored by the instrument or software. See Connecting and Disconnecting a
Gas Supply on page 76.
Note: The combination of temperature and CO2environment controls does not create
a true CO2incubator environment in the instrument.
Use a compressed gas supply in a well-ventilated area. The instrument is not air-tight, and so
gas can escape into the atmosphere surrounding the instrument. When using potentially
toxic gas, always observe the applicable cautionary procedures as defined by your safety
officer to maintain a safe working environment.
2
18 5014038 E
Chapter 2: Read Modes and Read Types
The detection capabilities of the SpectraMaxParadigm Multi-Mode Detection Platform are
determined by the installed detection cartridges. Up to six detection cartridges can be
installed in each of the two detection cartridge drawers. For information about detection
cartridges, see Available Detection Cartridges on page 93.
The software detects the installed cartridge configuration and does all measurement types
supported by the detection cartridges. Use the SoftMax Pro Software to define the
parameters for the read mode and read type of your assay. See the SoftMax Pro Software
application help or user guide.
Application notes with specific application protocol suggestions can be found in the
Information Center and the Knowledge Base on the Molecular Devices web site at
www.moleculardevices.com.
For more information on the supported read modes, see the following topics:
Absorbance Read Mode on page 23
Fluorescence Intensity Read Mode on page 28
Luminescence Read Mode on page 33
Time-Resolved Fluorescence Read Mode on page 38
HTRF Read Mode on page 45
FRET Read Mode on page 44
Fluorescence Polarization Read Mode on page 49
AlphaScreen Read Mode on page 53
Western Blot TRF Read Mode on page 57
2
Supported Read Types
For most read modes, endpoint, kinetic, multi-point well-scan, and spectrum microplate
applications can be set up and run with the SoftMax Pro Software.
For more information on the supported read types, see the following topics:
Endpoint Read Type on page 20
Kinetic Read Type on page 20
Well Scan Read Type on page 21
Spectrum Read Type on page 21
Membrane Read Type on page 22
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SpectraMax Paradigm Multi-Mode Detection Platform User Guide
Endpoint Read Type
In an Endpoint read, a reading of each microplate well is taken in the center of each well, at a
single wavelength or at multiple wavelengths. Depending on the read mode, raw data values
are reported as optical density (OD), %Transmittance (%T), relative fluorescence units (RFU),
or relative luminescence units (RLU).
Kinetic Read Type
In a Kinetic read, the instrument collects data over time with multiple readings taken in the
center of each well at regular intervals.
The values calculated based on raw kinetic data include VMax, VMax per Sec, Time to VMax,
and Onset Time. Kinetic readings can be single-wavelength or multiple-wavelength readings.
Kinetic analysis can be done for up to 99 hours. The kinetic read interval depends on the
instrument setup parameters selected in the SoftMax Pro Software.
Kinetic analysis has many advantages when determining the relative activity of an enzyme in
different types of microplate assays, including ELISAs and the purification and
characterization of enzymes and enzyme conjugates. Kinetic analysis is capable of providing
improved dynamic range, precision, and sensitivity relative to endpoint analysis.
Peak Pro™ Analysis functions provide advanced peak detection and characterization for
applicable kinetic reads. See the SoftMax Pro Software Formula Reference Guide.
20 5014038 E
Chapter 2: Read Modes and Read Types
Well Scan Read Type
A Well Scan read can take readings at more than one location within a well. A Well Scan read
takes one or more readings of a single well of a microplate on an evenly spaced grid inside of
each well at single or multiple wavelengths.
Some applications involve the detection of whole cells in large-area tissue culture plates. Well
Scan reads can be used with such microplates to permit maximum surface area detection in
whole-cell protocols. Since many cell lines tend to grow as clumps or in the corners of
microplate wells, you can choose from several patterns and define the number of points to
be scanned to work best with your particular application.
The following scanning patterns are available:
A horizontal line
A vertical line
A cross pattern
A fill pattern
The fill pattern can be either round or square to match the shape of the well. The image in
the Well Scan settings shows the shape of the well as defined for the selected microplate.
You can set the density of the well scan to determine the number of points to read in a line
pattern or the maximum number of horizontal and vertical points included in a cross or fill
pattern.
Depending on the read mode selected, the values are reported as optical density (OD),
%Transmittance (%T), relative fluorescence units (RFU), or relative luminescence units (RLU).
Spectrum Read Type
Depending on the read mode selected, a Spectrum read measures optical density (OD),
%Transmittance (%T), relative fluorescence units (RFU), or relative luminescence units (RLU)
across a spectrum of wavelengths.
Spectrum reads are available only for specific detection cartridges. See Available Detection
Cartridges on page 93.
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Membrane Read Type
The Membrane read type is used for a time-resolved fluorescence reading of a Western Blot
membrane. The selected area is read, and a TIFF image is generated with the results of the
read.
The Molecular Devices ScanLater™Western Blot Assay Kit is a novel system for protein
analysis that is incorporated into a SpectraMaxParadigm Multi-Mode Detection Platform.
Membranes are incubated with Eu-chelate labeled secondary antibodies or streptavidin that
bind specifically to the target protein-specific primary antibody. For more information,
contact your Molecular Devices representative or search the knowledge base for ScanLater
or Western Blot at www.moleculardevices.com/support.
For information about the detection cartridge for Western Blot membrane reads, see
ScanLater Western Blot (WB) Detection Cartridge on page 142.
22 5014038 E
Absorbance Read Mode
In the Absorbance (ABS) read mode, the instrument measures the Optical Density (OD) of
the sample solutions.
Absorbance is the quantity of light absorbed by a solution. To measure absorbance
accurately, it is necessary to eliminate light scatter. If there is no turbidity, then
absorbance=optical density.
A=log10(I0/I)=–log10(I/I0)
where I0is incident light before it enters the sample, I is the intensity of light after it passes
through the sample, and A is the measured absorbance.
For Absorbance reads, you can choose whether to display absorbance data as Optical
Density (OD) or %Transmittance (%T) in the Reduction dialog.
Optical Density
Optical density (OD)is the quantity of light passing through a sample to a detector relative to
the total quantity of light available. Optical Density includes absorbance of the sample plus
light scatter from turbidity and background. You can compensate for background using
blanks.
Chapter 2: Read Modes and Read Types
A blank well contains everything used with the sample wells except the chromophore and
sample-specific compounds. Do not use an empty well for a blank.
Some applications are designed for turbid samples, such as algae or other micro-organisms
in suspension. The reported OD values for turbid samples are likely to be different when read
by different instruments.
For optimum results, Molecular Devices recommends that you run replicates for all blanks,
controls, and samples. In this case, the blank value that can be subtracted is the average
value of all blanks.
% Transmittance
%Transmittance is the ratio of transmitted light to the incident light for absorbance reads.
T=I/I
0
%T=100T
where I is the intensity of light after it passes through the sample and I0is incident light
before it enters the sample.
Optical Density and %Transmittance are related by the following formulas:
%T=10
2–OD
OD=2–log10(%T)
The factor of two comes from the fact that %T is expressed as a percent of the transmitted
light and log10(100)=2.
When in %Transmittance analysis mode, the SoftMax Pro Software converts the raw OD
values reported by the instrument to %Transmittance using the above formula. All
subsequent calculations are done on the converted numbers.
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Applications of Absorbance
Absorbance-based detection has been commonly used to evaluate changes in color or
turbidity, permitting widespread use including ELISAs, protein quantitation, endotoxin
assays, and cytotoxicity assays. With absorbance readers that are capable of measuring in
the ultraviolet (UV) range, the concentration of nucleic acids (DNA and RNA) can be found
using their molar extinction coefficients.
For micro-volume measurements, you can use SpectraDrop 24-well Micro-Volume
Microplates and SpectraDrop 64-well Micro-Volume Microplates.
To do absorbance reads, the SpectraMaxParadigm Multi-Mode Detection Platform requires
the Absorbance Detection Cartridge, see page 94.
You can use the Protocol Manager in the SoftMax Pro Software to quickly find and open a
predefined protocol.
More protocols and updated protocols can be downloaded from the Knowledge Base on the
Molecular Devices support web site (www.moleculardevices.com/support) or from the
protocol sharing web site (www.softmaxpro.org).
PathCheck Pathlength Measurement Technology
The temperature-independent PathCheck® Pathlength Measurement Technology
normalizes your absorbance values to a 1cm path length based on the near-infrared
absorbance of water.
The Beer–Lambert law states that absorbance is proportional to the distance that light
travels through the sample:
A = εbc
where A is the absorbance, εis the molar absorptivity of the sample, b is the pathlength, and
c is the concentration of the sample. The longer the pathlength, the higher the absorbance.
Microplate readers use a vertical light path so the distance of the light through the sample
depends on the volume. This variable pathlength makes it difficult to do extinction-based
assays and also makes it confusing to compare results between microplate readers and
spectrophotometers.
The standard pathlength of a 1cm cuvette is the conventional basis for quantifying the
unique absorptivity properties of compounds in solution. Quantitative analysis can be done
on the basis of extinction coefficients, without standard curves (for example, NADH-based
enzyme assays). When using a cuvette, the pathlength is known and is independent of
sample volume, so absorbance is directly proportional to concentration when there is no
background interference.
24 5014038 E
Chapter 2: Read Modes and Read Types
Horizontal
light path
Vertical light path
Cuvette Microplate wells
In a microplate, pathlength is dependent on the liquid volume, so absorbance is
proportional to both the concentration and the pathlength of the sample. Standard curves
are often used to determine analyte concentrations in vertical-beam photometry of
unknowns, yet errors can still occur from pipetting the samples and standards. The
PathCheck technology automatically determines the pathlength of aqueous samples in the
microplate and normalizes the absorbance in each well to a pathlength of 1cm. This way of
correcting the microwell absorbance values is accurate to within ±4% of the values obtained
directly in a 1cm cuvette.
Figure 2-1: Cuvette and Microplate Well Light Paths
The 1cm values can be obtained by using the factory installed Water Constant. PathCheck
technology is used to normalize the data acquired from absorbance endpoint microplate
readings to a 1cm pathlength, correcting the OD for each well to the value expected if the
sample were read in a 1cm cuvette.
Water Constant
The Water Constant correction method is supported for absorbance endpoint reads.
The PathCheck technology is based on the absorbance of water in the near infrared spectral
region (between 900 nm to 1000 nm). If the sample is completely aqueous, has no turbidity
and has a low salt concentration (less than 0.5 M), the Water Constant is sufficient. The
Water Constant is determined for each instrument during manufacture and is stored in the
instrument.
Note: After you have read a plate with PathCheck technology turned on, PathCheck
information is stored permanently in the data file. You have the option of applying, or
not applying, PathCheck technology to the absorbance values. If you do not have
PathCheck technology turned on during the plate read, you cannot apply the
PathCheck Pathlength Measurement Technology feature after the read.
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SpectraMax Paradigm Multi-Mode Detection Platform User Guide
Eliminating the Pathlength-Independent Component
Raw OD measurements of microplate samples include both pathlength-dependent
components (sample and solvent) and a pathlength-independent component (OD of
microplate material). The pathlength-independent component must be eliminated from the
calculation to get valid results that have been normalized by the PathCheck technology. You
can do this using a plate blank or using a plate background constant.
Using a Plate Blank
This method can be used if all samples in the microplate are the same volume and you are
not depending on the PathCheck technology to correct for variability in volumes.
To use this method:
1. Designate a minimum of one well (preferably several) as Plate Blank.
2. Pipette buffer (for example, your sample matrix) into those wells and read along with
your samples. Do not use an empty well for a blank.
The SoftMax Pro Software automatically subtracts the average of the blank wells from
each of the samples. The OD of the microplate material is subtracted as part of the blank.
3. Make sure that Use Plate Blank is checked under Other Options in the Data Reduction
dialog.
Using a Plate Background Constant
If your sample volumes are not identical or if you choose not to use a Plate Blank, then you
must use a Plate Background Constant. Omitting a Plate Background Constant results in
artificially high values after being normalized by the PathCheck technology.
To determine the Plate Background Constant:
1. Fill a clean microplate with water.
2. Read at the wavelengths that you will be reading your samples.
The average OD value is the Plate Background Constant. If you intend to read your samples
at more than one wavelength, there should be a corresponding number of Plate Background
Constant values for each wavelength.
Note: It is important that you put water in the wells and not read a dry microplate for
the Plate Background Constant. A dry microplate has a slightly higher OD value than a
water-filled microplate because of differences in refractive indices. Using a dry
microplate results in PathCheck technology normalized values that are lower than
1cm cuvette values.
26 5014038 E
Chapter 2: Read Modes and Read Types
Interfering Substances
Material that absorbs in the 900nm to 1000nm spectral region could interfere with
PathCheck technology measurements. Fortunately, there are few materials that do interfere
at the concentrations generally used.
Turbidity is the most common interference. If you can detect turbidity in your sample, you
should not use the PathCheck technology. Turbidity elevates the 900nm measurement
more than the 1000nm measurement and causes an erroneously low estimate of
pathlength. Using Cuvette Reference does not reliably correct for turbidity.
Samples that are highly colored in the upper-visible spectrum might have absorbance
extending into the near-infrared (NIR) spectrum and can interfere with the PathCheck
technology. Examples include Lowry assays, molybdate-based assays, and samples
containing hemoglobins or porphyrins. In general, if the sample is distinctly red or purple,
you should check for interference before using the PathCheck technology.
To determine possible color interference, do the following:
Measure the OD at 900nm and 1000nm (both measured with air reference).
Subtract the 900nm value from the 1000nm value.
Do the same for pure water.
If the delta OD for the sample differs significantly from the delta OD for water, then it is
recommended to not use the PathCheck technology.
Organic solvents could interfere with the PathCheck technology if they have absorbance in
the region of the NIR water peak. Solvents such as ethanol and methanol do not absorb in
the NIR region, so they do not interfere, except for causing a decrease in the water
absorbance to the extent of their presence in the solution. If, however, the solvent absorbs
between 900nm and 1000nm, the interference would be similar to the interference of highly
colored samples as previously described. If you are considering adding an organic solvent
other than ethanol or methanol, you should run a Spectrum scan between 900nm and
1000nm to determine if the solvent would interfere with the PathCheck technology.
5014038 E 27
SpectraMax Paradigm Multi-Mode Detection Platform User Guide
500 550 600 650
0
0.5
1.0
Excitation maximum
Emission maximum
Relative Fluorescence
Wavelength (nm)
Absorption
Stokes
Shift
Fluorescence Intensity Read Mode
Fluorescence occurs when absorbed light is re-radiated at a longer wavelength. In the
Fluorescence Intensity (FL) read mode, the instrument measures the intensity of the reradiated light and expresses the result in Relative Fluorescence Units (RFU).
The governing equation for fluorescence is:
Fluorescence = extinctioncoefficient × concentration × quantumyield ×
excitationintensity × pathlength × emissioncollectionefficiency
Fluorescent materials absorb light energy of a characteristic wavelength (excitation), undergo
an electronic state change, and instantaneously emit light of a longer wavelength (emission).
Most common fluorescent materials have well-characterized excitation and emission
spectra. The following figure shows an example of excitation and emission spectra for a
fluorophore. The excitation and emission bands are each fairly broad, with half-bandwidths
of approximately 40nm, and the difference between the wavelengths of the excitation and
emission maxima (the Stokes shift) is generally fairly small, about 30nm. There is
considerable overlap between the excitation and emission spectra (gray area) when a small
Stokes shift is present.
Figure 2-2: Excitation and Emission Spectra
Because the intensity of the excitation light is usually many tens of thousands of times
greater than that of the emitted light, you must have sufficient spectral separation to reduce
the interference of the excitation light with detection of the emitted light.
28 5014038 E
Chapter 2: Read Modes and Read Types
500 550 600 650
0
0.5
1.0
Excitation maximum
of fluorophore
Emission maximum
of fluorophore
Relative Fluorescence
Wavelength (nm)
Excitation
reading wavelength
Emission
reading wavelength
Tip: If the Stokes shift is small, you should choose an excitation wavelength that is as
far away from the emission maximum as possible while still being capable of
stimulating the fluorophore so that less of the excited light overlaps the emission
spectrum, permitting better selection and quantitation of the emitted light.
The Spectral Optimization Wizard in the SoftMax Pro Software provides the best settings
for maximizing the signal to background window, (S-B)/B, while minimizing the optimization
time. You can use this wizard with a Tunable Wavelength (TUNE) Detection Cartridge installed
in the SpectraMaxParadigm Multi-Mode Detection Platform. See the SoftMax Pro Software
application help or user guide.
5014038 E 29
Figure 2-3: Optimized Excitation and Emission Reading Wavelengths
The previous figure shows that the best results are often obtained when the excitation and
emission wavelengths used for reading are not the same as the peak wavelengths of the
excitation and emission spectra of the fluorophore. When the reading wavelengths for
excitation and emission are separated, a smaller quantity of excitation light passes through
to the emission monochromator (gray area) and on to the PMT, resulting in a purer emission
signal and more accurate data.
SpectraMax Paradigm Multi-Mode Detection Platform User Guide
The instrument permits scanning of both excitation and emission wavelengths, using
separate tunable dual monochromators. One benefit of scanning emission spectra is that
you can determine more accurately whether the emission is, in fact, the expected
fluorophore, or multiple fluorophores, and not one generated by a variety of background
sources or by contaminants. One more benefit is that you can find excitation and emission
wavelengths that prevent interference when interfering fluorescent species are present.
For this reason, it is desirable to scan emission for both an intermediate concentration of
labeled sample, as well as the background of unlabeled sample. The optimum setting is
where the ratio of the sample emission to background emission is at the maximum.
Fluorescence intensity data is dependent on a number of variables. See Analyzing
Fluorescence Intensity Data on page 31.
Applications of Fluorescence Intensity
Fluorescence intensity is used widely in applications such as fluorescent ELISAs, protein
assays, nucleic acid quantitation, reporter gene assays, cell viability, cell proliferation, and
cytotoxicity. One more major application of this mode is to study the kinetics of ion release.
Some assays use a fluorescent label to selectively attach to certain compounds. The quantity
or concentration of the compound can then be quantified by measuring the fluorescence
intensity of the label, which is attached to the compound. Such methods are often used to
quantify low concentrations of DNA or RNA, for example.
You can use the Protocol Manager in the SoftMax Pro Software to quickly find and open a
predefined protocol.
More protocols and updated protocols can be downloaded from the Knowledge Base on the
Molecular Devices support web site (www.moleculardevices.com/support) or from the
protocol sharing web site (www.softmaxpro.org).
The following detection cartridges have fluorescence intensity read mode capability:
Tunable Wavelength (TUNE) Detection Cartridge, see page 97
Multi-Mode (MULTI) Detection Cartridge, see page 102
Fluorescence Intensity (FI) Detection Cartridges, see page 116
Fluorescence Intensity (FI) GeneBLAzer Detection Cartridge, see page 119
Fluorescence Intensity Dual Label (FI-DL) (MultiTox-Fluor) Detection Cartridge, see page
122
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