Molecular Devices SpectraMax M2 User manual

A MULTI-DETECTION MICROPLATE READER WITH TWO-MODE CUVETTE PORT
SpectraMax®M2/M2euser guide

SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers User Guide — 0112-0102 Rev. D
SpectraMax®M2
SpectraMax®M2e
Multimode Plate Readers
User Guide
Molecular Devices Corporation
1311 Orleans Drive Sunnyvale, California 94089
Part #0112-0102 Rev. D.

SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers User Guide — 0112-0102 Rev. D
Copyright
© Copyright 2006, Molecular Devices Corporation. All rights reserved. No part of this publication may
be reproduced, transmitted, transcribed, stored in a retrieval system, or translated into any language or
computer language, in any form or by any means, electronic, mechanical, magnetic, optical, chemical,
manual, or otherwise, without the prior written permission of Molecular Devices Corporation, 1311
Orleans Drive, Sunnyvale, California, 94089, United States of America.
Patents
The SpectraMax M2 and SpectraMax M2eand use thereof are covered by issued U.S. Patent nos.
5,112,134; 5,766,875; 5,959,738; 6,188,476; 6,232,608; 6,236,456; 6,313,471; 6,316,774; 6,320,662;
6,339,472; 6,404,501; 6,496,260; and foreign patents. Other U.S. and foreign patents pending.
Trademarks
PathCheck, SpectraMax and SoftMax are registered trademarks and Automix is a trademark of
Molecular Devices Corporation.
All other company and product names are trademarks of their respective owners.
Disclaimer
Molecular Devices Corporation reserves the right to change its products and services at any time to
incorporate technological developments. This user guide is subject to change without notice.
Although this user guide has been prepared with every precaution to ensure accuracy, Molecular Devices
Corporation assumes no liability for any errors or omissions, nor for any damages resulting from the
application or use of this information.

SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers User Guide — 0112-0102 Rev. D iii
Contents
Contents
1. Description
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. Principles of Operation
Absorbance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Optical Density. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Transmittance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
PathCheck®. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Time-resolved Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Luminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Functional Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3. Installation
Unpacking. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Setting up the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Installing the Drawer Adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Removing the Drawer Adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
4. Operation
Cuvette Read: Quick Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Microplate Read: Quick Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Preparing for a Cuvette or Microplate Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Read the Cuvette. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Read the Microplate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Optimizing Fluorescence Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Contents
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Contents
5. Maintenance
Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Moving a SpectraMax M2 or M2e. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
General Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Cleaning the Fan Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Changing the Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
6. Troubleshooting
Opening the Drawer Manually . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Error Codes and Probable Causes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
7. Specifications
SpectraMax M2 and M2ePerformance Specifications . . . . . . . . . . . . . . . . . . . . . . . . 45
A. Appendix
Cables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Accessories. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Cuvettes in SpectraMax M2 and SpectraMax M2e. . . . . . . . . . . . . . . . . . . . . . . . . . 52
Common Wavelengths for Fluorescence and Luminescence . . . . . . . . . . . . . . . . . . . 54
Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Time-Resolved Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Luminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
System Diagrams and Dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers Operator’s Manual — 0112-0115 Rev. D 1
1. Description
1. Description
1.1. INTRODUCTION
The SpectraMax M2 and SpectraMax M2eMulti-detection Readers are monochromator-
based microplate readers that have 6-well to 384-well microplate reading capability as well
as a built-in absorbance and fluorescence cuvette port. The spectrophotometric
performance of the SpectraMax M2 and SpectraMax M2eis similar to the SpectraMax
Plus, a dedicated absorbance plate reader. The fluorometric performance of the
SpectraMax M2 is similar to that of the Gemini XPS, a dedicated top-read fluorescence
microplate reader. The fluorometric performance of the SpectraMax M2eis similar, but
slightly superior to that of the Gemini EM, a dedicated top and bottom read fluorescence
microplate reader.
SpectraMax M2 and SpectraMax M2ereaders can acquire absorbance as well as
fluorescence data for samples by issuing a single read command in SoftMax®Pro. Dual
monochromators allow selection of any absorbance wavelength between 200 nm and
1000 nm, any excitation wavelength between 250 nm and 850 nm, and any emission
wavelength between 360 nm and 850 nm (SpectraMax M2) or 250 n and 850 nm
(SpectraMax M2e) for readings of both microplates and cuvettes.
1.1.1. APPLICATIONS
Endpoint, kinetic, spectrum, and multi-point well-scanning applications combining
absorbance and fluorescence in 6-well to 384-well microplates, as well as endpoint,
kinetic, and spectrum applications in absorbance and fluorescence using cuvettes, can be
run with little to no optimization.
The extreme flexibility and high sensitivity of the SpectraMax M2 and SpectraMax M2e
make them appropriate for applications within 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.
The SpectraMax M2 and SpectraMax M2ealso have two secondary modes that can be
used for limited development of glow luminescence or time-resolved fluorescence assays.
The performance of these two modes is not comparable to dedicated luminescence or
time-resolved fluorescence instruments, or multimode readers such as the
SpectraMax M5, Analyst HT or Analyst GT.

1. Description
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1. Description
1.1.2. OPTICS
Mirrored optics focus the light into the sample volume, and cutoff filters are used to
reduce stray light and minimize background interference. The light source is a high-
powered Xenon flashlamp; additional flexibility is provided by allowing a variable number
of lamp flashes per read.
1.1.3. DYNAMIC RANGE
The dynamic range of detection is from 10–6 to 10–12 molar fluorescein. Variations in
measured fluorescence values are virtually eliminated by internal compensation for
detector sensitivity, photomultiplier tube voltage and sensitivity, as well as excitation
intensity. The photometric range is 0–4 ODs with a resolution of 0.001 OD.
1.1.4. PATHCHECK
SpectraMax M2 and SpectraMax M2ewith PathCheck®Sensor allow normalization of
variable well volumes to 1-cm cuvette readings. PathCheck allows for multichannel
pipettor validation and for experiment comparison from different days.
1.1.5. AUTOMIX
Using SoftMax Pro, the contents of the wells in a microplate can be mixed automatically
by linear shaking before each read cycle, making it possible to perform kinetic analysis of
solid-phase, enzyme-mediated reactions (mixing is not critical for liquid-phase reactions).
1.1.6. TEMPERATURE CONTROL
Temperature in the microplate chamber is isothermal, both at ambient and when the
incubator is turned on. When the incubator is on, the temperature may be controlled
from 4°C above ambient to 45°C.
1.1.7. SUPPORTED PLATES
Microplates having 6, 12, 24, 48, 96, and 384 wells can be used in the SpectraMax M2
and SpectraMax M2e. Top detection is available for fluorescence detection on the
SpectraMax M2, while top and bottom reads are possible on the SpectraMax M2e. When
reading optical density at wavelengths below 340 nm, special UV-transparent, disposable
or quartz microplates and cuvettes that allow transmission of the far UV spectra must be
used.
One plate carrier adapter is provided with the instrument. The adapter is required for
optimum performance with standard 96-well and 384-well format microplates for all top-
read applications.
1.1.8. COMPUTER CONTROL
SpectraMax M2 and SpectraMax M2eare controlled by an external computer running
SoftMax Pro software which provides integrated instrument control, data display, and
statistical data analysis. Cuvette port functionality can be accessed using SoftMax Pro
software.

1.2. Components
SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers Operator’s Manual — 0112-0115 Rev. D 3
1. Description
The on-board microprocessor calculates and reports the absorbance, % transmittance,
and/or fluorescence for each well of a microplate or from the cuvette port. Data from
multiple wavelengths can be acquired and ratio analysis can be performed during a single
reading, if desired, and different calculations can be made based on this data using
SoftMax Pro software, including the subtraction of blanks, use of standard curves, etc.
For detailed reader specifications, refer to the chapter “Specifications” in this guide.
1.2. COMPONENTS
The main components of the SpectraMax M2 and SpectraMax M2eare:
>Control panel: for cuvette chamber control.
>Microplate drawer: used for absorbance and fluorescence intensity read modes for
endpoint, kinetic, well scan and spectrum scanning.
>Cuvette chamber: used for absorbance, fluorescence intensity read modes for endpoint,
kinetic, and spectrum scanning.
>Back panel: connections and power switch.
Figure 1.1: SpectraMax M2 and SpectraMax M2ecomponents.
1.2.1. THE CONTROL PANEL
The control panel consists of a 2x20-character LCD and eleven pressure-sensitive
membrane keys that can be used to control some functions of the instrument. When you
press a control panel key, the instrument performs the associated action. Note that
settings made in SoftMax Pro software override control panel settings.
The left side of the display shows the cuvette temperature, both actual and set point, and
whether or not the temperature is at the set point (enunciator blinks if not at set point).
To see the microplate chamber temperature, you must use SoftMax Pro software.
Control Panel Back PanelCuvette Chamber
Microplate Drawer

1. Description
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1. Description
The middle of the display shows the wavelengths for absorbance/excitation and emission.
The right side of the display shows the data received from the reading as absorbance,
percent transmission, fluorescence emission or excitation, or luminescence, and indicates
whether or not a reference measurement was made (enunciator blinks if no reference
reading was taken).
To change the contrast on the control panel, press and the temperature up or
down setting keys.
Figure 1.2: SpectraMax M2 and SpectraMax M2econtrol panel.
Temp On/Off Key
The key enables and disables the incubator that controls the temperature
within both the microplate chamber and the cuvette port.
>When the incubator is on, the set temperature and actual temperature are shown on the
front panel LCD display.
>When the instrument is performing a kinetic or spectral scan, the temperature keys on
the front panel are disabled.
Te m p K e y
The keys allow you to enter a set point at which to regulate the microplate
chamber temperature.
Pressing this key scrolls the temperature up or down, starting at the previous temperature
setting (or the default of 37.0°C, if no setting had been made):
>Pressing the up (S) or down (T) arrow once increments or decrements the displayed
temperature by 0.1°C.
>Pressing and holding either arrow increments or decrements the displayed temperature
by 1°C until it is released.
You cannot set a temperature beyond the upper (45°C) or lower (15°C) instrument limits.
%T/A/RFU
TEMP on/off
TEMP

1.2. Components
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1. Description
λKey
Selects the wavelength to be used for reading the microplate manually. The control panel
does not display the wavelength selected through SoftMax Pro.
Pressing the up or down arrow key scrolls up or down through the available wavelengths,
starting at the previous setting:
>Pressing the up (S) or down (T) arrow once increments or decrements the displayed
wavelength by 1 nm.
>Pressing and holding either arrow increments or decrements the displayed wavelength
by 10 nm until it is released.
Ref Key
A reading of buffer, water, or air taken in the cuvette that is used as I0to calculate
Absorbance or % Transmittance. If no reference reading is taken, the instrument uses the
I0values stored in the NVRAM (non-volatile memory) of the instrument.
Read Cuvette Key
Initiates the sample reading of the cuvette.
%T/A Key
A toggle switch used to display cuvette data as percent transmission or absorbance.
Drawer key
The key opens and closes the microplate drawer.
1.2.2. THE MICROPLATE DRAWER
The microplate drawer is located on the right side of the instrument and slides in and out
of the reading chamber. An internal latch positions the microplate in the drawer as it
closes (allowing for better robot integration—no springs or clips are used).
The drawer remains in the reading chamber during read cycles.
Figure 1.3: The microplate drawer.
DRAWER

1. Description
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1. Description
Microplate drawer operation varies, depending on the incubator setting:
>If the incubator is off, the drawer remains open.
>If the incubator is on, the drawer closes after approximately 10 seconds to assist in
maintaining temperature control within the microplate chamber.
Do not obstruct the movement of the drawer. If you must retrieve a plate after an error
condition or power outage and the drawer does not open, it is possible to open it
manually (refer to the chapter “Troubleshooting” in this guide).
1.2.3. MICROPLATES
The SpectraMax M2 and SpectraMax M2ecan accommodate SBS-standard 6-well to
384-well microplates and strip wells. When reading optical density at wavelengths below
340 nm, special UV-transparent, disposable or quartz microplates allowing transmission
of the deep UV spectra must be used.
Not all manufacturers’ microplates are the same with regard to design, materials, or
configuration. Temperature uniformity within the microplate may vary depending on the
type of microplate used.
Microplates currently supported by SoftMax Pro for use in this instrument are:
>96-well Standard, 96 Costar, 96 Greiner Black, 96 Bottom Offset, 96 Falcon, 96 BD
Optilux/Biocoat, 96 BD Fluoroblok MW Insert, 96 Corning Half Area, 96 MDC HE
PS
>384-well Standard, 384 Costar, 384 Greiner, 384 Falcon, 384 Corning, 384 MDC HE
PS
>48 Costar
>24 Costar
>12 Costar, 12 Falcon
>6 Costar, 6 Falcon
The SoftMax Pro plate list also includes half area and low-volume plates. SoftMax Pro can
always be used to define a new plate type using the manufacturer's specifications for well
size, spacing and distance from the plate edge.
1.2.4. THE CUVETTE CHAMBER
Located at the right front of the SpectraMax M2 and SpectraMax M2e, the cuvette
chamber has a lid that lifts up, allowing you to insert or remove a cuvette. The chamber
contains springs that automatically position the cuvette in the proper alignment for a
reading. The cuvette door must be closed before initiating a reading.

1.2. Components
SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers Operator’s Manual — 0112-0115 Rev. D 7
1. Description
Figure 1.4: The cuvette chamber.
Cuvettes
The SpectraMax M2 and SpectraMax M2ecan accommodate standard-height (45 mm),
1-cm cuvettes and 12 x 75 mm test tubes when used with the test tube cover (Figure 1.5).
Not all manufacturers’ cuvettes are the same with regard to design, materials, or
configuration. Temperature uniformity within the cuvette may vary depending on the
type of cuvette used.
Cuvettes used for absorbance readings are frosted on two sides. Be sure to handle cuvettes
on the frosted sides only. Place the cuvette into the chamber so that the “reading” (clear)
sides face left and right.
Fluorescence cuvettes are clear on all four sides and should be handled carefully. Place a
frosted cuvette into the chamber so that the “reading” (clear) sides face left and right.
Semi-micro and ultra-micro cuvettes can also be used with an adapter.
Figure 1.5: Test tube cover.

1. Description
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1. Description
1.2.5. THE BACK PANEL
The following components are located on the back panel of the SpectraMax M2 and
SpectraMax M2e:
>Power switch: a rocker switch, labeled I/O (for on and off, respectively).
>Power cord receptacle: plug the power cord in here.
>Fuse box cover: cannot be opened while the power cord is plugged in. When opened, it
provides access to the fuse box containing two fuses that are required for operation.
>Computer port (double-shielded 8-pin RS-232 serial, for use with an external
computer): plug one end of an 8-pin DIN serial cable into this port; the other end
attaches to the serial (modem) port of the computer.
>Printer port: not used in the SpectraMax M2 or SpectraMax M2e.
>Label: provides information about the reader, such as line voltage rating, cautionary
information, serial number, etc. Record the serial number shown on this label for use
when contacting Molecular Devices Technical Support.
Figure 1.6: Schematic of the back panel of SpectraMax M2 and SpectraMax M2e.
Power Switch
Power Cord
Receptacle
Label
Fuse Box
Cover
Computer
Port
Printer
Port

SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers User Guide — 0112-0102 Rev. D 9
2. Principles of Operation
2. Principles of Operation
2.1. ABSORBANCE
Absorbance is the amount of light absorbed by a solution. To measure absorbance
accurately, it is necessary to eliminate light scatter. In the absence of turbidity, absorbance
= optical density.
where I0is incident light, and Iis transmitted light.
In this user guide, we use the terms absorbance and optical density interchangeably.
2.2. OPTICAL DENSITY
Optical density is the amount of light passing through a sample to a detector relative to
the total amount of light available. Optical density includes absorbance of the sample plus
light scatter from turbidity.
2.3. TRANSMITTANCE
Transmittance is the ratio of transmitted light to the incident light.
where I0is incident light, and Iis transmitted light.
2.4. PATHCHECK
The Beer-Lambert law states that absorbance is proportional to the distance that light
travels through the sample:
where Ais the absorbance,
ε is the molar absorbtivity of the sample, bis the pathlength
and cis the concentration of the sample. In short, 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 perform extinction-
Alog I0I ⁄ ()=
TI
0I⁄()=
%T 100T=
Aεbc=

2. Principles of Operation
SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers User Guide — 0112-0102 Rev. D
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2. Principles of Operation
based assays and also makes it confusing to compare results between microplate readers
and spectrophotometers.
The standard pathlength of a cuvette is the conventional basis for quantifying the unique
absorbtivity properties of compounds in solution. Quantitative analyses can be performed
on the basis of extinction coefficients, without standard curves (e.g. NADH-based enzyme
assays). When using a cuvette, the pathlength is known and is independent of sample
volume, so absorbance is proportional to concentration.
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 arise from pipetting the samples and standards. The
PathCheck feature automatically determines the pathlength of aqueous samples in the
microplate and normalizes the absorbance in each well to a pathlength of 1 cm. This
novel approach to correcting the microwell absorbance values is accurate to within 2.5%
of the values obtained directly in a 1-cm cuvette.
Figure 2.1: Cuvette and microwell light paths.
Reference measurements made by reading the cuvette (Cuvette Reference) or using
factory-stored values derived from deionized water (Water Constant) can be used to
normalize the optical density data for microplate wells.
PathCheck pathlength correction is accomplished only when using the SoftMax Pro
software. PathCheck is patented by Molecular Devices and can be performed only on an
MDC plate reader.
The SpectraMax M2 and SpectraMax M2eoffer both the Cuvette Reference and the
Water Constant methods.
The actual pathlength, d, of a solvent is found from the following equation:
Horizontal
light path
Vertical light path
Cuvette Microplate wells
dcm()Sample OD1000 OD900
–()
k
---------------------------------------------------------------------
=

2.4. PathCheck
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2. Principles of Operation
When a Cuvette Reference is used for pathlength correction, the value of kis obtained by
taking optical density measurements on the fluid in the cuvette at two wavelengths, 1000
and 900 nm:
When the Water Constant is used for pathlength correction, the value of kis obtained
from the instrument. This constant is saved in the instrument in the factory and may
differ slightly from instrument to instrument.
Once the pathlength d is found, the following equation is used for the pathlength
correction:
PathCheck is applicable to almost all biological/pharmaceutical molecules in aqueous
solution because they have little or no absorbance between 900 nm and 1000 nm at
concentrations normally used. PathCheck can also be used with samples containing small
amounts of organics or high buffer concentrations by using the Cuvette Reference
(below).
2.4.1. WATER CONSTANT OR CUVETTE REFERENCE?
The PathCheck measurement is based on the absorbance of water in the near infrared
region (between 900 nm and 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
adequate. The Water Constant is determined during manufacture and is stored in the
instrument.
If the sample contains an organic solvent such as ethanol or methanol, we recommend
using the cuvette reference. It is important that the solvent does not absorb in the 900 nm
to 1000 nm range (to determine whether or not a given solvent would interfere, see the
discussion of interfering substances below). When a non-interfering solvent is added to
the aqueous sample, the water absorbance decreases proportionally to the percentage of
organic solvent present. For example, 5% ethanol decreases the water absorbance by 5%
and results in a 5% underestimation of the pathlength. You can avoid the error by putting
the same water/solvent mixture in a cuvette and using the Cuvette Reference.
To use the Cuvette Reference, place into the cuvette port a standard 1 cm cuvette
containing the aqueous/solvent mixture that is used for the samples in the microplate.
The cuvette must be in place when you read the microplate. When you click the Read
button in SoftMax Pro, the instrument first makes the 900 nm and 1000 nm
measurements in the cuvette, and then makes the designated measurements in the
microplate. The cuvette values are stored temporarily and used in the PathCheck
calculations for the microplate samples.
kCuvette OD1000 OD900
–()
=
OD
cm
---------ODSample
dcm()
---------------------------
=

2. Principles of Operation
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2. Principles of Operation
Use of Cuvette Reference with PathCheck is different from a reference reading of a cuvette
in a CuvetteSet section (by clicking the Ref button in the CuvetteSet section tool bar in
SoftMax Pro). The cuvette reference used for PathCheck calculations (measurements at
900 nm and 1000 nm) does not produce data that can be viewed in a CuvetteSet section
and is used only with data in microplates, not cuvettes.
2.4.2. BACKGROUND CONSTANT SUBTRACTION AND BLANKING
CONSIDERATIONS
Raw optical density measurements of microplate samples include both pathlength-
dependent components (sample and solvent) and a pathlength-independent component
(OD of microplate material). The latter must be eliminated from the PathCheck
calculation in order to get obtain PathCheck-normalized results. There are three ways to
accomplish this—plate blanks, plate background constants, and plate pre-reads—all of
which are described in the PathCheck section of the SoftMax Pro User Guide.
2.4.3. PATHCHECK AND INTERFERING SUBSTANCES
Any material that absorbs in the 900 nm to 1000 nm spectral region could interfere with
PathCheck measurements. Fortunately, there are few materials that do interfere at the
concentrations typically used.
Turbidity is the most common interference: if you can detect any turbidity in your
sample, you should not use PathCheck. Turbidity elevates the 900 nm measurement more
than the 1000 nm 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 may have absorbance
extending into the near infrared (NIR) and can interfere with PathCheck. 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 PathCheck.
To determine possible color interference, do the following:
>Measure the optical density at 900 nm and 1000 nm (both measured with air refer-
ence).
>Subtract the 900 nm value from the 1000 nm 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
advisable not to use the PathCheck feature. Use of Cuvette Reference does not correct for
the interference with the current calculation scheme in SoftMax Pro. Currently, Cuvette
Reference involves a single (automated) read at 900 nm and 1000 nm and the automated
calculations in SoftMax Pro do not compensate for color or solvent interference.
However, you could correct for such interference by taking two cuvette measurements and

2.5. Fluorescence
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2. Principles of Operation
using a different set of calculations. For further information, contact Molecular Devices
Te c h n ic a l Su p p o r t .
Organic solvents could interfere with PathCheck if they have absorbance in the region of
the NIR water peak. Solvents such 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. Their passive interference can be avoided by
using the Cuvette Reference. If, however, the solvent absorbs between 900 and 1000 nm,
the interference would be similar to the interference of highly colored samples described
previously. If you are considering adding an organic solvent other than ethanol or
methanol, you are advised to run a spectral scan between 900 nm and 1000 nm to
determine if the solvent would interfere with PathCheck.
2.4.4. MAKING ABSORBANCE MEASUREMENTS NORMALIZED TO A 1-CM
PATHLENGTH
SoftMax Pro automatically reports absorbance values normalized to a 1-cm pathlength.
The table below shows results obtained with 75 µL to 300 µL yellow reagent.
Optical pathlengths and raw absorbance values were directly proportional to well
columns. After normalization to a 1-cm pathlength, all absorbance values, regardless of
the volume in the wells, were within 1% of the value obtained by measuring the same
solution in a 1-cm cuvette.
2.5. FLUORESCENCE
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 figure below 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 40 nm, and the wavelength difference between the
Well Volume
(µL)
Pathlength
(cm)
Raw
Absorbance
Absorbance/
cm SD CV%
75 0.231 0.090 0.390 0.006 1.6
100 0.300 0.116 0.387 0.005 1.2
150 0.446 0.172 0.385 0.003 0.8
200 0.596 0.228 0.383 0.002 0.4
250 0.735 0.283 0.384 0.002 0.5
300 0.874 0.336 0.384 0.001 0.3
Absorbance in 1-cm cuvette = 0.386

2. Principles of Operation
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14
2. Principles of Operation
excitation and emission maxima (the Stokes shift) is typically fairly small, about 30 nm.
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, some type of spectral separation is necessary to
reduce the interference of the excitation light with detection of the emitted light. The
SpectraMax M2 and SpectraMax M2eincorporate many features designed to restrict
interference from reflected excitation light. Among these features is a set of long-pass
emission cutoff filters that can be set automatically by the instrument or manually by the
user. If the Stokes shift is small, it may be advisable to 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, allowing better selection and quantitation of the emitted light.
Wavelength (nm)
Relative Fluorescence
Absorption
Excitation
maximum
Emission
maximum
Stokes
Shift
500 550 600 650
0
0.5
1.0

2.5. Fluorescence
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2. Principles of Operation
Figure 2.3: Optimized excitation and emission reading wavelengths.
The figure above shows that the best results are often obtained when the excitation and
emission wavelengths used for reading are not the same as the wavelengths of the
excitation and emission spectra of the fluorophore. When the reading wavelengths for
excitation and emission are separated, a smaller amount 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.
The SpectraMax M2 and SpectraMax M2eallow scanning of both excitation and
emission wavelengths, using separate tunable monochromators. One benefit of being able
to scan emission spectra is that you can assess 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. Another benefit is that you may be
able to find excitation and emission wavelengths that avoid interference when interfering
fluorescent species are present.
For this reason, it may be 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.
For more information regarding optimizing excitation and emission wavelengths using
the spectral scanning capabilities of the SpectraMax M2 and SpectraMax M2e, refer to the
section “Optimizing Fluorescence Assays” of the chapter “Operation” in this guide.
Wavelen
g
th (nm)
Relative Fluorescence
Excitation
reading
wavelength
Emission
reading
wavelength
Fluorophore’s
excitation
maximim
Fluorophore’s
emission
maximim
500 550 600 650
0
0.5
1.0