Molecular Devices SpectraMax M3 User manual

0112-0115 G
October 2018
SpectraMax® M3
SpectraMax® M4
SpectraMax® M5
SpectraMax® M5e
Multi-Mode Microplate Readers
User Guide

SpectraMax M3, M4, M5, and M5e Microplate Reader User Guide
2 0112-0115 G
This document is provided to customers who have purchased Molecular Devices equipment, software,
reagents, and consumables to use in the operation of such Molecular Devices equipment, software,
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Portions of this document may make reference to other manufacturers and/or their products, which
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Each product is shipped with documentation stating specifications and other technical information.
Molecular Devices products are warranted to meet the stated specifications. Molecular Devices makes
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Molecular Devices, LLC is ISO 9001 registered.
©2018Molecular Devices, LLC.
All rights reserved.

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Contents
Safety Information 5
Warnings, Cautions, Notes, and Tips 5
Chapter 1: Introduction 9
Computer Integration 10
Supported Plates and Cuvettes 11
Shake 12
Temperature Regulation 12
Chapter 2: Setting Up the Instrument 13
Connecting Instrument Cables 14
Chapter 3: Getting Started 15
Control Panel 16
Cuvette Chamber 18
Plate Drawer 19
Plate Adapter 20
Chapter 4: Operating the Instrument 21
Temperature Settings 21
Wavelength Settings 22
Read a Plate 23
Read a Cuvette 24
Chapter 5: Read Modes and Read Types 25
Absorbance Read Mode 25
Fluorescence Intensity Read Mode 30
Luminescence 32
Time-Resolved Fluorescence Read Mode 33
Fluorescence Polarization Read Mode 34
Read Types 37
Chapter 6: Maintenance 39
Cleaning the Instrument 39
Replacing Fuses 40
Before You Move the Instrument 42

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Chapter 7: Troubleshooting 43
Opening the Drawer Manually 47
Obtaining Support 48
Appendix A: Specifications 49
System Diagrams and Dimensions 55
Appendix B: Accessories 57
Appendix C: Electromagnetic Compatibility 60

Safety Information
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Safety Information
Information about the safe use of the instrument from Molecular Devices® includes an
understanding of the user-attention statements in this guide, the safety labels on the
instrument, precautions to follow before you operate the instrument, and precautions to
follow while you operate 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.
Read and observe all warnings, cautions, and instructions. 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 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. Follow the
related safety information.
The following user attention statements might be displayed in the text of Molecular Devices
user documentation. Each statement implies the amount of observation or recommended
procedure.
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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Symbols on the Instrument
Each safety label found on the instrument contains an alert symbol that indicates the type of
potential safety hazard.
Symbol Indication
Consult the product documentation.
Potential electrical-shock hazard from a high-voltage source. All safety instructions
must be read and understood before you proceed with the installation, maintenance,
and service of all modules. Power off the instrument and disconnect the power cord
before you do maintenance procedures that require removal of a panel or cover or
disassembly of an interior instrument component.
Risk of electrical shock. Refer servicing to qualified personnel only.
Potential lifting hazard. To prevent injury, use a minimum of two people to lift the
instrument.
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, 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.
- - California proposition 65 requires businesses to provide
warnings to Californians about significant exposures to chemicals that cause cancer, birth
defects, or other reproductive harm.

Safety Information
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Electrical Safety
To prevent electrical injuries and property damage, inspect all electrical equipment before
use and report all electrical deficiencies. Contact Molecular Devices technical support for
equipment service 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. Read and understand
all safety instructions before you install, maintain, and service the instrument.
To prevent electrical shock, use the supplied power cord and connect to a properly
grounded wall outlet.
To protect against fire hazard, replace the fuses only with the same type and rating as the
original factory installed fuses.
To ensure sufficient ventilation and provide access to disconnect power from the
instrument, maintain a 20cm to 30cm (7.9in. to 11.8in.) gap between the rear of the
instrument and the wall.
Power off the instrument when not in use.
Chemical and Biological Safety
Normal operation of the instrument can involve the use of materials that are toxic,
flammable, or otherwise biologically harmful. When you use 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 required precautions when using pathological, toxic, or radioactive materials.
Splashing of liquids can occur. Take applicable safety precautions, such as using safety
glasses and wearing protective clothing, when working with potentially hazardous
liquids.
Observe the applicable cautionary procedures as defined by your safety officer when
using hazardous materials, flammable solvents, toxic, pathological, or radioactive
materials in or near a powered-up instrument.
WARNING! Never use the instrument in an environment where potentially
damaging liquids or gases are present.

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CAUTION! Use of organic solvents can cause harm to the optics in the instrument.
Extreme caution is recommended when you use organic solvents. Always use a plate
lid and do not place a plate that contains these materials in the plate chamber for
prolonged periods of time. Damage caused by the use of incompatible or aggressive
solvents is NOT covered by the instrument warranty.
CAUTION! When you use aggressive or corrosive reagents, you should have the plate
automatically move out of the instrument after a read.
Moving Parts Safety
The instrument contains moving parts that can cause injury. Under normal conditions, the
instrument is designed to protect you from these moving parts.
WARNING! If the instrument is used in a manner not specified by Molecular
Devices, the protection provided by the equipment might be impaired.
To prevent injury:
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 front of the instrument of 18 cm (7.1 in.) for the plate drawer.
CAUTION! To prevent damage to the instrument, the height of the plate must not
exceed 17mm, including the lid if the plate is lidded.
WARNING! Do not attempt to access the interior of the instrument unless
specifically instructed to do so. The moving parts inside the instrument can cause
injury. Do not operate the instrument with any covers or panels removed.
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.

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Chapter 1: Introduction
The SpectraMax® M3 Multi-Mode Microplate Reader, SpectraMax® M4 Multi-Mode
Microplate Reader, SpectraMax® M5 Multi-Mode Microplate Reader, and SpectraMax® M5e
Multi-Mode Microplate Reader are dual monochromator, multi-detection instruments with
a triple-mode cuvette port and a 6-well to 384-well plate read capability.
The flexibility and high sensitivity makes these instruments appropriate 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.
Read Modes SpectraMax
M3
SpectraMax
M4
SpectraMax
M5
SpectraMax
M5e
Absorbance
Fluorescence Intensity
Luminescence
Time-Resolved
Fluorescence
Fluorescence Polarization
Read Modes
The SpectraMax M5e is certified for use with Cisbio Bioassays’ HTRF (Homogeneous Time-
Resolved Fluorescence) technology. HTRF is a proprietary Time-Resolved Fluorescence
technology that overcomes many of the drawbacks of standard Fluorescence Resonance
Energy Transfer (FRET) techniques, such as the requirements to correct for autofluorescence
and the fluorescent contributions of unbound fluorophores.
Read types include: See Read Types on page 37.
Endpoint
Kinetic
Spectrum
Well Scan
The instrument light source is a high-powered Xenon flash lamp. Two holographic diffraction
grating monochromators allow for individual optimization of wavelengths for both excitation
and emission in the Fluorescence Intensity read mode. Mirrored optics focus the light in to
the sample volume, and cutoff filters reduce stray light and minimize background
interference. You can vary the number of lamp flashes per read to optimize the sensitivity or
read speed.
The instrument dynamic range of detection is from 10-6 to 10-12 molar fluorescein. Internal
compensation for detector sensitivity, photomultiplier tube voltage and sensitivity, as well
as excitation intensity, effectively eliminates variations in measured fluorescence values. The
photometric range is 0–4 ODs with a resolution of 0.001 OD.
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Dual monochromators enable you to select absorbance wavelength between 200 nm and
1000 nm and excitation wavelengths:
Between 250 nm and 850 nm for reads in:
Fluorescence Intensity read mode
Time-Resolved Fluorescence read mode (SpectraMax M4, SpectraMax M5, and
SpectraMax M5e)
Luminescence read mode
Between 350–750 nm for Fluorescence Polarization read mode (SpectraMax M5 and
SpectraMax M5e)
Computer Integration
Each Molecular Devices microplate reader is shipped with a license key for the SoftMax® Pro
Data Acquisition and Analysis Software that you install on the computer that you use to
operate the instrument. The SoftMax Pro Software provides integrated instrument control,
data display, and statistical data analysis.
You should install the SoftMax Pro Software on the computer before you set up the
instrument. Please be aware that some updates to the SoftMax Pro Software require a
purchase. Contact Molecular Devices before you update the software. To download the
latest version of the software, visit:
https://www.moleculardevices.com/products/microplate-readers/acquisition-and-analysis-
software/softmax-pro-software#Order.
Note: For information about the computer specifications that are required to run the
software, the software installation and licensing instructions, and the directions to
create the software connection between the computer and the instrument, see the
SoftMax Pro Data Acquisition and Analysis Software Installation Guide.
To prevent data loss, turn off all sleep and hibernation settings for the hard disk, the CPU,
and the USB ports. Disable automatic Windows updates. Update Windows manually when
you do not use the computer to control an instrument. You can set these options in
Windows Control Panel.

Chapter 1: Introduction
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Supported Plates and Cuvettes
The instrument accommodates both plates and cuvettes.
Note: The supplied plate adapter is required for some applications. See Plate Adapter
on page 20.
Supported Plates
You can use 6, 12, 24, 48, 96, and 384-well plates that conform to ANSI/SBS standard
microplate footprint and dimensions in the instrument. The plate list in the software includes
half area and low- volume plates. The software enables you to define a new plate type based
on the manufacturer's specifications for well size, spacing, and distance from the plate edge.
Use polystyrene plates for absorbance wavelengths above 340 nm.
To allow transmission of the deep UV spectra when you do a read at wavelengths below
340 nm, you must use special UV-transparent plates or quartz plates.
Endpoint, Kinetic, Spectrum, and Well Scan read types are available for the Absorbance
and Fluorescence read modes with any plate model.
Top and bottom reads are available for the Fluorescence, Time-Resolved Fluorescence,
and Luminescence read modes.
One plate drawer adapter is provided with the instrument. The plate adapter is required
for optimum performance with standard 96-well and 384-well format plates for all top-
read applications.
Note: Although not recommended, you can also use strip well plates in the
instrument. When you use strip-wells, use high quality support frames or holders. To
avoid warping, you should fill the frame or holder with empty strips.
Supported Cuvettes
For a cuvette read, you must ensure that the meniscus is above the light beam in a standard
cuvette and that the sample chamber is aligned properly with the beam in a microcuvette.
The light beam is 0.625 in (15.87 mm) above the cuvette bottom.
Use the cuvette port for Absorbance, Fluorescence, and Luminescence read modes.
To allow transmission of the deep UV spectra when you do a read at wavelengths below
340 nm, you must use special UV-transparent cuvettes or quartz cuvettes.
Endpoint, Kinetic, and Spectrum read types are available for the Absorbance and
Fluorescence read modes with cuvettes.
Note: You can use a 12 x 75 mm test tube with a test tube cover. You can order the
test tube cover from Molecular Devices. See Accessories on page 57.

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Shake
The software enables you to define settings to mix of the contents of the wells by shaking the
plate linearly along the long axis at preset intervals.
For the Endpoint read type, you can shake the plate for 0 to 999 seconds before the read
at each wavelength you set.
For the Kinetic read type, you can shake the plate for 0 to 999 seconds before the initial
read, and/or before each subsequent read.
Use of shake is recommended for ELISAs and other solid-phase, enzyme-mediated reactions
to enhance accuracy.
Temperature Regulation
The temperature in the plate chamber and cuvette chamber is isothermal, both at ambient
and when you turn on the incubator. When you turn on the incubator, you can set the
temperature from 2°C above ambient to 60°C. To achieve regulation at temperatures greater
than 50°C, the instrument’s ambient temperature may need to be increased to 25°C or
higher. The operating humidity must be non-condensing, and can range from 0 to 70%.

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Chapter 2: Setting Up the Instrument
Before you unpack and set up the instrument, prepare a dry, flat work area that is away from
direct sunlight, dust, drafts, vibration, and moisture. The work area must have sufficient
space for the instrument, host computer, and required cables. To provide access for
disconnecting power from the instrument, maintain a 20cm to 30cm (7.9in. to 11.8in.) gap
between the rear of the instrument and the wall. To ensure sufficient ventilation, do not
block the ventilation grids on the front and rear of the instrument.
The package contains the instrument and the following accessories:
SoftMax Pro Software, product key, and installation guide
Instrument installation guide
USB computer connection cable
AC power adapter
Plate adapter
For a complete list of the package contents, see the enclosed packing list.
Before you set up the instrument, you should install the SoftMax Pro Software on the
computer that you plan to use to operate the instrument. See the SoftMax Pro Data
Acquisition and Analysis Software Installation Guide.
Unpacking the Instrument
The packaging is designed to protect the instrument during shipment. Tape is placed on the
cuvette door to protect the instrument from damage during shipment.
CAUTION! Do not touch or loosen screws or parts other than those specifically
designated in the instructions. Doing so could cause misalignment and possibly void
the warranty.
The instrument is packed in a specially designed carton. Before you unpack the instrument,
check the box for any damage that might have occurred during transportation. If any
damage is noted, inform the supplier immediately and keep the damaged packaging.
Note: Retain the shipping box and all packing materials for future transport needs.
To unpack the instrument:
1. Open the top of the box.
2. Lift the accessories tool box and the instrument from the package, and then place the
instrument on a level surface.
3. Remove the cardboard divider from the top of the instrument.
4. Remove the packing material from both ends of the instrument, remove the instrument
from the plastic bag, and then set the instrument down carefully on a level surface.
5. Remove the tape from the cuvette door.
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Connecting Instrument Cables
The power cord and USB cable connect to the ports on the rear of the instrument.
Part Number Description
1-2100-0548 USB computer connection cable, 3meter (9.8foot)
4400-0002 Power cord, 1meter (3.3foot) See Accessories on page 57 for international
power cord part numbers.
Note: Before you connect or disconnect the power cord, make sure that the power
switch that is on the rear of the instrument is in the Off position.
To connect the instrument cables:
1. Turn the instrument around so that the rear of the instrument faces you.
2. Make sure that the power switch is in the Offposition.
3. Connect the appropriate end of the supplied USB cable to the USB port, and then
connect the other end to a USB port on the computer.
4. Connect the supplied power adapter to the power cord port, and then connect the other
end to a grounded electrical wall outlet.
5. Turn the instrument around so that the front of the instrument faces you.
Note: Ensure no cables run beneath the instrument.

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Chapter 3: Getting Started
Now that you have installed the SoftMax Pro Software software on the computer, removed
the tape from the cuvette port, and connected the cables, it is time to get started.
Turn on the instrument power switch located on the rear of the instrument. Wait for the
instrument to complete its diagnostic check and the plate drawer opens.
The following are the main components of the instrument:
Control panel: Enables you to open the plate drawer, control the read chamber
temperature, and set the wavelengths.
Cuvette chamber: Enables you to read the contents of a cuvette.
Plate drawer: Enables you to insert the plate to read.
CAUTION! Never touch the internal optic mirrors, lenses, filters, or cables. The optics
are extremely delicate, and critical to the function of the instrument.
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Control Panel
The instrument control panel works in conjunction with the SoftMax Pro Software to open
the plate drawer, adjust the temperature, and to set the wavelengths for cuvette reads. See
the SoftMax Pro Data Acquisition and Analysis Software User Guide.
To change the control panel display contrast, press Mode, and then press Temp por q.
The left side of the control panel displays the actual temperature inside the cuvette chamber
above the display of the temperature you set for the cuvette chamber. If the temperature
inside the cuvette chamber is not at the set temperature, the indicator blinks. The
temperature inside the plate chamber lags slightly behind the temperature inside the
cuvette chamber. The temperature inside the plate chamber displays in the software.
The center of the control panel displays the absorbance/excitation and emission
wavelengths the instrument uses for a cuvette read.
The right side of the control panel displays the data the instrument receives from a cuvette
reference read as percent transmission, absorbance, relative fluorescence units, or relative
luminescence units. If you do not do a reference read, the indicator blinks.
Note: Settings you enter in the software override the corresponding control panel
setting.
Temp On/Off
The Temp On/Off button enables or disables the incubator that controls the temperature
inside the cuvette chamber and the plate chamber.
When the incubator is on, the control panel displays the temperature inside the cuvette
chamber above the display of the temperature you set.
For the Kinetic and Spectrum read types, the temperature buttons on the control panel
are disabled.

Chapter 3: Getting Started
0112-0115 G 17
Temp
The Temp pand qbuttons enable you to enter the set point at which to regulate the
temperature inside the cuvette chamber and the plate chamber. The temperature for the
instrument chambers is isothermal at ambient temperature as well as at elevated
temperatures. The cuvette chamber temperature displays on the instrument control panel
and the plate chamber temperature displays in the software.
You cannot set a temperature beyond the upper (60°C) or lower (2°C above ambient)
instrument limits. To set the temperature at 60°C, the ambient temperature of the room
must be at least 20°C.
Wavelength (λ)
The λpand qbuttons enable you to set the wavelengths to read the cuvette. The control
panel contains two sets of λwavelength buttons. The left buttons enable you to set the
absorbance/excitation (fluorescence) wavelength and the right buttons enable you to set the
emission (fluorescence) wavelength.
Note: The control panel does not display the wavelength if you use the software to
set the wavelength.
Mode
The Mode button enables you to change the display of the cuvette reference read data
betweenpercent transmittance (%T), absorbance (A), relative fluorescence units (RFU), or
relative luminescence units (RLU).
Read Cuvette
The Read Cuvette button enables you to start a cuvette read.
Note: The Read Cuvette button is disabled when the software does a read.
REF
The REF button enables you to do a cuvette reference read of buffer, water, or air. The
software uses this value as I0to calculate Absorbance or % Transmittance. If you do not do a
reference read, the instrument uses the I0value that is stored in the instrument.
Note: The REF button is disabled when the software does a read.
Drawer
The Drawer button enables you to open and close the plate drawer.

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Cuvette Chamber
The cuvette chamber is located to the right of the control panel. Lift up the lid over the
chamber to insert or remove a cuvette. The chamber contains springs that position the
cuvette in the proper alignment for a read. You must close the cuvette door before you start
a read.
The instrument can accommodate standard height (45 mm), 1 cm cuvettes and 12 x 75 mm
test tubes when you use a test tube cover. You can also use semi-microcuvettes and ultra-
microcuvettes with an adapter. See Supported Plates and Cuvettes on page 11.
Cuvettes for the Absorbance read mode are frosted on two sides. Handle the cuvettes on the
frosted sides only. Place the cuvette in the cuvette chamber so that the read (clear) sides face
left and right.
Cuvettes for the Fluorescence read mode are clear on all four sides and you should handle
these cuvettes carefully.

Chapter 3: Getting Started
0112-0115 G 19
Plate Drawer
The plate drawer is located on the right side of the instrument and slides in and out of the
plate chamber. An internal latch positions the plate on the drawer as it closes. You can use 6,
12, 24, 48, 96, and 384-well plates that conform to ANSI/SBS standard microplate footprint
and dimensions in the instrument. See Supported Plates and Cuvettes on page 11.
Note: The supplied plate adapter is required for some applications. See Plate Adapter
on page 20.
The Drawer button enables you to open and close the plate drawer, or you can use the
software. The drawer closes for each read. Subsequent plate drawer operation is dependent
on the incubator setting:
When you open the drawer, if the incubator is off, the drawer remains open.
When you open the drawer, if the incubator is on, the drawer closes after approximately
10 seconds to maintain temperature control within the plate chamber.
Drainage ports protect the plate drawer mechanism and plate chamber from accidental
spills.
CAUTION! Do not obstruct the movement of the plate drawer. If you must retrieve a
plate after an error condition or power outage and the plate drawer does not open,
you can open the drawer manually. See Opening the Drawer Manually on page 47.
CAUTION! To prevent damage to the instrument, the height of the plate must not
exceed 17mm, including the lid if the plate is lidded.

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Plate Adapter
Before you read standard 96-well or 384-well plates from the top, you must insert the plate
adapter in the plate drawer. Before you do a bottom read, or use high profile (6-well, 12-well,
24-well, or 48-well) plates, you must remove the plate adapter from the plate drawer.
CAUTION! Incorrect insertion or removal of the plate adapter can cause damage to
the instrument plate drawer. To ensure that you do not damage the plate drawer, do
the following procedures.
To insert the plate adapter:
1. Open the plate drawer.
2. Hold the plate adapter so that the label is on the front side facing up.
3. Place the top back (Row A) of the plate adapter into the drawer first. The corner cutout
must be in the lower left corner where the plate pusher is located. While pushing against
the back edge of the adapter, lower the front of the plate adapter onto the drawer.
To remove the plate adapter:
1. Open the plate drawer.
2. In the lower left corner where the plate pusher is located, carefully lift the front of the
plate adapter at the corner cutout. As you lift the plate adapter, make sure to also lift
from the other end of the front edge, and then completely remove the plate adapter
from the plate drawer.