Molecular Devices SpectraMax GEMINI EM Operator's Manual
SpectraMax® GEMINI EM
Dual-Scanning
Microplate Spectrofluorometer
Operator’s Manual
1311 Orleans Drive
Sunnyvale, California 94089
Part # 0112-0088
Rev. A
Molecular Devices Corporation
®
SpectraMax
GEMINI EM Operator’s Manual
Copyright
© Copyright 2002, 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 GEMINI EM and methods have U.S. and International patents pending.
Trademarks
SpectraPlate and Automix are trademarks and SpectraMax and SoftMax are registered trademarks of
Molecular Devices Corporation.
Cliniplate is a registered trademark of Labsystems.
DELFIA is a registered trademark of Wallac Oy.
Emerald II is a trademark of Tropix, Inc.
All other company and product names are trademarks or registered 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 manual is subject to change without notice.
Although this manual 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.
Molecular Devices Corporation Instrument Warranty
Molecular Devices Corporation warrants this product against defects in material or workmanship as
follows:
1) All parts of the SpectraMax GEMINI EM Microplate Spectrophotometer are warranted for a
period of one (1) year from the original date of delivery.
2) All labor charges to repair the product for a period of one (1) year from the original date of
delivery will be paid by Molecular Devices Corporation.
3) This warranty covers the SpectraMax GEMINI EM instrument only and does not extend to any
computer, printer, software, reagents, or disposables used with the instrument.
ii SpectraMax GEMINI EM Operator’s Manual
Labor and Parts
To obtain warranty service during the applicable warranty period, you must take the product or deliver the product properly packaged in the original shipping materials and carton to an authorized
Molecular Devices Corporation service facility. You must call or write to the nearest Molecular
Devices Corporation service facility to schedule warranty service. You may call Molecular Devices
Corporation at the telephone number or address below to locate the nearest service facility. You must
schedule warranty service
questing warranty service, you must present proof of purchase documentation which includes the
date of purchase, and Molecular Devices Corporation must have the enclosed Warranty Registration
form completed, signed and returned by you within ten (10) working days of the date of delivery.
This warranty covers only defects arising under normal usage and does not cover malfunctions of failures resulting from misuse, abuse, neglect, alteration, modification, or repairs by other than an authorized Molecular Devices Corporation service facility.
Repair or replacement as provided under this warranty is the exclusive remedy to the purchaser (the
“Buyer”).
or consequential damages for breach of any express or implied warranty on this product, except to the extent required by applicable law. The Seller specifically excludes all express and
implied warranties including without limitation any implied warranty that the products sold
under this agreement are merchantable or are fit for any particular purpose, except such warranties expressly identified as warranties and set forth in the Seller’s current operating manual, catalog or written guarantee covering such product. The Seller also makes no warranty
that the products sold under this agreement are delivered free of the rightful claim of any
third party by way of patent infringement or the like.
Seller, the Buyer agrees to hold the Seller harmless against any claim which arises out of compliance
with the specifications.
Molecular Devices Corporation (the “Seller”) shall not be liable for any incidental
prior
to bringing or shipping the product for servicing. At the time of re-
If the Buyer furnishes specifications to the
Any description of the products contained in this agreement is for the sole purpose of identifying
them. Any such description is not part of the basis of the bargain and does not constitute a warranty
that the products shall conform to that description. Any sample or model used in connection with this
agreement is for illustrative purposes only, is not part of the basis of the bargain, and is not to be construed as a warranty that the products will conform to the sample or model. No affirmation of fact or
promise made by the Seller, whether or not in this agreement, shall constitute a warranty that the
products will conform to the affirmation or the promise.
For the name of the nearest authorized Molecular Devices Corporation service facility please contact
Molecular Devices at one of the following telephone numbers:
(408) 747-1700
(800) 635-5577 (U.S. and Canada)
~
SpectraMax GEMINI EM Operator’s Manual iii
iv SpectraMax GEMINI EM Operator’s Manual
Contents
Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Tables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii
Conventions Used in this Manual . . . . . . . . . . . . . . . . . .ix
Chapter 1: Instrument Description. . . . . . . . . . . . . . . . . . . . . . 1-1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-3
Component Description . . . . . . . . . . . . . . . . . . . . . . . . .1-4
The Control Panel . . . . . . . . . . . . . . . . . . . . . . . . . . .1-4
The Microplate Drawer . . . . . . . . . . . . . . . . . . . . . .1-6
The Optical System . . . . . . . . . . . . . . . . . . . . . . . . . .1-7
The Back Panel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-8
Principles of Operation . . . . . . . . . . . . . . . . . . . . . . . . . .1-9
Fluorescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-9
Luminescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-11
Functional Description . . . . . . . . . . . . . . . . . . . . . . . . .1-11
Modes of Operation . . . . . . . . . . . . . . . . . . . . . . . .1-11
Temperature Regulation . . . . . . . . . . . . . . . . . . . .1-12
Automix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-13
Computer Control. . . . . . . . . . . . . . . . . . . . . . . . . .1-13
Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-14
Glossary of Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-16
Chapter 2: Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
Installation Warnings . . . . . . . . . . . . . . . . . . . . . . . . . . .2-3
Installation Cautions . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-3
Unpacking. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-3
Setting Up the Instrument . . . . . . . . . . . . . . . . . . . . . . .2-3
Installing the Drawer Adapter. . . . . . . . . . . . . . . . . . . .2-4
Removing the Drawer Adapter . . . . . . . . . . . . . . . . . . .2-5
Chapter 3: Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1
Prepare for a Reading . . . . . . . . . . . . . . . . . . . . . . . . . . .3-3
Turn the Instrument and Computer On . . . . . . . .3-3
Set the Temperature . . . . . . . . . . . . . . . . . . . . . . . . .3-3
Read the Microplate . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-3
Operation Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-4
Optimizing Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-4
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-4
Using Spectral Scanning to Optimize Excitation
and Emission Wavelengths for Fluorescence
Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-5
SpectraMax GEMINI EM Operator’s Manual v
Chapter 4: Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-3
Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-3
Cleaning the Fan Filter . . . . . . . . . . . . . . . . . . . . . . . . . .4-4
Changing the Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-4
Installing the Drawer Adapter. . . . . . . . . . . . . . . . . . . .4-6
Removing the Drawer Adapter . . . . . . . . . . . . . . . . . . .4-7
Moving the SpectraMax GEMINI EM . . . . . . . . . . . . .4-7
Chapter 5: Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1
Error Codes and Resolutions . . . . . . . . . . . . . . . . . . . . .5-3
Opening the Drawer Manually . . . . . . . . . . . . . . . . . . .5-4
Appendix A: Cables and Accessories . . . . . . . . . . . . . . . . . . . .A-1
Cables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-3
Items Available for Use with the SpectraMax
GEMINI EM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-3
Appendix B: Common Wavelengths for Fluorescence and
Luminescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1
Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-3
Luminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-3
vi SpectraMax GEMINI EM Operator’s Manual
Figures
Figure 1.1 SpectraMax GEMINI EM. . . . . . . . . . . . . . . . . . . . . 1-4
Figure 1.2 Control Panel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4
Figure 1.3 Microplate Drawer (Shown with Adapter
Inserted) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6
Figure 1.4 Components of the SpectraMax GEMINI EM
Optical System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7
Figure 1.5 Components on the Back Panel of the
SpectraMax GEMINI EM. . . . . . . . . . . . . . . . . . . . . 1-8
Figure 1.6 Excitation and Emission Spectra . . . . . . . . . . . . . . 1-9
Figure 1.7 Optimized Excitation and Emission Reading
Wavelengths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10
Figure 2.1 View of Rear Panel . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
Figure 2.2 Adapter Inserted in Microplate Drawer.. . . . . . . . 2-4
Figure 2.3 Microplate Drawer without Adapter. . . . . . . . . . . 2-5
Figure 3.1 Plot of RFU vs. Wavelength . . . . . . . . . . . . . . . . . . 3-6
Figure 3.2 Effects of Cutoff Filters on Fluorescein . . . . . . . . . 3-8
Figure 4.1 Power Switch, Fuse Box, and Power Receptacle . 4-5
Figure 4.2 Removing the Fuse Box . . . . . . . . . . . . . . . . . . . . . . 4-5
Figure 4.3 The Fuse Box and Holder (with Fuses) Removed
from Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6
Figure 4.4 Adapter Inserted in Microplate Drawer.. . . . . . . . 4-6
Figure 4.5 Microplate Drawer without Adapter. . . . . . . . . . . 4-7
SpectraMax GEMINI EM Operator’s Manual vii
Tables
Table 3.1. : Emission Cutoff Filter Default Settings . . . . . . . . . 3-9
Table 5.1. : Error Codes and Resolution . . . . . . . . . . . . . . . . . . . 5-3
viii SpectraMax GEMINI EM Operator’s Manual
Conventions
Used in this
Manual
The names of keys that appear on the SpectraMax GEMINI EM control panel
are shown in boxed Helvetica type. Example:
Italic and boldface type are used for emphasis. Examples: “Press carefully to
engage,” “
NOTE:
CAUTION:
!
WARNING:
BIOHAZARD:
Do not press down. ”
A note provides information that will help you properly execute an
action or procedure.
Indicates an action or condition that could potentially damage the instrument or one of its components or could result in loss of
data.
Indicates a situation that could result in potential injury to a
person working with the system.
Indicates a condition involving potentially infectious
biological agents requiring that proper handling precautions be
taken.
[Setup]
.
SpectraMax GEMINI EM Operator’s Manual ix
xSpectraMax GEMINI EM Operator’s Manual
Chapter 1: Instrument Description
Introduction . . . . . . . . . . . . . . . . . . . . . . . . .1-3
Component Description . . . . . . . . . . . . . . .1-4
The Control Panel . . . . . . . . . . . . . . . . .1-4
The Microplate Drawer. . . . . . . . . . . . .1-6
The Optical System . . . . . . . . . . . . . . . .1-7
The Back Panel . . . . . . . . . . . . . . . . . . . .1-8
Principles of Operation . . . . . . . . . . . . . . . .1-9
Fluorescence . . . . . . . . . . . . . . . . . . . . . .1-9
Luminescence. . . . . . . . . . . . . . . . . . . .1-11
Functional Description . . . . . . . . . . . . . . .1-11
Modes of Operation. . . . . . . . . . . . . . .1-11
Temperature Regulation. . . . . . . . . . .1-12
Automix . . . . . . . . . . . . . . . . . . . . . . . .1-13
Computer Control . . . . . . . . . . . . . . . .1-13
Specifications . . . . . . . . . . . . . . . . . . . . . . .1-14
Glossary of Terms . . . . . . . . . . . . . . . . . . .1-16
Chapter 1
1-2 SpectraMax GEMINI EM Operator’s Manual
Instrument Description
Introduction
The SpectraMax™ GEMINI EM Dual-Scanning Microplate Spectrofluorometer can perform a variety of fluorescent applications as well as some luminescent assays. The extreme flexibility and high sensitivity of the SpectraMax
GEMINI EM make it appropriate for applications within the fields of biochemistry, cell biology, immunology, molecular biology, and microbiology.
The instrument features a bottom-read mode ideal for whole-cell-based
assays and may be automatically switched to a top read mode for solutionphase measurements.
The SpectraMax GEMINI EM uses two holographic diffraction grating
monochromators which allow for individual optimization of wavelengths for
both excitation and emission. The dual-scanning capability can also be used
to determine excitation and emission settings for new fluorescent probes.
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.
Microplates having 6, 12, 24, 48, 96, and 384 wells can be used in the
SpectraMax GEMINI EM. Top or bottom detection is available at the touch of
a button. Detection of species collected on membrane plates is also possible.
One plate carrier adapter is provided with the instrument. The adapter is
required for optimum performance with standard 96- and 384-well format
microplates when reading from the top of the microplate.
The dynamic range of detection is from 10
-6
to 10
-11
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.
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.
The contents of the wells in a microplate can be mixed automatically by shaking before each read cycle, which makes it possible to perform kinetic analysis
of solid-phase, enzyme-mediated reactions such as a kinetic ELISA.
The SpectraMax GEMINI EM is controlled by an external computer running
®
SoftMax
Pro software which provides integrated instrument control, data
display, and statistical data analysis. The SpectraMax GEMINI EM cannot be
operated without the computer and SoftMax Pro software.
SpectraMax GEMINI EM Operator’s Manual 1-3
Chapter 1
Component Description
Control Panel
The main components of the SpectraMax GEMINI EM described in this
manual are:
• The control panel
• The microplate drawer
• Optical system
• The back panel (connections and power switch)
Back Panel
Microplate
Drawer
Figure 1.1: SpectraMax GEMINI EM
The Control Panel
Figure 1.2: Control Panel
The control panel consists of an LCD and four pressure-sensitive membrane
keys which can be used to initiate and regulate the temperature and to open/
close the drawer. When you press a control panel key, the SpectraMax
GEMINI EM performs the desired action.
LCD
A 2-x-20-character liquid crystal display which shows the current instrument
settings.
1-4 SpectraMax GEMINI EM Operator’s Manual
TEMP
TEMP
on/off
Instrument Description
Keys
Allows 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 (
ments the temperature shown in the display by 0.1°C; pressing and holding
ther arrow increments or decrements the temperature shown in the display by
1°C until it is released. If you increment the setting to the highest limit (45°C)
and continue to press the up (
rement the setting to the lowest limit, 15°C, and continue to press the down (
arrow, the display will not change.
Enables/disables the incubator function. When the incubator is on, the set temperature and actual temperature are shown on the front panel LCD display.
▲
) or down (
▲
) arrow, the display will not change. If you dec-
▼
) arrow once increments or decre-
ei-
▼
)
NOTE:
When the instrument is performing a kinetic or spectral scan, the
temperature keys on the front panel are disabled.
Opens or closes the microplate drawer. Whether or not the drawer will remain
open depends on the incubator setting. If the incubator is off, the drawer will
remain open; if the incubator is on, the drawer will close after approximately
10 seconds to assist in maintaining temperature control within the microplate
chamber.
NOTE:
To add reagents during a kinetic read, it is necessary to open the
drawer by pressing the
[Drawer]
key. The drawer will only open,
however, if the interval between readings is equal to the minimum
read interval originally shown by SoftMax Pro software
tional 45 seconds.
If you plan to open the drawer during a kinetic read,
plus an addi-
first determine the minimum read interval allowed and then increase
the setting by a minimum of 45 seconds. The drawer will close automatically after this interval before the next read.
SpectraMax GEMINI EM Operator’s Manual 1-5
Chapter 1
The Microplate Drawer
Figure 1.3: Microplate Drawer (Shown with Adapter Inserted)
The microplate drawer, located on the right side of the SpectraMax
GEMINI EM, slides in and out of the microplate chamber. A small plastic
pusher, located in the front left corner of the drawer, holds the plate securely
in place when the drawer is closed. The drawer remains in the reading chamber during read cycles.
One plate carrier adapter is provided with the instrument. The adapter is
required for optimum performance with standard 96- and 384-well format
microplates in top read mode.
NOTE:
Microplate drawer operation varies, depending upon the incubator status.
When the incubator is off, the microplate drawer remains open at power up
and after a read. When the incubator is on, the drawer closes automatically to
assist in controlling the temperature of the microplate chamber. To open the
drawer, press the
mately ten seconds, after which a beeping sound will alert you approximately
two seconds before the drawer closes automatically. (The drawer will remain
open longer during intervals set for adding reagents during kinetic reads—
see the NOTE on page 1-5).
NOTE:
The adapter must be removed to read 6-, 12-, 24-, or 48-well plates.
[Drawer]
Do not obstruct the movement of the drawer. If you must retrieve a plate
after an error condition or power outage and the drawer will not open, it
is possible to open it manually (see Chapter 5, “Troubleshooting”).
key. The drawer will remain open for approxi-
Microplates
The SpectraMax GEMINI EM can accommodate standard 6-, 12-, 24-, 48-, 96-,
and 384-well microplates. Black-walled, clear-bottom or all black microplates
are generally recommended for fluorescence assays because they have lower
backgrounds than clear plates. White plates may be preferred for luminescence assays to optimize light collection.
Not all manufacturers’ microplates are the same with regard to design, materials, or configuration. Some plastics, most notably polystyrene, also have significant native fluorescence and can cause moderate to severe background
fluorescence, especially in the UV range. If high sensitivity is required, it may
be appropriate to use microplates that are designed to reduce background flu-
orescence.
1-6 SpectraMax GEMINI EM Operator’s Manual
The Optical System
Instrument Description
Excitation monochromator
movable
grating
Ex
cutoff
filter
wheel
2
3
1 mm
fiber
1
flash lamp
Figure 1.4: Components of the SpectraMax GEMINI EM Optical System
Emission monochromator
movable,
focusing
grating
4
microplate
5
6
Em
7
cutoff
filter
wheel
4 mm
optical
bundles
8
photomultiplier
tube
1) The excitation light source is a xenon flashlamp. (Note that the lamp is off
when luminescence mode is selected.)
2) The light passes through a band-pass filter which reduces the amount of
stray light to the excitation monochromator.
3) The holographic diffraction grating monochromator selects the desired
excitation wavelength.
4) The excitation beam is focused by a grating to a 1.0-mm diameter fiber
into the upper or lower optics read head (selectable) before entering the
sample in the microplate well. This focusing helps to prevent part of the
beam from striking adjacent wells.
5) The light beam enters the well and, if fluorescent molecules are present,
light of the emission wavelength is emitted back out to mirrors that focus
it and send it to an optical bundle.
6) The emission monochromator (also a holographic diffraction grating
monochromator) allows light of the chosen emission wavelength to pass
to the emission filter wheel.
7) A long-pass filter further conditions the light prior to detection by the
photomultiplier tube (PMT). This filter may be set automatically by the
instrument or manually by the user.
8) The PMT detects the emitted light and passes a quantitative signal to the
instrument’s electronics which then send the data to the computer.
SpectraMax GEMINI EM Operator’s Manual 1-7
Chapter 1
The Back Panel
Fuse Box Cover
Power Cord
Receptacle
Label
Power Switch
RS-232
Parallel Port
Serial Port
Figure 1.5: Components on the Back Panel of the SpectraMax GEMINI EM
The following components are located on the back panel of the SpectraMax
GEMINI EM:
Power switch : a rocker switch, labeled
•
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.
Printer port : Present but not used in this model of SpectraMax.
•
Serial port (double-shielded RS-232, 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.
Labels : provide information about the SpectraMax GEMINI EM, 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 Services.
I/O
(for on and off, respectively).
1-8 SpectraMax GEMINI EM Operator’s Manual
Instrument Description
Principles of Operation
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. Figure 1.6 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 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.
Excitation
maximum
1.0
0.5
Emission
maximum
Stokes
shift
Relative Fluorescence
0
500 550 600 650
Figure 1.6: 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 GEMINI EM incorporates many
features designed to restrict interference from reflected excitation light.
Among these features is a set of long-pass emission cutoff filters that may 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 will overlap the
emission spectrum, allowing better selection and quantitation of the emitted
light.
Wavelength (nm)
SpectraMax GEMINI EM Operator’s Manual 1-9
Chapter 1
Excitation
reading
wavelength
1.0
0.5
Relative Fluorescence
0
Fluorophore’s
excitation
maximum
500 550 600 650
Wavelength (nm)
Fluorophore’s
emission
maximum
Emission
reading
wavelength
Figure 1.7: Optimized Excitation and Emission Reading Wavelengths
Figure 1.7 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 will pass through to the emission monochromator
(gray area) and on to the PMT, resulting in a purer emission signal and more
accurate data.
The SpectraMax GEMINI EM allows 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 GEMINI
EM, please see “Optimizing Assays” on page 3-4.
1-10 SpectraMax GEMINI EM Operator’s Manual
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