Marshall Amplification Gemini EM, Gemini XPS User Manual
Gemini EM/XPS Dual-Scanning Microplate Spectrofluorometer User Guide — 0112-0128 Rev. A
Gemini EM
Gemini XPS
Dual Scanning Microplate
Spectrofluorometer User Guide
Molecular Devices Corporation
1311 Orleans Drive Sunnyvale, California 94089
Part #0112-0128 Rev. A.
Gemini EM/XPS Dual-Scanning Microplate Spectrofluorometer User Guide — 0112-0128 Rev. A
Molecular Devices Corporation
Gemini EM/XPS Manual
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 Gemini EM, Gemini XPS, and methods have U.S. and International patents pending.
Gemini EM Patents
6,097,025, 6,232,608, 6,236,456, 6,313,471, 6,316,774, and 6,693,709.
Gemini XPS Patents 6,097,025, 6,232,608, 6,236,456, 6,313,471, and 6,316,774.
Trademarks
SpectraPlate and Automix are trademarks and SoftMax are registered trademarks of Molecular Devices
Corporation.
DELFIA is a registered trademark of PerkinElmer Life Sciences.
Emerald II is a trademark of Applera Corp.
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.
Gemini EM/XPS Dual-Scanning Microplate Spectrofluorometer User Guide — 0112-0128 Rev. A
Questions?
Phone:1 (800) 6355577
Fax:+1 (408) 7473603
Web:www.moleculardevices.com
Gemini EM/XPS Dual-Scanning Microplate Spectrofluorometer User Guide — 0112-0128 Rev. A
Gemini EM/XPS Dual Scanning Microplate Spectrofluorometer User Guide — 0112-0128 Rev. A
iii
Contents
Contents
1. Description
Features. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. Principles of Operation
Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
TimeResolved Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Luminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Functional Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3. Installation
Unpacking. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Setting up the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Installing the Drawer Adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Removing the Drawer Adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4. Operation
Quick Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Preparing for a Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Read the Microplate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Optimizing Asays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5. Maintenance
Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Moving the Gemini. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Cleaning the Fan Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Changing the Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Contents
Gemini EM/XPS Dual Scanning Microplate Spectrofluorometer User Guide — 0112-0128 Rev. A
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Contents
6. Troubleshooting
Opening the Drawer Manually . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Error Codes and Probable Causes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
7. Specifications
Gemini EM Specifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Geminie XPS Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
A. Appendix
Cables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Accessories. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
B. Appendix
Common Wavelengths for Fluorescence and Luminescence . . . . . . . . . . . . . . . . . . . 45
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
System Diagrams and Dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Gemini EM/XPS Dual Scanning Microplate Spectrofluorometer User Guide — 0112-0128 Rev. A
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1. Description
1. Description
1.1. FEATURES
The Gemini EM and Gemini XPS DualScanning Microplate Spectrofluorometers can
perform a variety of fluorescent applications. The extreme flexibility and high sensitivity
of Gemini readers make them appropriate for applications within the fields of
biochemistry, cell biology, immunology, molecular biology, and microbiology.
1.1.1. DUAL MONOCHROMATORS
The right pair of excitation and emission wavelengths is always available because the dual
monochromators allow the selection of any wavelength in 1 nm increments. New
fluorophores can easily be evaluated without purchasing additional filters.
The Gemini EM and Gemini XPS microplate readers use two holographic diffraction
grating monochromators, which allow for individual optimization of wavelengths for
both excitation and emission. The dualscanning capability can also be used to determine
excitation and emission settings for new fluorescent probes.
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 flash lamp; additional flexibility is provided by allowing a variable
number of lamp flashes per read.
1.1.3. WAVELENGTH SCANNING
The most sensitive results are achieved by using optimal excitation and emission
wavelengths. Literature wavelengths are often based on results from wavelengthlimited,
filterbased readers. Wavelength scanning ensures that the most sensitive assay conditions
are used.
1.1.4. WELL SCANNING
Gemini EM and Gemini XPS can report a single point from the well center, or multiple
data points from the bottom of large well tissue culture plates to provide high sensitivity
for cellbased assays.
1.1.5. AUTO PMT GAIN
Because a single microplate often presents a range of fluorescence intensities greater than
three orders of magnitude, Gemini EM and Gemini XPS feature “Auto PMT Gain” to
1. Description
Gemini EM/XPS Dual Scanning Microplate Spectrofluorometer User Guide — 0112-0128 Rev. A
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1. Description
avoid saturating the photomultiplier tube. The signal is calibrated against an internal
standard, so the reported RFU values of individual samples can be accurately compared.
1.1.6. TOP AND BOTTOM READING OPTICS—GEMINI EM ONLY
The top/bottomreading optical design of the Gemini EM allows for measurements for
both solution and cellbased assays. With the click of a button, the Gemini EM can be
switched between top and bottomreading modes.
1.1.7. SUPPORTED PLATES
Microplates having 6, 12, 24, 48, 96, and 384 wells can be used in Gemini readers.
One plate carrier adapter is provided with the instrument. The adapter is required for
optimum performance with standard 96 and 384well format microplates when reading
from the top of the microplate.
1.1.8. DYNAMIC RANGE
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.
1.1.9. 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.
1.1.10. AUTOMIX
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 solidphase,
enzymemediated reactions such as a kinetic ELISA.
1.1.11. COMPUTER CONTROL
Gemini readers are controlled by an external computer running SoftMax® Pro software
which provides integrated instrument control, data display, and statistical data analysis.
Gemini readers cannot be operated without the computer and SoftMax Pro software.
1.1.12. SECONDARY MODES
The Gemini EM and Gemini XPS have two secondary modes that can be used for limited
development of glow luminescence or timeresolved fluorescence assays. The performance
of these two modes is not comparable to dedicated luminescence or timeresolved
fluorescence instruments.
1.2. Components
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1. Description
Figure 1.1: Gemini EM.
1.2. COMPONENTS
The main components of Gemini readers described in this manual are:
>
Control panel
>
Microplate drawer
>
Optical system
>
Back panel (connections and power switch)
1.2.1. THE CONTROL PANEL
The control panel consists of a 2×20character LCD and four pressuresensitive
membrane keys that can be used to initiate and regulate the temperature and to open and
close the drawer. When you press a control panel key, the Gemini performs the associated
action.
Figure 1.2: Control Panel.
1. Description
Gemini EM/XPS Dual Scanning Microplate Spectrofluorometer User Guide — 0112-0128 Rev. A
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1. Description
TEMP
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.
Tem p O n/ Of f
The key enables and disables the incubator.
>
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.
Drawer
The key opens and closes the microplate drawer.
1.2.2. THE MICROPLATE DRAWER
The microplate drawer, located on the right side of the Gemini, 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 96well and 384well format microplates in top read
mode. The adapter is required when using the SpectraTest FL validation plate to test the
Gemini XPS and when testing the Gemini EM top read optics. To test the Gemini EM
bottom reading performance, remove the purple adapter and then turn the validation
plate upside down by rotating it from toptobottom so that column 1 remains on your
left.
TEMP
TEMP on/off
DRAWER
1.2. Components
Gemini EM/XPS Dual Scanning Microplate Spectrofluorometer User Guide — 0112-0128 Rev. A
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1. Description
Figure 1.3: Microplate drawer (with adapter inserted).
The adapter must be removed to read 6well, 12well, 24well, or 48well plates.
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.
To add reagents during a kinetic read, it is necessary to open the drawer by pressing the
key. The drawer only opens, however, if the interval between readings is equal
to the minimum read interval originally shown by SoftMax Pro software plus an
additional 45 seconds. If you plan to open the drawer during a kinetic read, first
determine the minimum read interval allowed and then increase the setting by a
minimum of 45 seconds. The drawer closes automatically after this interval before the
next read.
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 (see Chapter 6, “Troubleshooting”).
1.2.3. MICROPLATES
Gemini readers can accommodate standard 6well, 12well, 24well, 48well, 96well, and
384well microplates. Blackwalled, clearbottom or allblack 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 fluorescence.
DRAWER
1. Description
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1. Description
1.2.4. THE OPTICAL SYSTEM—GEMINI EM
Figure 1.4: Components of the Gemini EM optical system.
1 The excitation light source is a xenon flash lamp. (Note that the lamp is off when
luminescence mode is selected.)
2 The light passes through a bandpass filter that 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 1.0mm 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 longpass 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 that then send the data to the computer.
movable
grating
flash lamp
1 mm
fiber
4 mm
optical
bundles
microplate
movable,
focusing
grating
photomultiplie
tube
1
2
3
4
5
6
7
8
Ex
cutoff
filter
wheel
Em
cutoff
filter
wheel
1.2. Components
Gemini EM/XPS Dual Scanning Microplate Spectrofluorometer User Guide — 0112-0128 Rev. A
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1. Description
1.2.5. THE OPTICAL SYSTEM—GEMINI XPS
Figure 1.5: Components of the Gemini XPS optical system.
1 The excitation light source is a xenon flash lamp. (Note that the lamp is off when
luminescence mode is selected.)
2 The light passes through a bandpass filter that 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 collimated by a mirror to a 1.0mm diameter fiber before
entering the sample in the microplate well. This focusing helps to prevent part of the
beam from striking adjacent wells.
movable
grating
flash lamp
1-mm
fiber
4-mm
optical
bundles
microplate
Excitation monochromator
Emission monochromator
Reading chamber
single channel upper
optics on linear stage
movable,
focusing
grating
photomultiplier
tube
1
2
3
4
5
6
7
8
Ex
cutoff
wheel
Em
cutoff
wheel
1. Description
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1. Description
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 longpass 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.
1.2.6. THE BACK PANEL
Figure 1.6: Schematic of the back panel of a Gemini reader.
The following components are located on the back panel of Gemini readers:
>
Power switc h: 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.
>
Parallel port: present but not used in this model of reader.
>
Serial port (doubleshielded RS232, for use with an external computer): plug one end
of an 8pin DIN serial cable into this port; the other end attaches to the serial (modem)
port of the computer.
>
Label: provides information about the Gemini, 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.
RS-232
Serial
Parallel Port
Power Switch
Fuse Box Cover
Power Cord
Receptacle
Label
Gemini EM/XPS Dual Scanning Microplate Spectrofluorometer User Guide — 0112-0128 Rev. A
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2. Principles of Operation
2. Principles of Operation
2.1. 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 wellcharacterized excitation and
emission spectra. Figure 2.1 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.
Figure 2.1: Excitation and emission spectra.
Excitation
maxim um
Emission
maxim um
Stokes
shift
1.0
0.5
0
Relative Fluorescence
Wavelength (nm)
500 550 600 650
2. Principles of Operation
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2. Principles of Operation
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
Gemini readers incorporate many features designed to restrict interference from reflected
excitation light. Among these features is a set of longpass 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.
Figure 2.2: Optimized excitation and emission reading wavelengths.
Figure 2.2 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 Gemini readers allow scanning of both excitation and emission wavelengths, using
separate tunable monochromators. One benefit of being able to scan emission spectra is
Excitation
reading
Emission
reading
Fluorophore’s
excitation
1.0
0.5
0
Relative Fluorescence
Wavelength (nm)
500 550 600 650
maximum
Fluorophore’s
emission
maximum
wavelength
wavelength
2.2. Time-Resolved Fluorescence
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2. Principles of Operation
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 Gemini, see “Optimizing Assays ” on page 21.
2.2. TIME-RESOLVED FLUORESCENCE
In normal fluorescence mode, readings are taken while the lamp is on. The most common
limitation to sensitivity in normal fluorescence is excitation energy or background
fluorescence that cannot be eliminated from the emission signal. Since the lamp is the
source of excitation energy, turning it off provides the best means of eliminating
background excitation.
Timeresolved fluorescence is performed by flashing the excitation lamp and, after it is off,
collecting the delayed emission for a period of time before the lamp is flashed again.
Lanthanide dyes are frequently used to delay the fluorescence long enough to measure it
after the lamp is turned off.
To assist with proper collection of data, you can also select when to start and end data
collection (within the limits of the system—the minimum is 50 μs and the maximum is
1450 μs in 200μs steps).
2.3. LUMINESCENCE
In luminescence mode, no excitation is necessary as the species being measured emit light
naturally. For this reason, the lamp does not flash, so no background interference occurs.
A dark estimate is done over a dark reference, and multiple readings are averaged together
into one reading per well.
You can choose the wavelength where peak emission is expected to occur. In addition,
multiple wavelength choices allow species with multiple components to be differentiated
and measured easily. In luminescence read mode, no emission cutoff filter is used. The
default setting for luminescence is the “zero order” position where the grating
monochromator acts as a mirror that reflects all light to the PMT detector.
The Gemini readers are microplate spectrofluorometers with photomultiplier tube
detection. Some luminescence applications, such as gene reporter assays, may require a
luminometer with photon counting detection for greater sensitivity.
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