The appeal of multidetection microplate readers
is
easy to understand: they combine multiple measurement
technologies such as absorbance, fluorescence,
and luminescence into one compact instrument.
This, along with both the extension of the microplate
format into new applications and the continued
development of new assay technologies, creates
a powerful research tool. This observation is substantiated
by the tremendous growth of the detection
platform over the last several years, leading one to
believe that the multidetection microplate reader
will become the de facto choice for most researchers.
Figure 1 - Synergy 4 multidetection microplate reader.
Given the great variations found in microplate-based
assays, the promise of multidetection microplate
readers is that they can accommodate new and unexpected
user requirements and applications when they
arise. This article reviews one of the most important
decisions to be made when purchasing such a reader,
that is, the type of wavelength selection system
available: filter-based or monochromator-based
. The
choice has a fundamental impact on what reagent
technology will be possible to perform on the instrument.
The author reviews the main drivers behind
the choice of filters versus monochromator
systems
and the significant benefits offered by the patent pending Synergy™ 4 Multidetection Microplate Reader with Hybrid Technology (BioTek Instruments ,
Winooski, VT) (Figure 1).
Monochromators use diffraction gratings to physically
separate the individual wavelengths present
in the white light coming from the instrument’s
light source. A series of slits allow for selecting a
specific wavelength to excite the sample. A similar
system is used to clean the signal coming from
the sample before it is measured. Monochromator-based readers offer several evident benefits
(summarized in Table 1). First, they are very convenient
to use. The wavelengths to be measured
are easily selected through the software and are
generally available in 1-nm increments. Manipulation
and storage of accessories are unnecessary,
contrary to filter-based systems. Adjustments for
new applications and wavelengths are relatively
simple, without an increase in time or cost for
extra optical elements.
Second, monochromators can run spectral scans that
can be used to characterize new fluorophores or study
spectral shifts in some assays. The usefulness of the scanning
function, however, is limited by the fact that microplate readers are optically different from dedicated analytical
cuvette-based spectrofluorometers. The 90° angle
between the excitation beam and the emission channel
found in cuvette-based readers (horizontal photometry)
is impossible to achieve in a microplate reader (vertical photometry). For this reason, microplate readers exhibit
higher background noise than cuvette-based readers and
cannot provide the sensitivity and resolution expected
from an analytical-grade instrument.
The third benefit of monochromator-based microplate
readers—flexibility—is mostly perceived. Because
these microplate readers are often more expensive
than filter-based microplate readers and allow reading
at most wavelengths, it is assumed that they can run
more assays than filter-based microplate readers. Further
discussion will reveal that the contrary is true.
Optical filters are characterized with a central wavelength
and a bandwidth. These two fixed parameters precisely define which wavelengths are going
to the sample on the excitation side, and from
the sample to the detector on the emission side.
Filter-based microplate readers have a number of
important advantages (see Table 1). First, they
are typically less expensive than monochromator-based microplate readers. This comes from
the fact that filter wheels are much less expensive
parts than monochromators, and the light
sources required to obtain the same level of sensitivity
do not need to be as powerful.
The second benefit is sensitivity. All things being
equal (quality of optical elements, power of light
source, detector used), a filter-based microplate
system will be more sensitive than a monochromator-based microplate system. Filter systems are
more efficient at delivering light to the sample.
They are also very efficient at providing correct
light blocking between the excitation channel
and the emission channel. Thus, the paradox
is that filter-based microplate readers will usually
be more sensitive than monochromator-based
microplate readers for less money.
A third advantage of filter-based systems is bandwidth
selection. A filter can be specifically tailored
to a particular assay (or fluorophore) to obtain maximum
sensitivity, and can have a bandwidth anywhere
between 5 nm to more than 100 nm, selectable
in 0.5–1 nm increments. Monochromator-based
microplate readers come with a fixed or limited range
of bandwidth selection. Given the fact that they
measure in epifluorescence mode (not at a 90° angle,
as found on cuvette-based readers), this bandwidth
limitation is a problem with low-level fluorescence,
where a large measurement bandwidth is necessary,
such as AlphaScreen® assays (PerkinElmer), which
require a strong excitation and the use of a 100-nm
emission bandpass.
The next advantage is that filter wheels allow quick
switching back and forth between two wavelengths.
Typical assays where this is required are ratiometric
fluorescent ion channel assays. In these assays, the
signal needs to be monitored at two wavelengths,
one being used to correct the other. The reactions
are extremely fast, triggered by automated injection,
and the assay kinetics must be monitored at two distinct wavelengths during the few seconds
that follow reagent injection. Typically, filter sets
can be switched back and forth in a fraction of a
second. Many high-throughput microplate readers
are equipped with dual-filter systems and detectors
to read these types of assays. Monochromator
microplate readers cannot run these ratiometric
assays because of the time it takes to move the
monochromator position from one wavelength to
the other. A secondary consequence of this difference
is that all filter-based multidetection microplate
readers are available with reagent injectors
to run these types of assays; most monochromator based
microplate readers are not. Very common
assays such as flash luminescence (e.g., flash luciferase,
or flash adenosine 5′-triphosphate [ATP]
assays) do require automatic injection.
Luminescence brings us to the final major advantage
of filter-based systems: efficient light transmission.
Because of the significant amount of light
lost through dual-monochromator systems during
wavelength selection, luminescence cannot be
read with adequate sensitivity. As a consequence,
light is typically channeled directly from the sample
to the detector, without going through the
monochromator system. This works very well for
whole light assays such as glow ATP and glow
luciferase assays, but does not allow for light filtration.
Some key applications of luminescence
involve the need for emission filtration: Bioluminescent
Resonance Energy Transfer assays (BRET,
BRET2) or Chroma-Glo assays are examples of
dual-wavelength assays that cannot be run on
monochromator-based microplate readers, unless
they are equipped with a separate dedicated filter-based
luminescence system.
The numerous advantages of filter-based systems
over monochromator-based systems explains why, as
previously mentioned, filter-based microplate readers
are in practical terms more versatile than monochromator-based microplate readers, since they will allow
the user to run more of the typical applications found
in the microplate format.
Hybrid Technology, introduced in the Synergy 4,
is a significant step forward in the design of multidetection
microplate readers. By combining the
benefits of both filter-based and monochromator based fluorescence detection, it brings to laboratories
a new level of flexibility and convenience. At
a price similar to monochromator-only systems,
the Synergy 4 bridges the gaps highlighted previously,
and provides a design that covers virtually all
microplate-based applications.
Summary
In the end, while it appears that multidetection
microplate readers are purchased on the promise
that they will meet future needs, both filter-only
and monochromator-only microplate readers have
important limitations in the type of assays they can
perform. Filter-based microplate readers do not allow
spectral scanning, and necessitate the use of specialized
filters for each new fluorophore used. Monochromator-based microplate systems have weaknesses
inherent to the nature of their optical design, which
prevent users from running some of the common
applications found in the microplate format. One
microplate reader that unites the two technologies
within a single footprint can truly satisfy current and
future laboratory needs and applications.
Mr. Amouretti is Product Manager, BioTek Instruments,
Inc., Highland Park, P.O. Box 998, Winooski, VT 05404-0998, U.S.A.; tel.: 888-451-5171; fax: 802-655-7941;
e-mail: [email protected].