May 1, 2012 (Vol. 32, No. 9)

Direct Optic Approach Enhances Fluorescent Protein and Other Microplate Analyses

When the Nobel Prize for Chemistry was awarded to Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien for the discovery and development of the green fluorescent protein (GFP) in 2008, researchers around the world were already using GFP (and the many other variants) in a wide range of cell-based assays.

One of the most important uses for GFP is monitoring a variety of cellular processes in real-time. The many applications that use fluorescent proteins include: intracellular transport, protein signaling, receptor desensitization, cell movement, migration, division, apoptosis, metabolism, differentiation, chemotaxis, transcription, translation, and many more.

As with any new, popular, Nobel Prize-winning research advancement, a correlative advancement usually occurs in analytical instrumentation. One instrument that all life science laboratories have access to because of the GFP discovery is the confocal microscope. Live, real-time pictures (and movies) of cellular processes, highlighted by different fluorescent proteins, are easily recorded using a confocal microscope.

Confocal microscopes have allowed researchers to obtain fantastic snapshots of biological processes using fluorescent proteins. One drawback of confocal microscopy, though, is that patience and time are needed to obtain reliable, reproducible data since only one cell or cell cluster is viewed at a time.

Another instrument that has increased in utility due to the GFP discovery is the flow cytometer. More specifically, specialized types of flow cytometers can perform Fluorescence-Activated Cell Sorting (FACS®), which is often used in high-throughput screening (HTS) and high-content screening (HCS) labs. FACS provides a way to sort heterogeneous cell populations into homogeneous subgroups, thereby counting and separating the cells that have a fluorescent protein from those that do not.

FACS lack one of the limitations of confocal microscopy in that it provides an automated method that counts one specific activated cell type. But like the confocal microscope, flow cytometers that perform FACS are still limited by the time it can take to obtain data from an experiment with many testable parameters. Hours can pass during a FACS experiment, which means that assay conditions may not be uniform across the entire test.

The fluorescent microplate reader is another instrument that has seen an expanded role in life science laboratories after the discovery of GFP. Used mainly in life science or HTS laboratories to study simple, homogeneous absorbance, luminescence, or fluorescence based experiments, microplate readers have evolved into multifunction instruments that can perform complex, heterogeneous cell-based assays. Being able to measure mL to nL volumes, and up to thousands of samples at once, microplate readers allow for all types of reproducible, cell-based assays to be measured in only minutes.

Until recently, though, there was a limitation to cell-based experiments performed in a microplate reader in that the same sensitivity obtained on a confocal microscope or a FACS could not be matched by a microplate reader. For live, real-time cell-based experiments, it is preferable to read from the bottom of the microplate.

Reading from the bottom allows for a cover or lid to be placed on top of the microplate to prevent cell contamination and liquid evaporation. One main reason for poorer sensitivity in microplate readers is that they use longer, more flexible fiber optics to reach the microplate bottom. Since fiber optics lose light, more signal (and thus sensitivity) is lost when measuring fluorescent proteins in a microplate reader.

To improve microplate cell-based assays, BMG LABTECH has advanced microplate reader technology by eliminating the need for fiber optics for bottom reading. Using a system analogous to the microscope, the PHERAstar FS incorporates a series of software-controlled, motor-driven mirrors to focus light through a free air optical path directly onto either the top or bottom of the microplate. Moreover, when switching between top and bottom reading modes a simple click in the software is all that is needed, there is no changing or installation of any additional hardware (i.e., optics, apertures, dichroics, filters, or mirrors). Thus direct-optic bottom reading is fully integrated into the PHERAstar FS optical system and it completely eliminates the need for fiber optics.

Figure 1. Direct-optic bottom reading (green columns) vs. fiber-optic bottom reading (red columns): A fluorescein dilution series in a 1,536-well microplate shows that even at picomolar concentrations the direct-optic bottom reading on the PHERAstar FS will give at least a 10 times higher signal-to-blank ratio than a fiber-optic bottom reading instrument.

Direct-Optic Bottom Reading

To demonstrate the overall improvement of direct-optic bottom reading on the PHERAstar FS as compared to a fiber-optic bottom-reading instrument, a fluorescein dilution series was measured from the bottom of a 1,536-well microplate. As shown in Figure 1, direct-optic bottom reading gives at least an 11-fold higher signal-to-blank ratio than fiber-optic bottom reading.

Furthermore, at lower concentrations of fluorescein, the difference increased to more than 12-fold higher. This will allow for more than 10 times less reagent to be used on the PHERAstar FS in noncell-based bottom reading assays, significantly saving on reagent costs.

To further demonstrate the overall improvement of direct-optic bottom reading on the PHERAstar FS, GFP-labeled BAE cells (37,500 cells/mL) were measured in a 384-well microplate. As shown in Figure 2, direct-optic bottom reading gives a threefold higher signal-to-blank ratio than fiber-optic bottom reading. This will allow for detection of fluorescent proteins at concentrations not possible on any other microplate reader.

Figure 2. Direct-optic bottom reading (green column) vs. fiber-optic bottom reading (red column). GFP labeled BAE cells in a 384-well microplate give a three times higher signal-to-blank ratio when using direct-optic bottom reading on the PHERAstar FS as compared to a fiber-optic bottom reading instrument.

With cellular fluorescent protein assays depending sometimes on only a 25% change in signal, a 300% higher signal to blank ratio will make experiments possible on a microplate reader that were not possible before. This also means that for secondary cell-based screening assays, fewer cells can be used and thus further miniaturization is possible on the PHERAstar FS than on other fiber-optic bottom-reading instruments.

In addition to an improved signal-to-blank ratio in fluorescent protein assays due to direct-optic bottom reading, the PHERAstar FS has a well-scanning mode that can create a high-resolution image of fluorescently labeled cells in each well. Figure 3 shows GFP-tagged HEK293 cells in a 384-well microplate, as visualized using a 20×20 well scanning matrix for each well. This feature enables a digital image to be created from an analog signal, which is complimentary to a microscope or CCD camera that creates analog numbers from a digital picture.

Figure 3. Well scanning (20×20 matrix) and direct-optic bottom reading were used to create a digital image of GFP labeled HEK293 cells in a 384-well microplate. Qualitative as well as quantitative data is easily collected on the PHERAstar FS.

E.J. Dell, Ph.D. ([email protected]), is the int’l marketing director for BMG LABTECH.

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