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Exploiting the dual nature of light

BioPhotonics
Oct 2008
Hank Hogan

In August, at the Drug Discovery & Development of Innovative Therapeutics conference in Boston, Kelly J. Cassutt, an applications scientist at Hamamatsu Corp. in Bridgewater, N.J., showcased light as an agonist. But in acting as an agent that binds to – and therefore alters the activity of – a receptor, light reveals its dual nature as a particle and a wave. “To activate the receptor, light behaves as a photon or particle. To activate the receptor, light must be at the correct wavelength, or acting as a wave,” he explained.

Although light’s duality may be an old idea, exploiting it solves a current problem. G protein-coupled receptors are a family of transmembrane receptors that trigger the response to molecules outside the cell. These receptors are involved in many diseases and so are the target of about half of all modern drugs. Hence, anything that acts as an agonist for G protein-coupled receptors could be important when screening for potential new drugs.

TSHamamatsu_Figure2.jpg

Continuous stimulation of cells loaded with Fluor-4 using light at 480 nm is shown. Because cells react to light, an assay using the calcium dye Fluor-4 does not allow the measurement of a baseline. This inability makes later readings difficult.

There is a problem in some model cell systems being investigated via high-throughput screening. The G protein-coupled receptors absorb light at 480 nm and undergo isomerization. Thus, a dye that absorbs at the same wavelength can make it difficult to truly gauge the receptor’s reaction. One such group of probes is the often used membrane-permeable Fluo-4-based dyes.

Cells loaded with these dyes immediately react to light, and this instant stimulation makes it impossible to collect a background signal. Cassutt noted that this immediate response to light also affects the ability to set exposure time, power or camera sensitivity before running an assay because the illumination needed to extract such information also would stimulate the cells.

TSHamamatsu_Figure1.jpg
When the act of observing changes things, using ultraviolet light can help. This graph shows what happens to cells when exposed to visible light. Using a UV ratiometric dye, researchers can read calcium mobilization independent of stimulating the cells, which takes visible light at 480 nm. Less than a 1-s exposure time to visible light leads to maximal response. Images courtesy of Kelly J. Cassutt, Hamamatsu Corp.

There is a second problem that arises from the light sensitivity of Fluo-4. It is common practice in high-throughput screening to add a library compound in an antagonist screen. As the name implies, an antagonist will suppress a response, not enhance it as an agonist would. The addition of the antagonist screening compound occurs while readings are taken, but the use of Fluo-4 precludes this. The stimulated cells recover too slowly, taking too long to be used in high-throughput screening.

Pulses to the rescue

Confronted with this inability to screen a cell line because of light activation, Cassutt and engineers at Hamamatsu came up with a solution and implemented it in the software running the company’s FDSS6000 imaging-based plate reader. The system has a CCD camera-based fluorescence sensor and a photon-counting luminescence sensor.

Illumination is provided by a xenon lamp with various excitation filters. Importantly, this source, the engineering team discovered, can be pulsed via software control. Thus, the developers added this capability to the system within the past six months or so. The ability to adjust the illumination time allows researchers to find the minimal exposure time needed for the maximal response.

In a demonstration, Cassutt showed how pulsing could be useful. He loaded cells with a new Fura-2 membrane-permeable-based dye and quencher from BD Biosciences of Franklin Lakes, N.J. Importantly, this dye kit and quencher are excited at 340 and 380 nm, spectrally far removed from the problem area. That is not the case with other dye kits with quenchers, which are single-wavelength-based and excited at 480 nm.

Cassutt measured the response at 340- and then at 380-nm excitation, using the ratio of the two intensity readings, as is standard for a Fura-2 assay. He then found the exposure time at 480 nm to get complete agonism, tracking the net change in the 340:380 ratio to obtain this information. He started with a 30-s, 480-nm illumination time. As his research progressed, it became clear that this was more than enough. In the end, he found that the time needed to achieve complete signal activity was only a few seconds.

A comparison showed that a Fura-2-loaded cell stimulated using 480-nm light for 3 s responded about the same as one loaded with Fluo-4 and bombarded with continuous light. Comparing the results of various dye groups revealed possible dye-independent antagonists, information that could be useful in an actual assay.

As for the future, one outcome of such work might be a tool for cell models that use light as a stimulus. Such models are a distinct possibility, given that many groups are researching light as an agonist because of the advantages it offers.

Chemical ligands that serve the same function suffer from dilution effects, solubility issues and diffusion delays, Cassutt noted. “By contrast, light is uniform, instantaneous, of controllable duration and wavelength-definable.”

Contact Kelly J. Cassutt, PhD, Hamamatsu Corp.; e-mail: kcassutt@yahoo.com.


GLOSSARY
light
Electromagnetic radiation detectable by the eye, ranging in wavelength from about 400 to 750 nm. In photonic applications light can be considered to cover the nonvisible portion of the spectrum which includes the ultraviolet and the infrared.
photon
A quantum of electromagnetic energy of a single mode; i.e., a single wavelength, direction and polarization. As a unit of energy, each photon equals hn, h being Planck's constant and n, the frequency of the propagating electromagnetic wave. The momentum of the photon in the direction of propagation is hn/c, c being the speed of light.
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