Fluorescence Lifetime-Based Whole Cell Bioassay

Cellular functions are regulated by hormones, neurotransmitters, and a variety of regulatory and growth-promoting factors. The agonists produce a host of physiological responses in their target tissues as a result of their interactions with specific cell surface receptors. One major class of cell surface receptors mediates their effects via an increase in cytosolic Ca2+. These are referred to as Ca2+-mobilizing receptors and they affect intracellular calcium levels, [Ca2+]i. In the unstimulated cell [Ca2+]i is approximately 0.1 *M, which is 105-fold lower than the mM extracellular [Ca2+], and following stimulus it rises to 1 to 10 *M. The source of Ca2+ could be either extracellular or intracellular.

Examples of cell functions that are controlled by Ca2+-mobilizing receptors include smooth muscle contraction by acetylcholine and norepinephrine; pancreatic secretion by acetylcholine; platelet aggregation by thrombin; glycogen breakdown in liver by vasopressin; prolactin secretion in pituitary cells by thyrotropin-releasing hormone; fluid secretion in salivary glands by serotonin; cell proliferation in fibroblasts by growth factors; neutrophil activation by chemotactic factors, and phototransduction by light. Exocytosis, neurotransmitter release, fertilization, intracellular transport and membrane topography all seem to be regulated by [Ca2+]i.

Because of the ubiquitous role of Ca2+ in signal transduction, the direct measurement of intracellular calcium within living cells offers a powerful approach to cell-based assays for high-throughput screening (HTS) and for biosensing applications. The most common approach for measuring intracellular calcium is based on the use of fluorescent chelator dyes that undergo a change in spectroscopic properties upon binding Ca2+. Fluorescent Ca2+indicators can be of two types: ratiometric and single-wavelength. In the latter, the fluorescence quantum yield increases upon calcium binding without a change in spectral characteristics. However, the measured fluorescence intensity is dependent on the amount of dye in the cell, which can vary as a result of unequal cytosol thickness, unequal distribution of dye within or between cells, and leakage and/or photobleaching of dye during an experiment. Therefore, quantitative measurement of [Ca2+]i using intensity-based detection and single-wavelength dyes is very difficult. Single-wavelength calcium indicator dyes are thus primarily used for qualitative imaging applications..

Quantitative determinations of [Ca2+]i typically employ "dual-wavelength" (or ratiometric) dyes. These fluorophores characteristically exhibit a shift in either their excitation or emission spectra when they bind to Ca2+ and are thus referred to as "dual-excitation" or "dual-emission" dyes. The key advantage of ratiometric dyes is that the measured ratio is independent of the amount of dye present but proportional to ion concentration. Under suitable conditions, fluorescence variations due to factors such as uneven cell thickness, unequal dye distribution, dye leakage or photobleaching are normalized using the ratio approach.

Despite this major advantage of ratiometric dyes, they suffer from two significant drawbacks, both related to their absorption in the UV range of the spectrum. The first problem is that with UV excitation many cellular components (as well as glass, plastics, etc) fluoresce and thus excitation in this range creates high non-specific fluorescence background. The second problem is that UV excitation is not convenient to laser- or LED-based instrumentation. As a result of these problems, a major effort has been devoted in recent years to the development of calcium indicator dyes with excitation at longer wavelengths. This has resulted in the commercial availability of several new dyes such as Calcium Green (Ex/Em at 488/535 nm), Calcium Orange (545/575 nm), and Calcium Crimson (570/610 nm). Unfortunately, none of the available long-wavelength dyes exhibit ratiometric behavior and therefore are not suited to quantitative analysis based on traditional intensity-based detection methods.

Fluorescence Lifetime Sensing solves this problem. The change in quantum yield that single-wavelength dyes exhibit upon binding Ca2+ results in a proportional change in fluorescent lifetime. However, the measured lifetime is independent of dye concentration, and therefore immune to photobleaching, cytosol thickness, dye loading, leakage, and other problems which affect intensity measurements. That is, with lifetime transduction of calcium binding, we can achieve the same benefits of a ratiometric approach with single-wavelength dyes. Furthermore, unlike an intensity ratio, fluorescence lifetime is an absolute quantity, and thus its measured value is independent of the instrument platform. This facilitates calibration issues and comparison of results from different instruments.

Ciencia is currently applying its patented lifetime sensing technology to the development of whole cell assays using intracellular calcium signal transduction as an analytical event marker.