TOOLS OF THE TRADE - Analytical Instrumentation | The Power of FCS

Samara Kuehne

ISS makes the Alba-FCS dual-channel spectrometer, which combines a confocal scanning microscope with FCS. Newer fluorescence correlation spectroscopy instruments focus on portability, affordability, and ease of use Fluorescence correlation spectro-scopy (FCS), originally developed in the early 1970s for use in physics and physical chemistry, has recently been applied to the fields of drug discovery and development, with powerful results. Early FCS equipment used in physics labs was large, with bulky lasers that were not necessarily user friendly. Since 2005, instrumentation with specific application to the drug discovery field has entered the marketplace, and products released since have increasingly focused on reducing the size of a unit’s footprint to make it easier to use in the lab. FCS Technology FCS is an extremely effective tool for ultrasensitive measurements. The technique measures and correlates fluctuations in fluorescence intensity within a very small volume element, allowing for single-molecule inspection. A sharply focused laser illuminates this small element, and single molecules diffusing through the illuminated confocal volume produce bursts of fluorescent light. Each burst is recorded by a single-photon detector and analyzed via autocorrelation. This autocorrelation provides data on concentration, the diffusion time of the individual molecules, and each molecule’s brightness, which subsequently allows differentiation of slower- and faster-diffusing particles. Ultimately, the binding and catalytic activity is calculated from the diffusion times and the ratio of faster and slower molecules.1,2 Pharmaceutical Uses Sensor Technologies’ QuantumXpert FCS Spectrometer includes optical and electronic components. In drug delivery and discovery, FCS is used to measure the quantity and distribution of a drug in the nanoparticles used to deliver it to its target and, ultimately, to determine how fast the binding is and how low a concentration of the drug is necessary to achieve an effective binding. Study results published in 2010 concluded that FCS, used in combination with a confocal microscope, could in fact determine diffusion constants and concentrations of fluorescent molecules.3 FCS can be used to study molecular and cellular interactions in homogeneous assays. In drug discovery and production, FCS can be used to develop assay targets for protein-protein interaction, protein-nucleic acid interaction, kinase activation by complexing, and host-cell contamination. The technology can be used to detect both direct binding and competitive inhibition of binding, a newer method of measurement that makes FCS useful in drug research and drug delivery analysis. Instrumentation There are several devices currently available on the FCS market. Carl Zeiss (Jena, Germany) offers the ConfoCor 3, which is designed to be paired with one of the company’s laser scanning microscopes. The unit’s software controls the detection module and can analyze single or multiple measurements and allow auto- and cross-correlation to be calculated at the same time as the current measurement. Software upgrades to the unit include options for photon-counting histograms and for defining start values and boundaries. The ConfoCor 3 by Carl Zeiss is designed to be paired with one of the company’s laser scanning microscopes. ISS (Champaign, Ill.) manufactures the Alba FCS dual-channel spectrometer, which combines a confocal scanning microscope with FCS. Its light sources are either single-photon or multi-photon lasers, and the device can be interfaced with Leica, Nikon, Olympus, or Zeiss epi-fluorescence microscopes. The Alba can acquire data either in time mode, which counts photons acquired in time intervals, or in photon mode, which measures the time delay between photons and builds a histogram. Sensor Technologies’ (Shrewsbury, Mass.) QuantumXpert FCS Spectrometer includes both optical and electronic components. The unit’s confocal microscope optics are built in to the system, so it doesn’t require them as external add-ons. The result is a much smaller and more portable system that does not require daily realignment exercises, one that can cost less than other traditional FCS systems. The device also features a data analysis software system designed to simplify the task of analyzing FCS measurement data, offering single and batch correction algorithms, along with curve-fitting methods for both fluorescence correlation data and photon-counting histograms. It is available with either a manual or an automated sample changer. Corrvus (Spokane, Wash.) is also developing a portable FCS system with a lower price tag than more traditional models. This unit, available in the next 12 months and designed for ease of use in the lab, will not need a dark room to develop the imaging. The Future of FCS Research over the past 10 years has shown FCS to be a powerful tool in drug discovery and delivery. It allows for single-molecule observation and can specifically measure how one molecule interacts with another. Parijat Sengupta, PhD, research assistant professor at Washington State University in Spokane and consultant to Corrvus, thinks “FCS might play a very important role in personalized medicine,” a model that emphasizes therapeutic care and treatment plans tailored to specific individuals. With the technology’s specific application to pharmaceuticals still in its relative infancy, its versatility and allowance for extremely sensitive measurements could pave the way for some exciting developments in drug formulation. References Seethala R. Homogeneous assays for high-throughput and ultrahigh-throughput screening. In: Seethala R, Fernandes PB, eds. Handbook of Drug Screening. New York: CRC Press; 2001:94-95. Schwille P, Haustein E. Fluorescence correlation spectroscopy: an introduction to its concepts and applications. Biophysics Textbook Online. 2003. Available at: www.dpi.physik.uni-goettingen.de/Praktika/ Biophysik/Versuche/2006w/Fluoreszenzkorrelationsspektroskopie-Literatur-Schwille_ Haustein.pdf. Accessed Oct. 16, 2011. Jung CC, Polier S, Schoeffel M, Drechsler M, Jerome V, Freitag R. Fluorescence correlation spectroscopy as a quantitative tool applied to drug delivery model systems. Nature Precedings. 2010. Available at: http://hdl.handle.net/10101/npre.2010.4140.1. Accessed Oct. 16, 2011