Improving flow cytometry with microspheres
cytometry has been developed. The technique, which is tipped to
become a new industry standard, promises to dramatically increase
the accuracy and speed of the technique, used in the pharmaceutical
industry for applications such as screening receptor binding
interactions and cell responses.
The new method is based on the use of microspheres coated with fluorescent dye. The tiny spheres, each about one-tenth the width of human hair, are used to calibrate flow cytometry instruments that measure fluorescence intensity. This has been the culmination of five years research by the NIST.
Fluorescence-based detection depends on the absorption of light by the cell and the subsequent re-emission of this light at a different frequency. Flow cytometers make use of this by employing filters to block the original light source from reaching the detector, while the fluorescence emission is allowed through for detection, something that allows only a very low background of stray light to reach the detector.
In flow cytometry experiments, fluorescence is achieved by deliberate labelling of a cellular component using a fluorescent marker, usually a type of dye. These dyes fluoresce only when light of the appropriate wavelength (the laser being designed to emit light at that wavelength) hits them, causing the emission of secondary light at a different wavelength. Detection of the second wavelength is used as a measure of the presence of the dye on the cell and thus the component it is labelling.
Biomolecules of interest such as antibodies, cancer cells and specific genes are often marked by a fluorescent dye. The brightness of the signal from a sample can indicate whether or not the biomolecular count has increased or decreased in number. This can be used to track events within cells in response to a stimulus, or even indicate the severity of a disease.
Amazingly, the accepted method of determining the intensity of a signal has been done visually or with measuring standards developed by individual manufacturers. Previous research has shown that measurements of the same samples can vary by more than 100 per cent, depending on the instrument used and a variety of experimental conditions.
The NIST plans to combine the new reference standard - called RM 8640 - with previously developed standards and measurement procedures in what could eventually become the authoritative national measurement scale for fluorescence flow cytometry.
Each kit contains six vials of microspheres that emit fluorescent light at six different intensity levels from zero to very bright. A flow cytometer is used to analyse the contents of all the vials and an unknown sample. By calibrating the cytometer to match the intensity values provided by NIST for the reference vials, the intensity of the unknown sample can be measured more accurately.
