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Authored by: Paul Taylor, Boehringer Ingleheim Inc.
Many readers today offer multimode capability so that a wide range of detection formats are available. Light sources are typically tungsten or xenon flash lamps and these provide white light that passes through fixed bandpass filters for generating the wavelength of interest. If higher sensitivities are required, lasers with fixed spectral lines are frequently used. Some readers offer tunable monochromators which allow excitation and emission scans to be performed and also allow fine-tuning of the desired wavelength. The instruments are divided across two main categories, PMT (photomultiplier tube) readers and plate imagers.
PMT readers usually have a single endpoint of detection per well with the plate indexing under a fixed location of the PMT. Typically, they are more sensitive than imagers, have a higher dynamic range, provide less crosstalk, have a smaller footprint and are also less expensive. Their disadvantage relative to imagers is that they are typically slower, particularly for higher plate densities using multiple read detection modes such as FP and time-resolved fluorescence.
Plate imagers comprise two main types: macro imagers for imaging an entire plate at a time (as used in HTS) and micro imagers most commonly used for monitoring cells (singularly or as multiples within a single well) in high content screening applications (HCS). Macro imagers commonly have charge coupled devices (CCD’s) that are cooled to -100°C to minimize backgrounds and enhance sensitivity. Being that a large telecentric lens is used for optical detection, parallax effects usually need to be corrected for at the edges of the plate and this is commonly called flatfield correction. This phenomenon has been avoided by some devices that use either a multi-lens array or paired optical fibers. The two types of HCS detectors use either CCD imagers (primary screening on entire well) or laser scanners (whole cell or bead analysis). In some cases, higher throughputs are initially run using a CCD imager followed by a more detailed investigation using laser scanning.
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