Detection Methods

Absorbance refers to the ability of a substance to absorb light of a specific wavelength.  In an microplate reader designed to measure absorbance, a light source illuminates the sample using a specific wavelength, selected by an optical filter or a monochromator, and a light detector located on the other side of the well measures how much of the initial light is transmitted through the sample: the amount of transmitted light will typically be related to the concentration of the molecule of interest, and the result is referred to as the Optical Density (OD) or absorbance of the sample. Click for more.

Fluorescence intensity detection has a much broader range of applications than absorbance detection. For fluorescence intensity measurements, an optical system (the excitation system) illuminates the sample using a specific wavelength (selected by an optical filter, or a monochromator), thereby exciting the sample. The excitation causes the sample to emit light (i.e. fluoresce) at a different wavelength. The emitted light is collected by a second optical system (emission system) and the signal is measured by a light detector such as a photomultiplier tube (PMT). Click for more.

Luminescence is the result of a chemical or biochemical reaction. Luminescence detection is simpler optically than fluorescence detection because luminescence does not require a light source for excitation or optics for selecting discrete excitation wavelengths. A typical luminescence optical system consists of a light-tight reading chamber and a PMT detector. Some plate readers offer filter wheel or tunable wavelength monochromator optical systems for selecting specific luminescent wavelengths. The ability to select multiple wavelengths allows for detection of assays that contain multiple luminescent reporters, the development of new luminescence assays, as well as a means to optimize signal to noise. Common applications include luciferase -based gene expression assays, as well as cell viability, cytotoxicity, and biorhythm assays based on the luminescent detection of ATP. Click for more.

Fluorescence polarization measurements are made using an optical system that includes polarizing filters in the light path. Samples in the microplate are excited using polarized light, and depending on the mobility of the fluorescent molecules found in the wells, the light emitted will either be polarized or not. For example, large molecules (e.g. proteins) in solution, rotate relatively slowly because of their size and will emit polarized light when excited with polarized light. The fast rotation of smaller molecules will result in a depolarization of the signal. The emission system uses polarizing filters to analyze the polarity of the emitted light. A low level of polarization indicates that small fluorescent molecules move freely in the sample. A high level of polarization indicates that fluorescent molecule is attached to a larger molecular complex.  Examples of FP-based assays include molecular binding assays, since they allow the detection of a small fluorescent molecule binding (or not) to a larger, non-fluorescent molecule: binding results in a slower rotation speed of the fluorescent molecule, and in an increase in the polarization of the signal. FP is widely used in research labs to study molecular binding or dissociation events and in screening labs to screen for drug candidates. There are many FP assay kits available for a wide variety of applications. Click for more.

Time-resolved fluorescence (TRF) measurement is very similar to fluorescence intensity (FI) measurement. The only difference is the timing of the excitation/measurement process. When measuring FI, the excitation and emission processes are simultaneous: the light emitted by the sample is measured while excitation is taking place. Even though emission systems are very efficient at removing excitation light before it reaches the detector, the amount of excitation light compared to emission light is such that FI measurements always exhibit fairly elevated background signals. TRF offers a solution to this issue; it relies on the use of very specific fluorescent molecules, called lanthanides, that have the unusual property of emitting over long periods of time (measured in milliseconds) after excitation, compared to most standard fluorescent dyes (e.g. fluorescein) that emit within a few nanoseconds of being excited. It is possible to excite lanthanides using a pulsed light source such as a 337 nm laser or a xenon flash lamp. and measure after the excitation pulse. This results in lower measurement backgrounds than in standard FI assays. The drawbacks are that the instrumentation and reagents are typically more expensive, and that the applications must be compatible with the use of these very specific lanthanide dyes. Click for more

AlphaScreen® is a bead-based assay technology used to study biomolecular interactions in a microplate format. The acronym “Alpha” stands for amplified luminescent proximity homogeneous assay. This platform provides a non-radioactive, homogeneous assay which has low background and high signal:background ratios.  The assay can be easily automated and miniaturized to a variety of well formats.  The assay incorporates two sets of beads, donor and acceptor beads, to generate a signal.  The donor bead is illuminated at 680 nm.  If the acceptor bead is in close proximity to the donor bead, energy is transferred to the acceptor bead, and light is produced in the 520-620 nm range.  If an acceptor bead is not in close proximity to the donor bead, no energy is transferred, and the donor bead falls back to its ground state. Click for more.

Intracellular calcium flux is a long accepted, tried and true measure of cellular activity. Calcium flux can be used as a measurement for a host of cellular processes including neurotransmitter release, GPCR activity, voltage or ligand gated ion channels, and cardiomyocyte beat patterns, among many others. The FLIPR Penta System is the drug discovery tool for evaluation of calcium flux in high and ultra-high throughput due to its ease of use, sensitivity and user configurability. Click for more.

Membrane potential. There are dozens of identified disorders caused by impaired ion channel function. Ion channels are important drug discovery targets because they are highly druggable and specific. The FLIPR® Membrane Potential Assay Kits provide a fast, simple and reliable fluorescence-based assay for detecting changes in voltage across the cell membrane mediated by ion channel to evaluate target or off-target effects based on the application. Click for more.

Microscopy: Fluorescence and Brightfield. Click for more.