Absorbance refers to the ability of a substance to absorb light of a specific wavelength. In a 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. Absorbance is calculated as shown in the equation:
A λ = log10 (I0/I)
Where I is the intensity of light at a specified wavelength λ that has passed through a sample (transmitted light intensity) and I0 is the intensity of the light before it enters the sample.
In a typical colorimetric assay, an enzyme/substrate reaction
causes a color change in the samples in the microplate.
Light at a specific wavelength is passed through the sample to a detector.
The amount of light absorbed by the sample is referred to as the Optical Density (OD) or absorbance.
Many conventional colorimetric analyses have been, or can be miniaturized for measurement in a microplate reader. BioTek’s microplate readers and Gen5 software can facilitate assay miniaturization to microplates by providing optional cuvette ports and automated pathlength correction. Absorbance detection in microplate readers is used for assays such as ELISA assays, protein & nucleic acid quantification and purity assays, endotoxin analysis and many others.
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).
Samples are chemically bound with fluorescent tag
Light at a specific wavelength excites the sample
Excited samples emit light at a different wavelength
The advantages of fluorescence detection over absorbance detection are increased sensitivity and a broader application range, given the wide selection of fluorescent labels available today. For example, a technique known as calcium imaging measures fluorescence intensity of calcium-sensitive dyes to assess intracellular calcium levels.
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.
Sample in the well before the chemical or biochemical reaction.
Reaction causes the sample to emit light that can be measured by a PMT detector
The ability to select multiple wavelengthsallows 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. Luminescence reactions can be slow (glow) or fast (flash). Flash reactions typically require a rapid dispense and measure sequence on a well-by-well basis.
Common applications include luciferase -based gene expression assays, as well as cell viability, cytotoxicity, and biorhythm assays based on the luminescent detection of ATP.
Time Resolved Fluorescence
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, when most standard fluorescent dyes (e.g. fluorescein) emit within a few nanoseconds of being excited. As a result, it is possible to excite lanthanides using a pulsed light source (Xenon flash lamp or pulsed laser for example), 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 have to be compatible with the use of these very specific lanthanide dyes.
In a typical TRF assay, samples emit at one wavelength (here in red) before they react
When a reaction occurs, the sample’s emission shifts (here to orange)
The main use of TRF is found in drug screening applications, under a form called TR-FRET (time-resolved fluorescence energy transfer). TR-FRET assays are very robust (limited sensitivity to several types of assay interference) and are easily miniaturized. Robustness, the ability to automate and miniaturize are features that are highly attractive in a screening laboratory.
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.
Molecules are excited with light. Unbound small
molecules emit depolarized light.
Small molecules bound to larger ones emit polarized light.
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.
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