While the names may be similar, a luminometer has to be distinguished from a photometer and a light meter:
- Photometer is a very broad term that includes any device that measures light, including luminometers, light meters, spectrophotometers and others.
- Light meter is also a broad term but it normally refers to a small device used in photography to measure ambient light.
To be able to measure the smallest quantities of light, luminometers use a photomultiplier tube (PMT). A photomultiplier tube greatly amplifies incoming signals, making weak signals detectable. The working principle of a PMT is the following one:
- Photons striking a photocathode at the entrance window of the PMT produce electrons, as a consequence of the photoelectric effect.
- Those electrons are then accelerated by a high-voltage field and multiplied in number within a chain of dynodes, by the process of secondary emission.
- The amplified electrons finally reach the anode, which is connected to an output processing circuit.
- The circuit translates the incoming signal to an output signal, that can be a pulse (if the PMT works in photon counting mode) or an analog current (if the PMT works in analog mode, also known as current mode). Photon counting mode gives the best sensitivity and is the best mode for luminescence measurement, while analog mode works better with high intensities of light and is popular for fluorescence measurements.
To catch the highest possible quantity of photons, the PMT has to be placed very close to the sample and in an optimized position that reduces the loss of photons coming from the sample, but that at the same time (in the case of) keeps away photons coming from adjacent wells ( ).
In addition to the PMT, other important parts of a luminometer are:
- The dark chamber: the area where samples are measured has to be absolutely protected from external light. Another requirement of the dark chamber is that it has to be suitable for the sample format to be measured: tubes, plates, or other vessels.
- Injectors: they are not always a needed but, as luminescence is provided by a chemical reaction, some reagents have to be added to the sample. If kinetics of the reaction is slow, reagents can be added manually, but in the case of fast kinetics reagent injectors can be beneficial (to accurately control the timing between reagent dispense and measurement) or even required (if the reaction lasts only one or a few seconds, as it’s the case of flash assays).
Most luminometers do not need filters or a monochromator, as light from all wavelengths is pooled and measured together. However, some assays such asneed filters to separate luminescence emitted by different proteins in the sample.
A useful way to classify luminometers is by the sample format they measure, as this has an impact on throughput, flexibility, reliability and price.
can measure a sample contained in a , microcentrifuge tube or similar. They are simple instruments that measure samples one by one, which the operator has to insert manually. This limits throughput but provides high sensitivity and reliability, as there are no compromises in the positioning of the PMT.
Some tube luminometers can be equipped with injectors, which are required to dispense reagents in flash assays (luminescent assays with fast kinetics).
There are several types of tube luminometers:
Benchtop Tube Luminometers
They are the most sensitive and reliable type, but they are relatively bulky and heavy and need to be plugged into a wall socket. They are the instrument of choice for laboratories with low numbers of samples. Theand the are high-performance benchtop tube luminometers.
They are light enough to be portable and can be operated on battery power to be used on the field. They are less sensitive than benchtop luminometers, but still provide enough performance for demanding applications. Theis an excellent portable luminometer.
They can be considered a subclass of portable luminometers. They are even smaller and lighter, but they have very low performance and are normally only used for qualitative assays (e.g. to discriminate if a surface is contaminated or not). As its main application is hygiene monitoring of surfaces, they often use special swabs for measurement, which are used to swipe the surface and inserted directly in the luminometer for measurement.
measure samples contained in the wells of , typically 96- or 384-well microplates. This greatly increases throughput, but makes the instruments more complex, as a system to move the measurement position from well to well has to be implemented, and light coming from adjacent wells has to be blocked. Hence, they are more expensive than tube luminometers.
While sensitivity of plate luminometers is normally lower than that of benchtop tube luminometers, theis the most sensitive plate luminometer in the market and has a comparable sensitivity.
are microplate readers that can measure luminescence but, in addition, can use other detection technologies, such as fluorescence and absorbance. This increase in complexity makes some compromises necessary in order to balance performance of all detection methods, but provides a great flexibility, including the possibility to use filters or, in some cases, monochromators. They are usually more expensive than microplate luminometers, but modular multimode readers such as the can be configured to fit any budget.
In Vivo Imaging Systems
While its working principle is very different from that of a luminometer,are also a popular instrument to measure luminescence. They use a camera instead of a photomultiplier tube; this results in a lower sensitivity, but enables the spatial localization of light emission and the measurement of luminescence in living animals and plants.