What is a Luminometer and what do they do?
A luminometer is a device that uses a photomultiplier tube to measure faintly visible light emissions emanating from a sample. Luminometers are highly sensitive instruments that can measure extremely tiny amounts of light, often just a few photons. Typically, they work with tiny samples—sometimes as little as a few microliters—such as protein solutions or suspensions of cells in microcentrifuge tubes, microplate wells, or protein solutions.
Because samples evaluated by a luminometer emit light on their own (due to a chemical reaction), rather than requiring excitation light, they often do not require a lamp or excitation optics, which is how they vary from fluorometers.
A luminometer must be distinguished from a photometer and a light metre despite the terms seeming similar:
- A photometer is a fairly general term that refers to any instrument that measures light, such as luminometers, light metres, spectrophotometers, and other similar instruments.
- Although the word "light metre" is general, it typically refers to a small camera accessory used to measure ambient light.
PRINCIPLE OF A LUMINOMETER
Luminometers use a photomultiplier tube to be able to measure the smallest amounts of light (PMT). Weak incoming signals can be detected thanks to a photomultiplier tube's significant amplification. A PMT operates on the following principle:
1. The photoelectric effect occurs when photons strike a photocathode at the PMT's entrance window and create electrons as a result.
2. Following that, a high-voltage field accelerates those electrons, and secondary emission causes them to multiply within a series of dynodes.
3. The anode, which is coupled to an output processing circuit, is where the amplified electrons finally arrive.
4. The circuit converts the input signal to an output signal, which can either be an analogue current or a pulse (if the PMT operates in photon counting mode) (if the PMT works in analogue mode, also known as a current mode). While analogue mode performs better with high light intensities and is frequently used for fluorescence measurements, photon counting mode offers the best sensitivity and is the optimum mode for luminescence measurement.
The PMT must be placed very close to the sample and in an ideal position to minimise photon loss from the sample while also keeping photons from adjacent wells out of the way (in the case of microplate luminometers) in order to capture the most photons feasible (crosstalk).
Besides the PMT, the following components of a luminometer are crucial:
The dark chamber: Complete protection from outside light is required for the area where samples are measured. The dark chamber must also be appropriate for the sample format being measured, such as tubes, plates, or other vessels.
Injectors: are not always required, however since a chemical reaction produces luminescence, reagents must be supplied to the sample. Reagents can be manually added if the reaction's kinetics are slow, but when the reaction's kinetics are fast, reagent injectors can be helpful (to precisely control the timing between reagent dispense and measurement) or even necessary (if the reaction only lasts a few seconds, as in the case of flash assays).
Since light of all wavelengths is gathered and measured collectively, the majority of luminometers do not require filters or a monochromator. Filters are required for some tests, such BRET, to distinguish between the light produced by various proteins in the sample.
PRINCIPLE OF ATP TESTING FOR HYGIENE MONITORING
The total ATP content of the sample is calculated for hygienic testing. Eukaryotic and microbial ATP are included in this. Due to the prevalence of ATP on various surfaces, caution must be taken to prevent contamination of the sample or substance.
The user may use an ATP-free buffer, water, or extractant to moisten an ATP-free swab that has been provided pre-moistened. ATP is successfully released from the surface when extractant is used, which can aid in sampling. Testing the swab is typically done immediately using portable luminometers. On the contrary, some products' swabs remain stable for several hours, allowing the user to return to the instrument at a "workstation" if they so want.
Utilizing selective extraction, the microbial ATP level is determined. non-microbial ATP is first removed using a non-ionic detergent (Triton X-100), and it is then destroyed for 5 minutes by being exposed to a high concentration of potato ATPase. The microbial ATP is then extracted using cationic detergents, trichloroacetic acid (5%) or an organic solvent (ethanol, acetone, or chloroform), which needs to be diluted afterwards to prevent luciferase suppression. The final stage involves adding luciferase and luciferin and measuring luminescence with a luminometer. To allow for luciferase inhibition, the timing of mixing and reading must be carefully considered (Simpson & Hammond, 1991).|Since eukaryotic cells have three orders of magnitude more ATP than bacterial cells, it can be challenging to accurately measure the ATP levels of the two types of cells individually.
To make the technique simpler and quicker, several manufacturers provide all required reagents and supplies as ready-to-use kits.
So do we! With our Hygiena Luminometers! Perfect for any application it's widely used in a healthcare settings, hospitality, food manufacturing and many more!
Hygiena ATP Luminometer & Accessories (anaeron.com.au)