RLUs are the units used for most luminescence measurements. RLUs do not have any physical meaning and are often not comparable between different instruments. This is due to the way light is measured in a luminometer. Luminometers use a photomultiplier tube (PMT) photons striking a photocathode at the entrance window of a PMT produce electrons which are then accelerated by a high-voltage field and multiplied within a chain of dynodes by the process of secondary emission. Finally, they reach the anode connected to an output processing circuit and this is where it translated to an output signal. It can be a pulse if the PMT works in photon counting mode or an analog current if the PMT works in analog mode

The output is affected by the PMT mode, the voltage applied, the efficiency of the PMT and other parameters. This makes it difficult to relate the output to any physical magnitude depending on the quantity of light emitted by the sample. Consequently, some instruments can give values between 1 and 10 million and others between 0.0001 and 100. Hence, light units are kept relative to other measurements taken in the same instrument.

While the use of relative units can be a problem in some areas it is absolutely fine in most life science applications, since they are mostly related to a control and they treat all results in relation to that value (e.g. the values in condition B can be 50% of the control value, while the values in condition C are 12 times higher than those of the control).

It is possible to measure a sample of a known intensity in two different instruments and to use the value as a reference to compare measurements in both instruments. This is easy and quite reliable if the instruments are similar (for example, same manufacturer and same type of PMT), but caution has to be exercised comparing instruments which are very different (for example, with PMT working in different modes and with different dynamic ranges.). It is not recommended to use biological or chemical samples as reference as there is an degree of variability and sometimes it is not possible to get the same measurement from the same sample (for example, in flash reactions the sample could be measured only once and in glow reactions there could be a noticeable decay in luminescence between the first and the second measurements).

Instead electronic light sources which are periodically calibrated and tested are recommended. Examples of such sources are our Luminescence Test Plates and Luminescence Test Tubes as they are often used to verify the performance of microplate luminometers and tube luminometers.

Our tube luminometers and microplate readers provide RLU values that are very similar and comparable with each other. We use PMTs that work in photon counting mode and implement an internal RLU factor to calibrate our instruments to a stable light source of known intensity.

RLU or RLU/s?

Many instruments express the measured values as RLU, while others take the measurement time into account and use RLU/s instead. At Berthold Technologies we use RLU/s by default, as this makes values comparable independently of the measurement time. In addition, it is really easy to convert RLU/s to RLU: just multiply by the measurement time.

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