RLU 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: briefly, 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 in number within a chain of dynodes by the process of secondary emission, and finally reach the anode connected to an output processing circuit. Then, this is translated 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).

The output is affected by the PMT mode, the voltage applied, the efficiency of the PMT and other parameters, that make it really difficult to relate the output to any physical magnitude depending on the quantity of light emitted by the sample.  As a consequence, some instruments can give values between 0 and 10 million, others between 0.0001 and 100, others something completely different. 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 handle all results related 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, with different dynamic ranges, and so on). It is not recommended to use biological or chemical samples as reference, as there is an important 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 (for example, LEDs) which are periodically calibrated are recommended. Examples of such sources are our Luminescence Test Plates and Luminescence Test Tubes, and they are often used to verify the performance of microplate luminometers and tube luminometers, respectively.

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 stable light sources 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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