For a device to monitor gene expression using bioluminescence, highly sensitive luminescence detector is used. A typical units are a PhotoMultiplier Tube (PMT) and a coolded CCD. AB-2550 Kronos Dio is equipped with a PhotoMultiplier Tube.
PMT’s detectable wavelength area is determined by the material of a photoelectric surface that converts the incident light to electron and the material of the window that keeps it in a vaccum state. In the case of bioluminescence, the luminescence is in the visible region, so the detection efficiency is decided mainly by the material of the photoelectric surface. In a photomultiplier tube equipped device, when the luminescence has a different wavelength even if the amount of light is the same (the number of photons is the same in this case), the signal value obtained various. This is due to the wavelength dependency of the electronic conversion efficiency (quantum efficiency) on the photoelectric surface.
Firefly luciferase changes the color of its luminescence from yellow green to red according to a change in pH. Care must be taken when trying to assay using this luciferase without lysing a cell because the signal intensity changes according to changes in pH in the cell.
Along with the popularization of digital cameras, CCDs have become well known optical sensors, which no longer require explanation. This sensor’s quantum efficiency is not low in the red luminescence though PMT is low in red. Although it is expensive, a back illuminated CCD has a high quantum efficiency of about 90% and is suitable for measurement of weak light.
Instantaneous luminescence and stable luminescence
Depending on the reaction conditions, light emission may finish within a few seconds or it may be relatively stable for about 60min. In the case of stable emission, the amount of luminescence can be measured with the luminescence measurement device after starting a luminescent reaction, so it is not necessary for the device to be equipped with a injection pump. In AB-2550 Kronos Dio, the luciferase assay that uses a stable luminescent reaction with a luminescent protein as a reporter will be the main target. On the other hand in the case of instantaneous emission, luminescence at dispensing reagent is often the strongest, therefore to measure the amount of luminescence just after dispensing, a injection pump must be equipped. So AB-2550 Kronos Dio is not suitable for this usage.