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.
30mM Ca(NO 3) 200 µ L was dispensed to 0.1ng/ µ L aequorin 10 µ L using a pump and luminescence was measured for 20 seconds (using Lminessensor JNRII). The graph above shows kinetics for 5 seconds
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.
35mm culture dishes
Luminescence measurement device for real-time reporter assay
AB-2550 Kronos Dio
AB-2550 Kronos Dio is a luminescence measurement device (luminometer) that equips a photomultiplier tube as a detector. This device is suitable to monitor gene expression of culture cells and cultured tissue slice at fixed intervals over a period of several hours to several days using 35mm diameter culture dish as a sample container.
For the long term cultures, it is possible not only to control temperature in the cabinet by the air circulation but also to introduce CO. Additionally, because an optical filter is built in, it is possible to monitor up to three kinds of gene expression using luciferase with different colors of luminescence.
Flow of sample measurement using Kronos Dio
Kronos Dio measures luminescence of the cells in the culture dish.
To measure living cells
In the chamber, the temperature is kept at a constant level and CO2 gas can be introduced. For general cultures of cells, constant temperature conditions of 30 to 40 degrees are essential. AB-2550 Kronos Dio is equipped with a constant temperature device using Peltier device, so it is possible to maintain the temperature in the chamber at a constant level (25 to 45 degrees:room temperature 25 degrees). Moreover, for cells that need to be cultured with CO2 introduced, it is possible to maintain CO2 density at 5% (CO2 sensor and regulator equipped)
There is a turntable in the chamber of Kronos Dio, and eight 35mm diameter culture dishes can be set. There is a detector (condenser lens + photomultiplier tube) under position A in the photo.
Applicable to long term measurements and multicolor luminescence measurement
Kronos Dio can hold up to eight 35 mm diameter culture dishes in the chamber. The turntable has a structure which no stray light can cause interference between each dish. Because dishes can be exchanged easily by opening the door of the chamber, stimulation with drugs can easily be performed. After the door is closed, temperature rises promptly, so a stable culture condition can be obtained. Luminescence is measured for long time at regular intervals. This is applicable to multicolor assay measurement to obtain luminescence of three colors using one substrate because a color separation mechanism is built-in.
The inner filters of Kronos Dio are switched sequentially and luminescence permeating through the filter is measured by the photomultiplier tube.