Various luminescence measurement devices like Kronos Dio

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.

Various luminescence measurement devices like Kronos Dio by Gentaur PMT CCD kronos gene expression bioluminescence PhotoMultiplier Charge CoupledDevice

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

PhotoMultiplierTube (PMT)
Various luminescence measurement devices like Kronos Dio by Gentaur PMT CCD kronos gene expression bioluminescence PhotoMultiplier Charge CoupledDevicePMT’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.

CCD(Charge CoupledDevice)
Various luminescence measurement devices like Kronos Dio by Gentaur PMT CCD kronos gene expression bioluminescence PhotoMultiplier Charge CoupledDeviceAlong 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.

Kronos Dio – Gene expression analysis and bioluminescence

Pacific Jellyfish Reprintedfrom Bioscience Saizensen(1998) ATTO Corp.“Discovery of ae quorin and GFP” by Osamu Shimomura.

Pacific Jellyfish Reprintedfrom Bioscience Saizensen(1998) “Discovery of ae quorin and GFP” by Osamu Shimomura.

The mutual adjustment of the expression of various genes within a cell a fundamental control process at the work within all living bodies, which is to say that t her e is a gene expr ession network. To analyze this kind of adjustment mechanism, the amount of mRNA, which is a transcript product or the amount of protein, which is a translation product can be masured directly by Kronos Dio. However, the reporter gene assay using luciferin-luciferase reaction is widely used because the expression can be quantitatively monitored more easily and more sensitively.



GFP and firefly luciferase are used as a reporter protein. GFP is fluorescent protein and it is widely used as a reporter of the gene expression. The search results of PubMed in 2005 are shown in the table above. One characteristic point is that in comparing total number of hit, the number of GFP is larger but, luminescent protein is more frequently used for studies related to transcription factors. GFP is an extremely stable protein that absorbs light of 470 to 490 nm and emits green fluorescence. It does not require a luminescent substrate such as luciferase, so it is advantageous in terms of cost, and it is a very convenient reporter when confirming the presence or localization of expression over time with Kronos Dio.

Luciferin shows specific luminescent reactions with luciferase and has few nonspecific reactions, therefore its S/N is satisfactory and it has excellent quantitativeness. It is also considered that cell toxicity from luciferin is small, so that it is possible to monitor fluctuation of transcriptional activity in real-time for extend periods by just resolving luciferin in the medium. However, when measuring fluctuations of expression over time, the fact that GFP is a stable protein becomes a disadvantage and it becomes difficult to monitor subtle difference. (It has become possible to obtain GFP with a shorter life in order to compensate for this disadvantage.) Moreover, for measuring the expression amount of the gene over time, it is necessary to assume that excitation light irradiation may easily damage the cell. Firefly luciferase is a protein with a molecular-size of approx. 62kDa, and it is normally considered as having a shorter half-life of around 3 hours. Luciferase with an even shorter life can also be used. Luciferin, the luminescent substrate is sold as an item enclosed in a kit together with a crystal product, cytolysis agent and reactive reagent, so it is utilized according to different needs.

2 color luminescence of railroad worm

2 color luminescence of railroad worm

As mentioned at the beginning, the method of measuring the amount of mRNA is also used normally. When this method is used, a cell needs to be crushed to extract RNA, so when measuring temporal changes, it is necessary to prepare cells of the same quality for the amount equivalent to the number of measurement times. Besides, RNA needs to be extracted within a limited time, so it becomes quite a serious task. It is also known that there is not necessarily a correlation between the amount of RNA and the amount of the translation when comparing the amounts of the two kinds of RNA.