Azure BiosystemsDensitometry is the quantitative measurement of optical density. Optical density is a measure of the “darkness” of a light sensitive material over a defined area.
Densitometry is measured by transmitting light through a material and comparing the intensity of the light that is transmitted to the intensity of the incident light. A larger optical density means less of the incident light was transmitted.
Densitometry is often used to quantify spots or bands on colorimetrically stained protein gels or on X-ray films used to image chemiluminescent Western blots or radiolabeled Northern or Southern blots. The optical density of bands on the gel or film is measured, and the grey values of the bands are associated with the amount of material in each band.
To carry out densitometric analysis, the gel or film must be scanned or digitally imaged. The scanning can be direct, where light is sent straight through the gel or film towards a detector, or reflected, using a densitometry plate. The Azure Imaging Systems and Sapphire FL Biomolecular Imager can directly image stained gels or exposed films. The Sapphire is can come equipped with DensitoMetrics™, a specialized densitometry package that uses a densitometry plate.
For sensitive densitometric quantification, combine the DensitoMetrics package with a Sapphire equipped with the green channel.
In contrast to sending white light through an X-ray film or a colorimetrically stained gel, with DensitoMetrics light travels through the sample twice, as shown in the figures below. The densitometry plate emits fluorescent light when illuminated by the laser. The plate is placed over the sample, and the laser light travels through the sample towards the plate. Any fluorescence emitted by the plate passes back through the sample to the detector. The sample can block some of the laser light from reaching the screen, and also block some of the resulting fluorescent light from reaching the detector. Any reduction in the amount of light is proportional to the amount of material in the band on the gel or film.
Using an imager, such as the Sapphire FL, presents multiple advantages for densitometry:
AzureSpot Pro Analysis Software includes tools for 2D densitometry. It is able to facilitate the identification and definition of bands, as well as the analysis of band optical density for images captured by imagers, like the Azure Imagers or the Sapphire FL.
Portable, personal imager capable of densitometry, and chemiluminescent imaging
Able to directly image stained gels or exposed films
Specialized densitometry package that uses a densitometry plate which emits fluorescent light when illuminated by the laser
Analysis software for the analysis of gels, blots, cell-based assays, and more
Check out this publication where gel images taken with an Azure c600 were quantified by densitometry using AzureSpot software at Scripps Research Institute.
Looking for a full list of applications?
Far from being an “is-it-there-or-not” technique, modern digital detection instruments can make Western blotting reproducible and quantitative. By working within the linear dynamic range of
Proanthocyanidins (PAs) are found in plants and have a role in protecting plants against herbivores and fungal pathogens. They are also used in many industrial
In a recent publication in Nature Communications, Caldwell et al used the multi-color fluorescence imaging capacity of the Azure Sapphire Biomolecular Imager to study the
