What is densitometry?

Densitometry is the quantitative measurement of optical density. Optical density is a measure of the “darkness” of a light sensitive material over a defined area.

How is densitometry measured?

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.

How densitometry applies to molecular biology

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.

How do you do a densitometry analysis?

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.

Densitometry absorbance allows light to travel through the sample twie
The laser light of the point scanner excites the densitometry plate placed on top of the sample. The plate’s fluorescent emission provides the light that passes through the sample towards the detector. The absorbance of light by the colorimetric stain in the sample is proportional to the concentration of the stain bound to the target

Advantages of using an imager for densitometry

Using an imager, such as the Sapphire FL, presents multiple advantages for densitometry:

  • Large imaging area: the Sapphire FL can image samples as large as 25 cm x 25 cm and 4 cm high.
  • Even illumination over scan area: the Sapphire FL provides even, consistent illumination across the entire scan area, required for accurate data
  • High resolution: the Sapphire FL’s 5 micron resolution allows detection of small spots or features
  • Large linear dynamic range: the Sapphire FL’s dynamic range of 3.6 OD allows detection of weak and strong bands without saturation of signal
  • Automated Z-scan: the Sapphire FL has an adjustable focal plane for capture of the best image of thick samples or gels
  • SmartScan: the Sapphire software detects and helps you avoid saturated spots during capture so you only collect and analyze appropriate images for reliable quantitative data

Which image processing software is the best 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.

Related Products
Azure Biosystems chemiSOLO personal chemiluminescent imager with lid open
Azure chemiSOLO

Portable, personal imager capable of densitometry, and chemiluminescent imaging

Azure Sapphire FL Biomolecular Imager with lid open
NEW! Azure Sapphire FL Biomolecular Imager

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

AzureSpot Pro analysis Software

Analysis software for the analysis of gels, blots, cell-based assays, and more

Featured Publication

Check out this publication where gel images taken with an Azure c600 were quantified by densitometry using AzureSpot software at Scripps Research Institute.

Resources for Densitometry

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