Densitometry

Table of Contents

What is densitometry?

Densitometry is the quantitative measurement of optical density. It is the process of quantitating sample amounts based on their absorbance of light.

How does optical density relate to densitometry?

Optical density is a measure of the “darkness” of a light sensitive material over a defined area. The optical density of a sample is related to the percent of incident light that is transmitted through the sample.

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.

Densitometry's application in molecular biology

Quantifying spots or bands on protein gels and films

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.

How to perform densitometric analysis using instruments

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.

Using digital imagers, such as the new Sapphire FL or chemiSOLO, presents multiple advantages for densitometry. Keep reading and we’ll go over these advantages and expand on how the Sapphire FL is the ultimate choice for densitometry.

Both the new chemiSOLO and Sapphire FL Biomolecular Imager are imagers capable of directly imaging stained gels or exposed films. Purchase a  DensitoMetrics™ kit, a specialized densitometry package that uses a densitometry plate, for the Sapphire FL or a Densitometry Package [CS1004] for the chemiSOLO before performing densitometric 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 Figure 1.

Densitometry absorbance allows light to travel through the sample twie
Figure 1. 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

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.

Advantages of using an imager for densitometry

Table 1. Advantages of using the new Sapphire FL Biomolecular Imager for densitometric imaging

Why use an imager
for densitometry
Advantages of using
Sapphire FL for densitometry
Access to a large imaging areaWith a broad scanning area, the Sapphire FL can image samples as large as 25 cm x 25 cm and 4 cm high.
Even illumination over scan areaThe Sapphire FL will provide even, consistent illumination across the entire scan area, which is required for accurate data
Higher resolutionHigh resolution is everything! Luckily, the Sapphire FL has 5 micron resolution, which allows for detection of small spots or features
Large linear dynamic rangeSapphire FL has dynamic range of 3.6 OD. This allows detection of weak and strong bands without saturation of signal
Automated Z-scanSapphire FL has an adjustable laser focus plane from -1 mm below to +6 mm above the glass surface tp capture the best images of thick samples or gels. The adjustable focal plane allows for optimal imaging of your sample, even when offset from the glass.
Access to additional featuresThe Sapphire software has SmartScan, which detects and helps you avoid saturated spots during capture. You will only collect and analyze appropriate images for reliable quantitative data

Digital imaging of and documenting stained gels allows for quick, qualitative analysis of protein samples. These gels can then be quantitatively analyzed using densitometry. To see how we demonstrated densitometric analysis using the chemiSOLO with two SDS-PAGE gels, scroll down to check out our latest app note.

Which kind of analysis software is the best for densitometry?

Imaging and documenting stained gels allows for quick, qualitative analysis of protein samples. The same gels can then be analyzed quantitatively using densitometry. Using analysis software, such as AzureSpot Pro, the concentration of a sample band can be determined by comparing its optical density to the optical densities of known standards.

Coomassie-stained gel with serial dilution of BSA with bands and region used to define Ti highlighted in AzureSpot Pro software in preparation for densitometric analysis.
Figure 2. Coomassie-stained gel (imaged using chemiSOLO) with serial dilution of BSA with bands and region used to define Ti highlighted in AzureSpot Pro in preparation for densitometric analysis.

AzureSpot Pro is a Western blot image analysis software for advanced analysis of Western blots, gels and plates. It includes tools for 2D densitometry (Figure 2). AzureSpot Pro is also able to easily facilitate the identification and definition of bands, as well as the analysis of band optical density for images captured by imagers, like chemiSOLO or the new Sapphire FL. If you’re interested in analyzing the software for yourself, we offer a free trial of AzureSpot Pro.

Products for Densitometric Analysis
Sapphire FL Biomolecular Imager

Able to directly image stained gels or exposed films using customizable laser modules

chemiSOLO

Portable, personal Western blot imager capable of both densitometry and chemiluminescent imaging

A specialized densitometry package which uses a densitometry plate to emit fluorescent light when illuminated

AzureSpot Pro analysis Software

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

Featured Publication

This publication from Scripps Institute features gel images from an Azure c600 quantified by densitometry using AzureSpot.

Resources for Densitometry

Related Applications

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