There are different digital imaging technologies available for Western blot protein detection. How do you choose between using a CCD camera and a laser scanner?
Whether you’re using chemiluminescence or fluorescence, digital imaging options are available to take a snap of your Western blot membrane. Two commonly-used technologies are charge-coupled device (CCD) cameras and laser scanners.
There are benefits and drawbacks to both imaging technologies. In this post, we discuss when you might use one option over the other.
What’s the difference between CCD camera and laser scanner systems?
As the name implies, laser scanners have a scan head that passes over the gel or membrane section by section. The light is detected as the scan head moves to produce the individual pixels of the image.
CCD camera-based imaging, on the other hand, works by detecting the entire sample at once, either with chemiluminescence or fluorescence. Varying the exposure time can improve detection or reduce noise.
When would you use a laser scanner?A laser scanner generates high-quality data thanks to a number of beneficial factors:
- Detection of even the lowest protein levels (as low as 3 pg)
- Linear dynamic range of >five orders of magnitude
- High resolution (as low as 10 µm wavelength).
Laser scanners, can image at near-infrared wavelengths and perform phosphorimaging. These features make it possible to detect light at longer wavelengths than systems that only work in UV or visible light, and detect signal from radioisotopes.
Protein detection using a laser scanner lets you carry out high-throughput analysis of even the lowest protein levels (as low as 3 pg). Laser systems can have a linear dynamic range of more than five orders of magnitude and typically a resolution of around 10 µm.
Although some laser scanning systems have a mode to detect chemiluminescent signals—known as a dark scan—this type of system is generally not the preferred choice for chemiluminescence. CCD-based imaging systems are more sensitive and therefore more suitable for chemiluminescent samples.
The principles of the moving-head laser scanner are shown in Fig 1.
Fig 1. Moving head scanning mechanism. The light from the laser beam passes a series of mirrors before hitting the sample. The sample is illuminated across its width as the scan head moves along the scan head rail. The entire sample is illuminated by the scan head, laser, and mirrors tracking along the length of the sample.
When would you use a CCD camera?
CCD cameras are a fast, sensitive, and affordable option for chemiluminescence, colorimetry, and UV and visible fluorescence imaging on a single platform.
Systems such as the Amersham Imager 600 series give highly linear responses and enable precise protein quantitation across four orders of magnitude.
Multipurpose CCD imagers bring high-performance imaging to chemiluminescence, fluorescence, and colorimetric applications. However, CCD cameras are not normally suitable for infrared detection and can’t be used for phosphorimaging.
CCD cameras are also sensitive to radiation—heat in particular—but often have cooling measures in place to minimize background noise and improve sensitivity.
The components of a CCD camera-based imaging device are shown in Fig 2.
Fig 2. Components of a typical CCD camera-based imaging device. The sample can be illuminated in a variety of ways depending on the nature of the labels to be analyzed. The sample is then viewed by the camera. The camera includes focusing optics to accommodate samples at different heights. Emission filters can be inserted in the light path to select specific wavelengths and significantly reduce background.
In summary, if you’re looking to use fluorescence or phosphorimaging, a laser scanner is most well suited. For chemiluminescence, try a CCD camera system. Both technologies are suitable for quantitation, but it’s worth considering that fluorescence detection is stable and repeatable for comparison across different blots.