Syngene combines innovation and excellence in life sciences, genomics, molecular biology, and proteomics.
As a division of Synoptics Ltd, Syngene is dedicated to pushing the limits of scientific research using its image analysis technology.
From gel documentation to fluorescence and chemiluminescence imaging equipment, Synoptics’ products are precisely designed to fulfill the stringent criteria set by regulatory organizations and accreditation bodies. Cameras are critical in this mission, demonstrating Synoptics’ commitment to offering unparalleled quality and performance.
Western blot imaging: Which CCD camera should be used for maximum sensitivity?
When studying proteins, the most common approach for protein detection is Western blot, whether using chemiluminescence or fluorescence. There are two popular ways of detecting chemiluminescence signals:
- Exposing the western blot to X-Ray film
- Use a digital imaging system with a cooled charged coupled device (CCD) camera
CCD cameras use a light-sensitive silicon chip to convert photons to digital signals. Cutting-edge technologies, such as the G:Box chemi series, offer digital imaging capabilities that outperform film detection techniques.
These enhancements include increased sensitivity, better linearity, and expanded dynamic range, allowing for accurate protein measurement across a broad range of concentrations spanning four orders of magnitude (4OD).
What should you consider when choosing a CCD camera?
One of the most important aspects is the sensitivity of the CCD camera. When evaluating the sensitivity of the camera, you should consider several important specifications, such as:
- Signal-to-noise ratio (SNR)
- Quantum efficiency (QE) percentage
- Pixel size
- Dynamic range
Understanding these essential parameters will help you to evaluate the imaging system that best meets your needs.
Signal-to-noise (SNR) ratio
A researcher performing Western blots wants to increase the signal from the protein band while reducing the membrane background. This is referred to as the signal-to-noise ratio (SNR).
To obtain the best SNR ratio, you need to capture as much of the signal as possible (highest quantum efficiency percentage) and reduce the sources of noise to a minimum.
The quantum efficiency will be explained later, but to comprehend the SNR ratio, you must know the various types of noise produced by the CCD sensor. Readout noise is one of the main sources of noise.
Readout noise is a collection of noise sources generated during the process of amplifying and converting the photoelectron to a voltage. The faster the camera readout, the louder the noise.
The second source of noise is dark noise, which is caused by heat generated without any signal applied. Cooling the camera minimizes the dark signal and allows for longer integration periods, up to several hours.
To prevent excessive dark noise generation in chemiluminescent imaging, it is important to use a cooled camera. All G:Box chemi range cameras are Peltier cooled, which reduces dark noise while capturing images with extended exposure times.
Incoming photons inherently generate noise called photon shot noise. When measuring the signal from a calibrated and constant light source, the values fluctuate, appearing as camera noise. However, this fluctuation is due to the variability of the signal itself.
Quantum efficiency (QE)
Image Credit: Synoptics Ltd
Quantum Efficiency (QE) refers to the sensor’s ability to convert incoming photons into a detectable electronic signal. QE is commonly stated as a percentage; therefore, a QE of 80 % suggests an 80 % chance that a photoelectron will be emitted for every incident photon.
QE is wavelength-dependent (photon energy), and therefore, it is crucial to verify the camera sensor’s QE curve at the wavelength of interest.
The G:Box Chemi range cameras employ the sensor ICX674 and have a high QE of 73 % at 425 nm, making them suitable for capturing chemiluminescence Western blots, as chemiluminescence emits at a wavelength of approximately 425 nm (see graph).
When imaging fluorescence, it is important to examine the QE curve at the wavelength of interest for the fluorescent dye being imaged to guarantee optimal sensitivity from the sensor camera.
Pixel size
A pixel in a camera sensor gathers photons of light and transforms them into an electrical signal. The signals are digitized, and the camera can create a picture using the values received at each pixel.
Smaller pixels can be binned, which means that the signals from nearby pixels on a sensor can be merged to create a single, bigger, effective pixel (also called a superpixel), boosting sensitivity and, as a result, reducing exposure times.
The disadvantage of binning is that it causes a reduction of resolution. The three-bucket image demonstrates this, with rain gathered in buckets. Larger buckets can collect a greater volume of raindrops; similarly, larger pixels can capture more photons.
The demand for high-megapixel sensors has resulted in advances in sensor design and enabled ever-smaller pixels to be packed into sensors.
While having more pixels might be advantageous, there are tradeoffs since pixel size can affect signal-to-noise ratio and dynamic range. As a result, it is essential to consider the application when selecting the pixel size for the gel doc.
Image Credit: Synoptics Ltd
Dynamic range
Image Credit: Synoptics Ltd
The dynamic range of a CCD camera refers to the span of intensities it can capture in a single image, exhibiting a linear response from the weakest to the strongest signal (as illustrated in the line graph).
Many researchers want to analyze both low- and high-expressing proteins in the same Western blot sample. Imaging these two items concurrently can be difficult for imaging systems with a limited dynamic range.
Long-exposure imaging allows for the identification of low-expressing proteins, resulting in saturated or ‘blown out’ bands.
Systems with a broad dynamic range can capture images of both the low and high-intensity bands, allowing for the analysis of weakly and strongly expressed proteins.
Understanding dynamic range is critical for correctly assessing and interpreting the Western blotting results. Precise quantification requires that the signal from protein bands be within the imaging system’s linear range.
The connection between band intensity and protein quantity within the linear range is both linear and proportional. Signals beyond the linear range can be influenced by noise if they are faint or overly strong, causing the imaging system to lose accuracy in recording the increasing signal (saturation), as illustrated in the image.
Image Credit: Synoptics Ltd
When evaluating the dynamic range of a CCD camera, the full capacity of the sensor must also be considered.
Full-well capacity is the largest charge a pixel can retain before saturation, which causes signal degradation. When the charge in a pixel exceeds the saturation level, it begins to cover adjacent pixels, a process called blooming, which causes image smearing. When the pixel is saturating (blooming), the CCD’s linear response begins to diverge, compromising quantitative performance.
Image Credit: Synoptics Ltd
In conclusion, various factors influence the camera’s sensitivity. When determining the sensitivity of a CCD camera, the following specifications must be considered:
- Dark noise
- Readout noise
- The quantum efficiency percentage
- The pixel size
- Dynamic range
- Full-well capacity
About Synoptics Ltd
Synoptics is a prominent Cambridge-based company that has been at the forefront of manufacturing scientific-grade digital imaging systems, water purification systems, and vacuum ovens for over 30 years. The company is comprised of three divisions: Syngene, Synbiosis, and Fistreem, each specializing in specific areas of expertise. With a strong commitment to innovation and quality, Synoptics has established itself as a trusted provider of cutting-edge solutions in the scientific research and laboratory equipment industry.
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