How to choose an available imaging system

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Only 1 minute to let you know the secret of imaging systems

Foreword


In recent years, the development of Western blot technology has entered a mature stage and has become a major technology in the direction of protein research. With the rapid advancement of digital imaging technology, many researchers have walked out of the darkroom, got rid of X-ray film, and used a more sensitive and reliable chemiluminescence imaging system, which also enabled Western blot technology to enter the "semi-quantitative" era.
However, it is troublesome for many biological professionals to choose the suitable imaging products from diverse brands. Here we will make a summary and explain the key parameters of imaging instruments in a simple way, so as to avoid choosing the instrument like choosing your mobile phone and putting the resolution in the first place.

1. Digital camera imaging principle

Firstly, let's take a brief look at the imaging principle, which is roughly divided into three steps (as shown in the figure below).
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1)Photon to electron conversion

The optical signal emitted by the chemiluminescent sample through the lens is collected by photographic chip (CCD or CMOS), the chip will convert photons into electrons.

2)Electronic to digital conversion
Electrons are converted to digital signals via an ADC analog-to-digital converter.

3)Digital imaging

The digital signal is processed by software to obtain the image.

2. Sensitivity of the Camera

Since Western blot experiments require the instrument to capture the weak fluorescence signal of the sample, the most important parameter of the scientific CCD camera is its sensitivity.

How to evaluate a camera's sensitivity? The indicator most directly related to the sensitivity is "quantum efficiency." (QE),It refers to the efficiency with which the photographic chip converts photons into electrons, as shown in the figure below:
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Certainly, just as our human eye can feel the wavelength of light at 380nm ~ 780nm (we cannot see ultraviolet and infrared ray), different chips for different wavelengths of light sources, its conversion rate is not the same. The popular chips used in chemiluminescent imaging systems on the market provide a quantum efficiency up to 75%.

3. Resolution

Resolution is completely familiar to everyone and refers to the number of pixels on the chip; the higher the resolution, the more delicate the image will be. The following example shows the images with high and low resolution.

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For resolution, we have to bring up the concept of pixel binning mode. All the chemiluminescence imaging systems on the market today support such modes (e.g. binning 2x2, 4x4). Pixel binning What is pixel binning? is a clocking scheme used to combine the charge collected by several adjacent CCD pixels. For example, a “2x2 Binning” mode is equivalent to using 4 pixels jointly as a single pixel (as shown below).

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With pixel binning, we can increase the sensitivity and the output speed of the image. However, since the pixels will be united and used as one pixel, the output resolution will be reduced. With 2x2 Binning mode, the resolution of the image will be reduced by 75%, but the sensitivity of the camera will be increased by about 4 times, and our exposure time will be greatly reduced.

When do we need high resolution, and when can we forego high resolution in favor of high sensitivity? The answer is our practical application. Just as we usually watch movies, if we watch a movie on a HDTV, the resolution 1920×1080 is available, because the screen size of the TV is wide, low resolution movies can be seen similar to the "mosaic" effect, but scaled to the mobile phone screen, the resolution 1280x720 is more than enough.

For the same reason, if we just need to observe the results of an experiment or to write papers, we can forego the high resolution and use pixel binning (2x2 or 4x4) to get the results quickly. However, if the results are going to be printed as a poster, we can choose high resolution to get a high definition image. Most of the chemiluminescent imaging systems on the market today adopt cameras with a resolution of 6.05 megapixels.

4. Signal-to-noise ratio(SNR)

When it comes to signal-to-noise ratio, we can start by comparing the following three results (figure).

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Most people would find the right picture is the most satisfying. We could also say that the right image has the best signal-to-noise ratio!

 

What's actually the signal-to-noise ratio? As its name,it refers to the signal to noise ratio (signal/noise, abbreviated as SNR).

 

To improve the signal-to-noise ratio (the image quality), we can:

1) Improving the signal, e.g., by using better luminescence reagents, improving camera sensitivity, etc.

2) Reduce the noise

Noise caused during the imaging process is simply the interference to the effective signal. Reducing such noise is the "life's work" of every camera manufacturer. The noise, which can be reduced is usually divided into two types: readout noise and thermal noise.

Readout noise

Returning to the digital imaging principle, readout noise refers to the noise that occurs when electrons are converted to digitals through an analog-to-digital converter (ADC). It is generally related to the frequency of the readout, and the higher the frequency, the higher the readout noise. Cameras commonly used in chemiluminescence imaging systems on the market generates the readout noise of around 5.5- Electrons.

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Certainly, just as our human eye can feel the wavelength of light at 380nm ~ 780nm (we cannot see ultraviolet and infrared ray), different chips for different wavelengths of light sources, its conversion rate is not the same. The popular chips used in chemiluminescent imaging systems on the market provide a quantum efficiency up to 75%.

Thermal noise

Thermal noise (also known as dark current noise) refers to the noise generated by the silicon lattice inside the camera sensor due to heat generation, its unit is e-/p/s (electron/pixel/second), which refers to the noise generated by the chip per second, it is accumulated over time. The longer the exposure time, the greater the interference from thermal noise. Such interference for chemiluminescence is obvious, since the sample signal is utter weak and requires longer exposure time.

However, we can reduce the thermal noise effectively by camera cooling.

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If we compare the two images above, the left image adopts a cooled camera, whereas the right image absorbs only a normal camera. This would explain the fact that all chemiluminescence image systems use a cooled camera! Most brands on the market have a cooling temperature of -55 ℃ below ambient , which means that at a environment temperature of 25 ℃, the actual temperature of the camera can be as low as -30℃ to reduce thermal noise, improve the signal-to-noise ratio and better the image quantity.

Questions for summary:

1. Which camera is more available for Chemiluminescence application?
a. Lowest cooling temperature of Camera A is -25℃. It generates 0.0005 e-/p/s thermal noise at -25℃.
b. Lowest cooling temperature of Camera B is -30℃. It generates 0.0008 e-/p/s thermal noise at -30 ℃.

Tips:Camera cooling is a method to reduce the thermal noise. The key point is generating lower the thermal noise instead of lower cooling temperature.

2.Is a high binning mode able to increase signal-to-noise ratio (SNR)?

Tips: To improve the SNR, we can increase the signal and reduce the noise simultaneously.

If we activate 2x2 binning mode, as we know, the camera can provide more signals (theoretic 4 times) at the same exposure time than the one, which uses the normal mode.

On the other hand, to capture equivalent signals from the same sample, the binning mode enables the camera to reduce to exposure time, which means the thermal noise should be decreased.

If you have more technical questions, please feel free to contact us!

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About SHST

Shenhua Science Technology Co., Ltd. (SHST) has a mature team including research and development, production, sales and after-sales service in biochemical imaging field. Inheriting the domestic leading imaging technology and absorbing the international advantages of similar products, SHST adopts high-standard core components for imaging systems such as gel documentation, fluorescence and chemiluminescence imaging systems and continuously optimizes the software to improve the user's operating experience.

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