Technical Information
Key Question and Features of an Imaging System
Here we have written down some of our knowledge and experience to help you decide on the system that suits your needs. If you have any questions you can contact us.
In Vitro Gel Documentation, Fluorescent and Western Blot Detection
The Main Components of an imaging system…
Lighting and other applications…
The Camera
An imaging camera composes two important parts:
The optical lens system, which gathers and focuses emitted photons and the sensor device or CCD (charge-coupled device) which collects and processes the light into data. A number of factors affect the overall sensitivity of the camera and ultimately the ability to obtain experimental data.
Optical Components
The lens is by far the most important part of the optical system. A good quality lens is essential to obtain uniform light from the sample and maximise sensitivity.
The term used to describe the lenses ability to gather light is called the Aperture or F-Stop. This is usually expressed as a number. The lower the F-Stop number, the more light that can access the lens and by definition, the more light that can be collected. Below are a number of different F-Stop lenses and as can be seen the sensitivity of these lenses can differ by up nearly 10 fold.
Home digital cameras often incorporate a less sensitive lens with a bigger f stop number to maximise focusing ability from distant to close objects. However in scientific instruments, focus distances are relatively short and do not vary, meaning high sensitivity lenses can be used. The quality of the lens is also important, all AMV instruments use lenses from the well established Schneider Company of Germany.
The CCD Chip
A standard CCD chip for in vitro imaging is made of light capturing pixels. The number of pixels in a camera is expressed usually as the number of mega pixels and like a home camera this describes how many million of pixels are on the chip. While home digital camera may have a similar number of pixels to a scientific camera, you must look at pixel size as this is another factor affecting sensitivity.
Pixel Size
Pixels can be compared to buckets gathering rain, the larger the bucket the more rain that can be gathered, and the more sensitive the camera is to low light. However the large pixels generally give poor resolution and suffer from a 'lego brick' effect which was observed in early imaging systems. For in vitro applications a pixel size of around 7um is commonly used as this benefits sensitivity and resolution. For low light applications such as in vivo imaging, pixel size can be as high as 13um as greater sensitivity is needed. Sensitivity can also be enhanced by the addition of microlenses which focus light to the pixel sensor which would otherwise be lost to the side or borders of a pixel. Similarly, the true in vivo systems often possess 'backthinned' (or back illuminated) chips. These are incredibly thin chips that allow the maximum light to penetrate the light sensitive areas of the chip. Using either micro lenses or back thinning can increase efficiency of a camera to the 90% mark compared with about 30% for standard chips (See figure below)
Bit Depth
The light, once collected is converted into electrical signal. An 8 Bit camera converts the light into 256 intensities (or grayscales) while a 16 Bit converts to 65536 intensities. Therefore an 8 bit camera gives only linear data slightly over only 2 orders of magnitude, while a 16 Bit Camera gives nearly 5 orders of linearity. Therefore the higher the Bit Depth, the more linear your data will be. 16 Bit imaging is essential for getting the maximum linearity from a signal but one important factor is the linearity of the experiments, ethidium bromide fluorescence is generally only linear over a 3 log range. So in that respect lower bit cameras are used for Gel Doc and Protein Gel Imaging as these offer economic advantages over true 16 bit cameras.
Cooling and Background Noise (Dark Current)
All CCD chips have a background activity in the absence of light. This essentiality creates a baseline threshold which must be overcome to see a sample signal. This is hugely important when detecting low light signals as a high level of noise swamps the signal and makes imaging difficult. Cooling the CCD chip can overcome this problem. Background noise is reduced approx by half for every 6°C drop in temperature.
For example a camera cooled to -30°C has approximately 2 times more noise that a camera cooled to -36°C. Therefore the cooler camera will be 2 times more sensitive picking up low signals. This is exemplified further for detection of bioluminescence in organisms where luminous signals can be a low as just a few photons/cm2/sec. For example the dark current in a camera cooled to as low as -80°C is over 200 times less than one cooled to -30°C.
Which Transilluminator?
For general imaging of DNA, all the AMV Gel Doc systems come with a mid range (312nm) UV transilluminator which works well for a range of UV fluorescent agents such as ethidium and SYBR. As UV light is damaging to DNA, if you are intending to use the DNA from your gel for cloning you may wish to consider a longer wavelength illuminator such as the 366nm transilluminator. For true lab and sample safety you may also wish to invest in a blue or green transilluminator which keeps both the DNA and the user safe from damaging rays and are also more compatible with modern stains such as SYBR. All the AMV transilluminators give minimal artefacts and some like our LED range offer a range of light sources for 2D imaging. For documentation of coomassie or silver stained gels, use a UV to White Light converter or else a white light table. These give even illumation and clear crisp images.
What filter should I use for my fluorescence application?
AMV has a range of filters available which fit the emission spectra for most dyes with a spectra between 400nm-850nm . A useful website for a detailed examination of your fluorophore spectra is below.
What ECL substrate should I use with my image for western blotting?
As the Chemi Image system is highly cooled and incredibly sensitive it is suitable to use with most if not all commercial ECL kits and also home made kits. Side by side against film, our systems more than make the grade.
Fluorescent Imaging
The AMV range of instruments works well with ECF, tagged antibodies and a range of fluorescent dyes and protein such as GFP. Transillumination is suited for agarose or SDS page gels and can give clean crisp images. For imaging of samples on opaque membranes applications such as fluorescent western blotting and Southern blotting or in cells detection of fluorescent proteins, we recommend the reflective imaging provided by the xenon light source in the Chemi Image FL. This allows a broad range of excitation filters to be used and a very flexible fluorescent option.
Analysis of the image
The AMV range of software allows the user to simply take a picture, optimise and record in the lab book. When you need to quantify, count or analyse our software comes into it's own and gives you a host of features. For specialist applications we also have a range of software available for fingerprinting, 2D imaging, profiling and more complex analysis. Contact us for more information.