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Image Capture: CCD Cameras

General Considerations | Software | Suppliers

Introduction

CCD stands for Charge Coupled Device which is an array of image sensors (single pixel) made using silicon chip technology. Traditionally sold for security applications, the use of CCD cameras has now broadened considerably. CCD video cameras are particularly useful for capturing fluorescent images, or images that require UV illumination. They may also be used for capturing images of small to medium sized standard gels, blots, autoradiograms. Video imaging is also ideal for capturing chemi- and bioluminescence in non-isotopic detection of biomolecules. For small objects the CCD video camera systems have the advantage of both speed and high resolution. However the resolution capabilities become lessened when the object is larger. Typical applications include UV trans-illuminated nucleic acid gels and TLC plates and general low light imaging. The important factors to consider are the resolution of the camera (should be more than 512 x 512 pixels), the sensitivity (normally expressed in lux units), the degree of "noise" that is acceptable and whether or not time integration facilities are required. The camera will require linkage to an image capture board inside the computer which should be able to handle 8 bit images of 512 x 512 pixels or greater.

A number of companies have put together complete systems for video photography. In recent years these systems have become quite popular for two main reasons:

They provide an inexpensive alternative to Polaroid instant photography by making use of video printing technology.

They provide a means of capturing images for further analysis using programs such as Phoretix 1D

Generally these systems include: CCD Video Camera and Power Supply Camera Stand, Lenses and Filters, Video Printer (Generally Mitsubishi or Sony) ,Time Integration Unit (or Computer with an image capture card and time integration software), Monitor and Connecting Cables.

Advice on CCD Camera Systems
In recent years there has been a massive expansion in the use of CCD Video Camera systems and digitising cameras for gel photography. There are two main reasons for this: - They provide an inexpensive alternative to Polaroid instant photography by making use of video printing technology. They provide a means of capturing images for further analysis using gel analysis programs such as Phoretix 1D. It is fast becoming recognised that there are real benefits to using good quality gel analysis and reporting software.

CCD stands for Charge Coupled Device, which is an array of sensing pixels made using silicon chip technology. Traditionally CCD Cameras were sold for security applications, but now that imaging technology has developed their use has broadened considerably. In fact imaging technology is moving almost as fast as computer technology. In electrophoresis applications CCD video cameras are particularly useful for capturing fluorescent images such as UV illuminated nucleic acid gels and TLC plates. The important factors to consider are the resolution of the camera (should be more than 512 x 512 pixels), the sensitivity (normally expressed in lux units), the degree of "noise" that is acceptable and whether or not time integration facilities are required. The camera will require linkage to an image capture board inside the computer, which should be able to handle 8 bit images of 512 x 512 pixels or greater.

Time Integration
Time integration is a process which mimics the effect of prolonged exposure. With conventional photography it is possible to let more light onto the film in two ways. The iris can be opened and the amount of time the iris is open can be controlled. Opening the iris too much when taking pictures of fluorescent gels can have the effect of allowing too much light through for brightly fluorescing bands and just enough light for very faint bands. It is therefore better to keep the iris small and increase exposure time. This will allow faint bands to be seen without overexposing the bright bands. The same is true with CCD cameras. However, under normal circumstances, CCD video cameras are processing a number of frames per second. Time integration effectively allows frame summation, thereby mimiking the effect of increased exposure time.

CCD Cameras
At the low cost end of the market there are those relatively insensitive cameras that are common in the security markets and could be used for capturing images that do not require sensitivity for very low light levels. Fluorescent applications generally require better sensitivities and frequently require the use of computer controlled time integration. Companies such as Sony, Hitachi and COHU produce typical CCD cameras with reasonable sensitivity and time integration. Without time integration these cameras will pick up most bands that can easily be seen by the eye. With correct time integration they can be made to reveal very faint bands without over exposing the bright bands. However the draw back is that at high integration times (more than 4 seconds) the noise levels and quantity of "bad" pixels are high. Nevertheless, it is in fact unusual to require integration times of more than 4 seconds for DNA/Ethidium bromide applications. In addition good image capture software, which is generally required to control time integration cameras, can minimise the effects of noise and “bad” pixels. If much higher integration times are required for special applications then it might be prudent to opt for a cooled CCD camera such as those that can be supplied by Photonic Science, Photometrics and AstroScan.

History of Purpose Built Systems (A Personal Perspective)
To my knowledge, the first purpose built system for the DNA gel electrophoresis market was produced by a German company, Cybertech, who mainly sold through UVP. This was a time integration system, but used a low resolution camera. UVP and Vilber Lourmat found that the higher resolution, low light COHU cameras produced a far better quality image even without time integration and therefore produced systems based on the COHU camera. As technology improved a time integration COHU camera formed the basis of the UVP system, whilst Vilber Lourmat went for the more compact Sony camera. UVP went to great lengths to educate the market on the benefits of using these systems and soon became recognised throughout the World as the major supplier. As market awareness of the benefits expanded, so did the number of competitors. Now there are quite a number of companies have put together complete systems for CCD video photography,

Generally these systems include:

  • Time Integration CCD Video Camera and Power Supply
  • Camera Stand, Lenses and Filters
  • Video Printer (Generally Mitsubishi, Sony or Fuji)
  • Time Integration Unit (or Computer with an image capture card and image capture software)
  • Light exclusion cabinet
  • Video Monitor
  • Connecting Cables

Suppliers of such systems include:

  • Analysis Software
  • UVP (Ultra Violet Products) GelWorks
  • Pharmacia ImageMaster
  • Bio/Gene Phoretix
  • Biocom Phoretix
  • Photometrics
  • Phoretix
  • Photonic Science
  • Bio-Rad Laboratories Molecular Analyst
  • Stratagene
  • Desaga
  • CAMAG

NB. This list is not meant to be complete.

Video Printers

In the early days nearly all systems included the Mitsubishi video printer and the main motivation for purchase was to reduce the cost of Polaroid instant photography. There was (and still is) a greater than ten fold saving per print and much lower wastage with regard to badly exposed pictures. This amounts to a considerable saving in consumable costs for most busy laboratories. Nowadays, Mitsubishi are currently loosing favour as they are not keeping up to date with the needs of scientists in this market. Sony video printers are becoming much more popular, partly because they have had the sense to produce a Windows driver, albeit not a very good one (Sony – please read this! We are waiting for a decent upgrade!). Pharmacia have opted for the Fuji printer as the prints look nicer since a different process is employed. However their competitors say that it is not without its problems, but then they would.

IMPORTANT

Image Capture Vs Image Analysis
It is often a good idea to have Image Analysis located on separate computers away from the image capture for several reasons:

Image Capture is generally performed in a laboratory area whilst good quality Image Analysis that facilitates proper reporting tends to be an office based function (i.e. On a computer containing a Word Processor, a Spreadsheet, a Database and a Slide Design package which is linked to a proper printer.)
Image Capture is not always followed by Image Analysis. Image Capture by CCD video photography is often a multi-user facility and is often used as a low cost replacement for instant photography.
Image Capture may only require a low cost computer, whereas good quality Image Analysis requires a higher specification computer.
The Image Capture board inside the computer may prevent the use of high-resolution screens (1024 x 768 x 256 colours) that are important during Image Analysis. Hopefully this situation will change in the future.
A user of Phoretix software may want to link several images together in one experiment for the purposes of comparison and reporting. When the gels are run at different times, possibly weeks apart, a combination system offers no advantage and can suffer disadvantages (particularly if space management on the Hard Drive is a problem).
Good quality Image Analysis packages, such as all Phoretix products, will be able to import and analyse images from a wide variety of image capture devices. This means that the analysis computer can be linked to document scanners such as the Epson GT8000 or the Sharp JX330 which are both excellent for scanning blots, gels and autorads at far higher resolutions than is possible with CCD cameras. Other devices such as radiation imaging equipment could also be networked to the same analysis station.

Image Analysis Software
The Video camera systems on the market at the present time have all tended to concentrate on the quality and flexibility of their image capture systems. Some of these companies have added some image analysis capabilities. The image analysis software they provide falls into one of the following categories:
Software that is completely dependent on the image acquisition card and is therefore restricted to the image size dictated by the card.
Basic software purchased from outside the company that offers only basic image analysis solutions.
We therefore recommend that all users of CCD Video based Image Capture systems purchase Phoretix software for their analysis requirements.

Sensitivity

Low cost devices

Relatively low cost, low-sensitivity (security-type) CCD cameras may be suitable for many routine gel or TLC image capture applications. Fluorescent applications generally require higher sensitivity and, frequently,the use of computer controlled time integration. When bands are sufficiently intense, low cost cameras without time integration can be quite effective. Such cameras can be forced to image faint bands by opening the camera shutter to maximum. However most gels have both bright and weak bands. When the shutter is open wide this will tend to let in too much light for sharp imaging of the bright bands. Thus, because the standard CCD video camera is limited to a set number of frames per second, it becomes very difficult to acquire images with both high sensitivity and the required sharpness of image for all bands on the gel.

Without time integration, a good low level light CCD camera (<0.1 lux sensitivity) will pick up most bands that can easily be seen by the eye in UV illuminated ethidium bromide-stained gels. With good quality time integration hardware and software such cameras can be made to reveal very faint bands which can be captured together with fairly bright bands. Generally only small time integration periods (0 to 3 seconds) are required to get good quality images. This is useful because much higher integration times can require higher cost equipment to avoid problems associated with increased background noise and the appearance of CCD pixel problems.Cooling the CCD device alows longer integration times (up to 30 minutes0 with a linear response, allowing detection of extremely weak signals (see below).

Moderate to high cost devices

A camera that may be considered as better suited for very low light level applications that require longer time integration is the CoolView camera system from Photonic Science Ltd. This camera is cooled with a Peltier device and is supplied with an image capture card, a board, operating software, cables and manuals. Because the camera is cooled, the background levels and the number of "bad" pixels are low which makes the camera better suited for very low light level situations and applications that require good densitometry analysis on the captured images. However the price for the CoolView basic system is over £7000 without accessories, computer or printer. Good cameras may offer binning Binning combines the charge from adjacent pixels on the CCD chip improving sensitivity and dynamic range at the expense of spatial resolution.

Where extremely low light level sensitivities are required for new fluorescence applications, then Astrocam Ltd of Cambridge have some excellent cameras developed for astronomy applications.

Resolution

Standard 1D gel images can be read at 100dpi (i.e. at a resolution of 254 microns per pixel). Indeed some imaging systems are limited to resolutions of over 400 microns. However, gels with many close and tight bands, 2D gels and miniature gels do require higher resolutions. Pharmacia Phast gels (35mm square) captured by standard CCD video cameras have a resolution of 68 gm (35mm/512 pixels) which is very good. However a 20cm gel will only have a resolution of 390ptm which is OK for some applications. Desktop scanners such as the Epson GT9000 and the Sharp JX330 can scan better than 600dpi (42gm per pixel).

Image Capture vs Image Analysis

It is often not advisable to have Image Capture and Image Analysis located on the same computer for several reasons:

Image capture is generally performed in a laboratory area whilst good quality Image Analysis that facilitates proper reporting tends to be an office based function (i.e. On a computer containing a Word Processor, a Spreadsheet, a Database and a slide design package which is linked to a proper printer.)

Image capture is not always followed by image analysis. Image capture by CCD video photography is often a multi-user facility and is often used as a low cost replacement for instant photography.

Image capture only requires a low cost computer, whereas good quality Image Analysis requires a higher specification computer.4. The image capture board inside the computer may prevent the use of high resolution screens (1024 x 768 x 256 colours) that are important during image analysis. Hopefully this situation will change in the future.

A user of Phoretix software may want to link several images together in one experiment for the purposes of comparison and reporting. When the gels are run at different times, possibly weeks apart, a combination system offers no advantage and can suffer disadvantages (particularly if space management on the hard drive is a problem).

Good quality image analysis packages, such as all Phoretix products, will be able to import and analyse images from a wide variety of image capture devices. This means that the analysis computer can be linked to document scanners such as the Epson GT9000 or the Sharp JX330 which are both excellent for scanning blots, gels and autoradiographs at far higher resolutions than is possible with CCD cameras. Other devices such as radiation imaging equipment could also be networked to the same analysis station.

Image Analysis Software

Currently available video camera systems tend to concentrate on the quality and reliability of their image capture hardware. Some products include image analysis capabilities:

Software that is completely dependent on the image acquisition card and is therefore restricted to the image size dictated by the card.

Basic software, often purchased from third party suppliers, that offers only basic image analysis solutions for electrophoresis applications.

Higher quality software, often supplied by Phoretix or one of its distributors.

When examining software systems it is important to consider your present and also future analytical needs and insist on high quality, supported software. Some companies offer sofware from more than one supplier. Check the options available and choose the system which is best suited to your present and likely future needs


Suppliers

Amersham Pharmacia Biotech

Supply software from two different sources. For 1D applications ImageMaster Prime or ImageMaster Elite is available:

These are fully integrated units which offer simple sample location with a built-in transilluminator, video monitor and thermal printer. The system has a high sensitivity for ethidium bromide stained gels and an optional PC interface for image transfer as a tiff files.

Pharmacia Biotech (UK)

Tel: +44 (0) 1727 814000
Fax: +44 (0) 1727 814001


Photometrics Gmbh products
Sollner Strasse 61, D-81479 Munich, Germany
Tel: +49 89 79 95 80
Fax:+49 89 79 97 15
www: http://www.photomet.com

Advanced Gel Documentation (AGD)
Photometrics offer package consisting of a "back thinned" 512x512 CCD from SITe with up to 85% quantum efficiency (as against a more common 40%). and a dynamic range of 16bits (65,535 counts). Phoretix Windows gel analysis software (1D and/or 2D) and a tabletop darkroom (Dark Top®) designed for long exposures of bio-luminescence and chemiluminescence probes.

Table top darkroom
This is a modular table dark room for long-term exposures of chemiluminescence and bioluminescence probes without the need for darkroom operating. This has an open base to accept most transilluminators, UV interlocks and filter holder

Sensys air-cooled camera system
Thermoelectric cooled CCD, 768x512 - 1536-1024 pixel area, PC 486/Pentium ISA Interface/Mac OS SCSI interface;Imaging software

CCD Camera control & Image acquisition software

Lightview software from Photometrics has been designed to operate with Phoretix gel analysis software.This is native 32-bit software operating under Windows 95 (or Windows 3.x) supporting Photometrics CCD cameras.

Lightview Features

32 bit image arithmetic with constant or images Cosmic ray filter Histogram plot, X and Y line plot and X, Y area plot Static display for image min. max, stnd and sigma
Read/write 16-bit PMIS files Read/write FITS image files Read/write 8-,16- and 32-bit gray scale TIFF files Image zoom, squeeze & panning
Interactive display scaling Real time cursor X, Y coordinate & Intensity display Real time min/max intensity display Grey-level, false colour log & inverted image display
Full camera control Interactive fast focus region readout Real time dark current correction Full support for the AT200 Controller TTL I/O port

Bio/Gene - supply Phoretix software with their CCD Camera Systems.

See also radiation and phosphoimaging section


A number of companies with a long background in image acquisition offer much useful information on conventional camera systems (film, use of filters, image capture and analysis and so on). A number of these manufacturers have significant sales in the bioscience market and so offer information specific to biochemists:

Fuji

Polaroid

CCD equipment suppliers addresses

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