The imaging and analysis of proteins and DNA, in both gels and Western blot membranes, are key applications for digital charge-coupled device (CCD) imagers. The sensitivity and broad dynamic range of modern CCD cameras offer confidence in quantitation and convenience that cannot be matched by developing films in a darkroom.
To improve CCD imaging further, the Amersham ImageQuant 800 biomolecular imager offers enhanced image quality with reduced noise and better resolution.
Image noise limits the range of signal intensities that can be analyzed accurately from an exposure. Poor resolution limits the viewing of fine details in the image, such as closely-spaced bands on a gel. The ImageQuant 800 biomolecular imager overcomes these challenges with a novel automated exposure setting, the signal-to-noise optimization watch (SNOW) mode, and a high-resolution imaging option with no binning.
Another challenge with CCD imagers and laboratory imaging is the variety of sample types that researchers need to image and the illumination modes these sample types require. Laboratories often have multiple imaging systems dedicated to different applications, including chemiluminescent Western blots with color markers, DNA gels that require UV illumination, colonies in petri dishes, and assays in multi-well plates.
The ImageQuant 800 system offers flexibility across a variety of sample types and high-quality image generation that stands out across a range of applications. The system is well suited for both a quality control laboratory that needs calibrated optical density measurements of Coomassie™ stained gels and the R&D lab that requires the broadest possible dynamic range for ECL Western blot experiments.
Automating signal-to-noise optimization for the best image quality
Many laboratory CCD imaging systems have automatic exposure modes. For Western blots, these modes attempt to find an exposure time that is a favorable compromise between saturating the more intense bands and underexposing the weaker bands on a blot. As a result, the dynamic range is limited, and it is challenging to quantitate low intensity bands in the presence of high intensity bands. This difficulty is seen when a target protein is in the presence of a highly expressed protein.
The ImageQuant 800 system uses the proprietary intelligent SNOW imaging algorithm to automatically find the optimal signal-to-noise ratio (S/N) with minimal input or guesswork from the user. This approach provides the sensitivity needed to detect faint bands that could not be visualized by conventional imaging without saturating stronger signals.
The SNOW imaging mode process involves the following steps:
- A pre-capture exposure to identify an optimum exposure time
- Selection of the region of interest and background
- Multiple automatic exposures, continuously averaged to find the optimal S/N
The SNOW imaging mode is distinct from other auto-exposure modes in that it works by capturing multiple images and continuously averaging the signal. This image averaging process effectively minimizes random noise, thereby improving the signal-to-noise ratio (Fig 1).
Fig 1. Image capture and averaging by SNOW algorithm reduces noise to maximize S/N. (A) Line profiles from continuously averaged 7.5 s exposures with SNOW algorithm, from first capture to the average after capture 73, shown by (B) a reduction in noise and stable signal and resulting in (C) an increase in S/N.
The only user input required for the SNOW imaging process is selection of background and region of interest during the initial pre-capture. The user can then watchthe signal-to-noise ratio improve in real time for their region of interest until the system reaches an optimum S/N. Figure 2 demonstrates how the SNOW process reduces background noise and improves the S/N.
Fig 2. ImageQuant 800 system control software and SNOW exposure mode in progress. The image of a dilution series for a Cy5-labeled antibody transferred to a Western blot membrane was continuously updated during the averaging process. In this image, the signal-to-noise ratio is close to its maximum value. The SNOW mode subsequently stopped automatically when the S/N started to decrease, and the software saved the image with maximum S/N.
The SNOW algorithm removes the guesswork from the conventional trial-and-error approach to finding optimal exposure times. This automated mode improves the efficiency and productivity of Western blot or DNA gel workflows by increasing the chance that all bands of interest will be within the broad linear dynamic range and removing the need to repeat experiments or develop multiple blots and DNA gels. Figure 3 compares different exposures for imaging a Western blot, showing the various effects of these approaches on image background noise.
Fig 3. Comparison of conventional and SNOW imaging approaches for Western blots in chemiluminescence mode. (A and B) Conventional imaging with short exposures. (C) 93 s SNOW detection mode run on ImageQuant 800 CCD imager, consisting of multiple short exposures with image averaging to reduce noise. (D) Single 93 s exposure without the use of the SNOW algorithm. Compared to conventional imaging approaches, SNOW detection mode results in a broader linear dynamic range and minimal background noise, enabling the visualization and quantitation of protein bands from a wider range of signal intensities without saturation.
Achieving this level of S/N optimization requires not just the SNOW algorithm, but also robust optics and hardware in the form a high-resolution, 8.3-megapixel CCD camera with a large aperture F 0.74 Fujifilm™ lens. The ImageQuant 800 system represents the culmination of 10 years of ongoing partnership, bringing Fujifilm’s expertise in optics together with our expertise in life science imaging.
See deeper into protein levels with Western blot analyses
Another common challenge with both digital CCD imaging and X-ray film is the ability to resolve closely-spaced and multiple different protein bands on the same blot. This challenge would typically require the following strategies to resolve:
- An extended polyacrylamide gel electrophoresis (PAGE) run in the hope that closely-spaced bands separate sufficiently for resolution
- Duplicate Western blots
- Stripping and reprobing with additional primary antibodies
For a total protein stain, distinguishing bands on a Coomassie stained gel can be challenging; however, epi- and trans-white illumination combined with the high-resolution camera of the ImageQuant 800 CCD imager provides the ability to resolve proteins of similar molecular weights with as little as 0.5 mm spacing (Fig 4A).
For targeting multiple proteins, fluorescent Western blots provide a straightforward alternative to chemiluminescence. Supported by IR short and IR long illumination capabilities, the ImageQuant 800 system enables multiplexed detection of several proteins on the same blot (Fig 4B).
Fig 4. Differentiating between closely-spaced bands and multiplexed imaging of different proteins by fluorescence on an ImageQuant 800 CCD imager. (A) High-resolution colorimetric imaging of Coomassie stained gel demonstrates the ability to resolve bands on a gel 0.5 mm apart. (B) Three-color multiplexed overlay image of Western blot nitrocellulose membrane. Target ERK proteins detected using IR long (red) LED-filter combination and GAPDH detected using IR short (green) on the same blot.
Taking CCD imagers beyond the immunoblot
Analysis of Western blots is the primary application of CCD imagers in many laboratories. While some CCD systems make it possible to image for other applications, these imagers might not have been designed with these applications in mind. This accommodation could require changes to hardware and settings that complicate workflows. A compromise in image quality or sensitivity is also possible.
By comparison, the ImageQuant 800 CCD imager simplifies and delivers high-quality images for a broad range of applications with flexibility in illumination and versatility in allowed sample type built in. The light modes, for example, include:
- Epi-white (470 nm to 656 nm)
- Epi-UV (360 nm)
- Epi-RBG (635 nm, 460 nm, and 535 nm)
- Epi-IR short (660 nm)
- Epi-IR long (775 nm)
These illumination options, as well as a variety of customizable filters, enable users to image a variety of samples across the full spectrum using a single instrument. This flexibility allows users to complete a range of useful research functions and applications while saving space and simplifying workflows.
Colony counting and analysis
Counting cell colonies can be tedious and time consuming. A colony counting system takes up bench space while providing one very specific function. Additionally, reproducing the same image and analysis across different petri dishes with a standard hand-held camera can present a challenge.
The ImageQuant 800 CCD imager software takes advantage of illumination flexibility, enabling automatic colony analysis using the optical density (OD), fluorescence, or UV modes (Fig 5). An additional non-parallax (NP) lens accessory allows chemiluminescence imaging to be used for petri dishes and multi-well plates without introducing optical artifacts.
Fig 5. Colony imaging options for petri dishes with the ImageQuant 800 system. (A) Optical density measurements providing a direct measurement of the OD of each colony. (B) Full-color imaging. (C) Epi-UV fluorescence imaging to capture the auto-fluorescence of the cells. Chemiluminescence imaging is also possible using the NP lens accessory tray.
Host cell protein analysis
Host cell protein (HCP) levels provide a purity indicator for biologics, enabling manufacturers to evaluate their purification strategies and meet pharmacopeia recommendations. HCP analysis represents a critical step in gaining regulatory approval.
Analyzing an HCP ELISA usually requires a separate microplate spectrophotometer. With ImageQuant TL analysis software on the ImageQuant 800 system, however, HCP ELISAs can be analyzed without the need for a dedicated plate reader (Fig 6).
Fig 6. Quick evaluation of HCP ELISA using the HCPQuant ELISA kit and the ImageQuant 800 CCD imager. (A) White-light image shows yellow color change upon detection of HCP proteins from Chinese hamster ovary (CHO) cells. (B) Array analysis image where suitable reference samples were available, and (C) the standard curve generated by software for quantitation.
As an extension of its chemiluminescent and fluorescent Western blot imaging modes, the ImageQuant 800 CCD imager provides straightforward imaging of HCP coverage assays. These assays can be from a traditional Western blot, differential in-blot electrophoresis (DIBE), or difference in-gel electrophoresis (DIGE) approach.
DNA gel visualization and macroscopic imaging applications
The conventional method of imaging a DNA gel uses a dedicated UV platform with a connected computer, camera, and printer. The UV illumination mode on the ImageQuant 800 removes the need for a separate device and generates an image file that is easily accessible locally and remotely using ImageQuant CONNECT software.
The versatility of the ImageQuant 800 system continues beyond laboratory gels, blots, and plates. The UV illumination function, for example, has been used to study structures at the macroscopic level, such as vein-like patterning on flowers revealed under fluorescence excitation (Fig 7).1
Fig 7.Dendrobium nobile orchid imaged in the ImageQuant 800 system using different LED and filter combinations. The imager revealed how parts of the flower fluoresced under different excitation wavelengths, showing vein-like patterns.
Getting the most out of a CCD imager
Combining multiple imaging instruments into one CCD imaging system might raise concerns about instrument availability, especially in multi-user laboratories. If an instrument is unavailable or in high demand, there is risk of restricting researchers and creating bottlenecks relating to instrument access.
The ImageQuant 800 CCD imager and associated ImageQuant CONNECT software are designed with these challenges in mind. Connected to the local network, the system software enables both local and remote scheduling, as well as access to images from previous runs. This accessibility eliminates the need to physically transfer images with a USB drive.
CCD imagers can grow with a lab’s research needs
A CCD imager is a substantial investment for any lab or research facility. It can be challenging to strike the optimum balance between meeting a laboratory’s ever-changing research needs and avoiding redundancy in equipment.
While the ImageQuant 800 biomolecular imager provides a considerable array of functionality, laboratories that do not require the full suite of options can still benefit from the high-resolution, high-quality, and high-confidence images provided. The system is available in four configurations: ImageQuant 800, ImageQuant 800 UV, ImageQuant 800 OD, and ImageQuant 800 Fluor. Applications and light sources for each configuration are described in Table 1. Each option provides a straightforward upgrade path to expand and adapt to accommodate the unique and changing needs of a research group over time.
Table 1. ImageQuant 800 system configurations and applications
|Chemiluminescence with color marker overlay||n/a||√||√||√||√|
|Optical density (OD) measurements||Trans-white||√||√|
|RGB fluorescence imaging of blots||Epi-RGB||√|
|IR short and IR long fluorescence imaging of blots||Epi-IR short,|
We provide a range of solutions for life science imaging applications and workflows. Through a combination of robust hardware and innovative software, including the unique SNOW imaging mode, the ImageQuant 800 CCD imager enables flexible and versatile imaging across multiple applications. To learn more about the SNOW imaging algorithm or for support with any other aspect of the imaging workflow, contact our Scientific Support team.
- ImageQuant 800 biomolecular CCD imager
- Amersham ECL Prime Western Blotting Detection Reagent
- Amersham ECL Prime Blocking Reagent
- Amersham ECL Select Western Blotting Detection Reagent
- Amersham ECL Plex Western Blotting Combination Packs
- Amersham ECL rainbow molecular weight markers
- Amersham ECL Plex fluorescent rainbow molecular weight markers
- Amersham QuickStain Protein Labeling Kit
- Amersham CyDye NIR secondary antibodies
- Amersham Protran 0.45 NC nitrocellulose Western blotting membranes
- Amersham HCP DIBE CHO kit
- Koski, M., Ashman, T. Dissecting pollinator responses to a ubiquitous ultraviolet floral pattern in the wild. Functional Ecology 28, 868-877 (2014).