Figure 1. Increased dynamic range with CCD imaging. HeLa lysates were serially diluted, transferred to nitrocellulose membrane and probed for cyclophilin B, β-tubulin or DDX3. Blots were exposed to X-ray film or imaged using Smart Exposure on the iBright FL1000 Imaging System. Signals were quantified from a single captured image. In all experiments, strong signals quickly reached a plateau when detected with film. The signal from the iBright system yielded a wider linear response range than film for each of the targets analyzed. Linear range indicated by linear regression.
Take full advantage of our comprehensive offering of western blotting reagents by pairing them with the right detection tool for your needs. Explore our x-ray film and CCD-based imaging instrument options below to find which solution is best for you.
Invitrogen iBright Imaging Systems
Simplify western blot and gel data capture and analysis with our iBright Imaging Systems. The iBright Imaging Systems streamline the imaging experience with a combination of powerful hardware, automated features, and an easy to use interface.
iBright CL1500 Imaging System
Core imaging applications:
- Chemiluminescent western blot
- Stained protein gels and blots
- Stained nucleic acid gels
iBright FL1500 Imaging System
Core imaging applications:
- Fluorescent western blot
- Chemiluminescent western blot
- Stained protein gels and blots
- Stained nucleic acid gels
Feature overview of the iBright Imaging Systems:
- 9.1 MP cooled CCD camera: high sensitivity and dynamic range to help enable the detection of subtle differences in samples
- Five fluorescence channels: multiplex and capture up to four proteins in a single blot for more meaningful and representative experiments (FL1500 model only)
- Smart Exposure technology: provides rapid determination of optimal exposure time to help minimize the need to repeat exposures to acquire the desired signal
- Simple interface: clear layout of functions and features combined with a 12.1-inch capacitive touchscreen for a smooth imaging experience
- Advanced automated features: automatic sample rotation, automatic zoom, and automatic focus help streamline image capture
- 22.5 x 18.0 cm field of view: large field of view for high-throughput imaging (image up to four mini or two midi blots at a time)
- Green LED-based transilluminator: effectively excite popular DNA dyes such as ethidium bromide and Invitrogen SYBR Green dyes with an alternative to UV-based transilluminators
- Flexible connectivity: export captured images via ethernet connection, Wi-Fi (with optional accessory), USB, or directly to Connect cloud-based platform
- Multiple image analysis options: perform densitometry, quantitation, and normalization directly using the on-instrument software, or for more in-depth analysis use iBright Analysis Software (available in both desktop and cloud-based versions)
Thermo Scientific CL-XPosure Film is an excellent photographic film for use with enhanced chemiluminescence (ECL) substrates for horseradish peroxidase (HRP) or alkaline phosphatase (AP).
| Color of film | Blue |
| Detection | Chemiluminescence |
| Isotopes | 35S, 32P, 14C |
| Applications | Chemiluminescent western blotting, isotopic DNA and RNA methods, including Southern and northern blotting and gel-shift assays (EMSA) |
Available sizes
Comparison between image capture options for chemiluminescent detection
| Film | CCD cameras | ||
|---|---|---|---|
| Advantages | Limitations | Advantages | Limitations |
| Extended exposures -unlimited exposure times can be used to capture extremely low abundant targets | 1.5 orders of magnitude dynamic range | Greater than 4 orders of magnitude dynamic range | Extended exposures with weak signals can result in increased background from camera noise, affecting the overall signal-to-noise ratio |
| Low entry cost | Can require multiple exposures to obtain ideal image | Auto-exposure algorithms help determine optimal exposure time (less time needed optimizing exposure for the best balance of signal to noise) | Initial instrument costs |
| Requires dedicated darkroom space and film processor. Film processor can require ongoing maintenance. | Digital capture of data streamlines archiving of results | ||
| Qualitative | Qualitative | ||
| Film requires processing step before signal can be visualized | Instant visualization of results | ||
Chemiluminescent western blotting continues to be a fundamental application to characterize proteins. X-ray film has traditionally been used to capture the chemiluminescent signal generated by enzyme (horseradish peroxidase and/or alkaline phosphatase) conjugated antibodies. However, today CCD (charged-coupled device) camera-based imaging instruments are rapidly replacing X-ray film.
CCD-based cameras utilize light-sensitive silicon chips that convert photons to digital signals. Improvements in chip design have enabled the development of sensitive, cooled-CCD-based cameras with higher light-capturing performance than X-ray film. Powerful high-resolution cooled-CCD cameras in instruments such as the iBright Imaging Systems enable capture and analysis of western blots with greater signal sensitivity, linearity and dynamic range than X-ray film.
Quantitative analysis with CCD imaging
Signal linearity is an important factor for quantitative measurement. When the relationship between signal intensity and sample quantity is linear, unknown sample amounts can be calculated using a simple linear regression model. Visually, western blot exposures on X-ray film may appear to have a broad range of signal linearity; however, densitometry analysis indicates signals on film have narrow linear dynamic range. CCD camera-based instruments have an increased linear dynamic range compared to X-ray film (which has 1.5 orders of dynamic range). The cameras in iBright Imaging Systems and other equivalent 16-bit CCD-based imaging systems, display at least a 4-fold dynamic range, permitting the capture of strong chemiluminescent signals without sacrificing the detection of faint bands. This broad dynamic range improves the potential to accurately quantitate western blot results.
Automatic exposure algorithms for finding the optimal exposure time
New western blot documentation systems are being equipped with algorithms to assist in rapidly determining the optimal exposure time for chemiluminescent and fluorescent signals. Chemiluminescent signals can easily be saturated on film due to the limited dynamic range. Digital imaging has made it possible to visualize saturation and adjust exposure time accordingly, whereas this information is not evident when using film. The iBright Imaging systems feature Smart Exposure technology that rapidly predicts optimal exposure time, helping minimize the potential for over- or underexposed images, and thus the need to repeat exposures to get the desired signal.
Figure 2. Smart Exposure provides an optimal image without the need to capture multiple images. Smart Exposure and manual exposure on an iBright Imaging System were used to analyze HeLa lysates serially diluted and probed for HDAC1. Using Smart Exposure, a wide range of signal is captured where only the highest load of HeLa is saturated, providing the greatest range of signal from the highest load to the lowest load in one image. Smart exposure prevented overexposure and underexposure resulting in data that can provide more meaningful quantitation. Saturation is indicated in red.
Invitrogen iBright Imaging Systems
Simplify western blot and gel data capture and analysis with our iBright Imaging Systems. The iBright Imaging Systems streamline the imaging experience with a combination of powerful hardware, automated features, and an easy to use interface.
iBright CL1500 Imaging System
Core imaging applications:
- Chemiluminescent western blot
- Stained protein gels and blots
- Stained nucleic acid gels
iBright FL1500 Imaging System
Core imaging applications:
- Fluorescent western blot
- Chemiluminescent western blot
- Stained protein gels and blots
- Stained nucleic acid gels
Feature overview of the iBright Imaging Systems:
- 9.1 MP cooled CCD camera: high sensitivity and dynamic range to help enable the detection of subtle differences in samples
- Five fluorescence channels: multiplex and capture up to four proteins in a single blot for more meaningful and representative experiments (FL1500 model only)
- Smart Exposure technology: provides rapid determination of optimal exposure time to help minimize the need to repeat exposures to acquire the desired signal
- Simple interface: clear layout of functions and features combined with a 12.1-inch capacitive touchscreen for a smooth imaging experience
- Advanced automated features: automatic sample rotation, automatic zoom, and automatic focus help streamline image capture
- 22.5 x 18.0 cm field of view: large field of view for high-throughput imaging (image up to four mini or two midi blots at a time)
- Green LED-based transilluminator: effectively excite popular DNA dyes such as ethidium bromide and Invitrogen SYBR Green dyes with an alternative to UV-based transilluminators
- Flexible connectivity: export captured images via ethernet connection, Wi-Fi (with optional accessory), USB, or directly to Connect cloud-based platform
- Multiple image analysis options: perform densitometry, quantitation, and normalization directly using the on-instrument software, or for more in-depth analysis use iBright Analysis Software (available in both desktop and cloud-based versions)
Thermo Scientific CL-XPosure Film is an excellent photographic film for use with enhanced chemiluminescence (ECL) substrates for horseradish peroxidase (HRP) or alkaline phosphatase (AP).
| Color of film | Blue |
| Detection | Chemiluminescence |
| Isotopes | 35S, 32P, 14C |
| Applications | Chemiluminescent western blotting, isotopic DNA and RNA methods, including Southern and northern blotting and gel-shift assays (EMSA) |
Available sizes
Comparison between image capture options for chemiluminescent detection
| Film | CCD cameras | ||
|---|---|---|---|
| Advantages | Limitations | Advantages | Limitations |
| Extended exposures -unlimited exposure times can be used to capture extremely low abundant targets | 1.5 orders of magnitude dynamic range | Greater than 4 orders of magnitude dynamic range | Extended exposures with weak signals can result in increased background from camera noise, affecting the overall signal-to-noise ratio |
| Low entry cost | Can require multiple exposures to obtain ideal image | Auto-exposure algorithms help determine optimal exposure time (less time needed optimizing exposure for the best balance of signal to noise) | Initial instrument costs |
| Requires dedicated darkroom space and film processor. Film processor can require ongoing maintenance. | Digital capture of data streamlines archiving of results | ||
| Qualitative | Qualitative | ||
| Film requires processing step before signal can be visualized | Instant visualization of results | ||
Chemiluminescent western blotting continues to be a fundamental application to characterize proteins. X-ray film has traditionally been used to capture the chemiluminescent signal generated by enzyme (horseradish peroxidase and/or alkaline phosphatase) conjugated antibodies. However, today CCD (charged-coupled device) camera-based imaging instruments are rapidly replacing X-ray film.
CCD-based cameras utilize light-sensitive silicon chips that convert photons to digital signals. Improvements in chip design have enabled the development of sensitive, cooled-CCD-based cameras with higher light-capturing performance than X-ray film. Powerful high-resolution cooled-CCD cameras in instruments such as the iBright Imaging Systems enable capture and analysis of western blots with greater signal sensitivity, linearity and dynamic range than X-ray film.
Quantitative analysis with CCD imaging
Signal linearity is an important factor for quantitative measurement. When the relationship between signal intensity and sample quantity is linear, unknown sample amounts can be calculated using a simple linear regression model. Visually, western blot exposures on X-ray film may appear to have a broad range of signal linearity; however, densitometry analysis indicates signals on film have narrow linear dynamic range. CCD camera-based instruments have an increased linear dynamic range compared to X-ray film (which has 1.5 orders of dynamic range). The cameras in iBright Imaging Systems and other equivalent 16-bit CCD-based imaging systems, display at least a 4-fold dynamic range, permitting the capture of strong chemiluminescent signals without sacrificing the detection of faint bands. This broad dynamic range improves the potential to accurately quantitate western blot results.
Figure 1. Increased dynamic range with CCD imaging. HeLa lysates were serially diluted, transferred to nitrocellulose membrane and probed for cyclophilin B, β-tubulin or DDX3. Blots were exposed to X-ray film or imaged using Smart Exposure on the iBright FL1000 Imaging System. Signals were quantified from a single captured image. In all experiments, strong signals quickly reached a plateau when detected with film. The signal from the iBright system yielded a wider linear response range than film for each of the targets analyzed. Linear range indicated by linear regression.
Automatic exposure algorithms for finding the optimal exposure time
New western blot documentation systems are being equipped with algorithms to assist in rapidly determining the optimal exposure time for chemiluminescent and fluorescent signals. Chemiluminescent signals can easily be saturated on film due to the limited dynamic range. Digital imaging has made it possible to visualize saturation and adjust exposure time accordingly, whereas this information is not evident when using film. The iBright Imaging systems feature Smart Exposure technology that rapidly predicts optimal exposure time, helping minimize the potential for over- or underexposed images, and thus the need to repeat exposures to get the desired signal.
Figure 2. Smart Exposure provides an optimal image without the need to capture multiple images. Smart Exposure and manual exposure on an iBright Imaging System were used to analyze HeLa lysates serially diluted and probed for HDAC1. Using Smart Exposure, a wide range of signal is captured where only the highest load of HeLa is saturated, providing the greatest range of signal from the highest load to the lowest load in one image. Smart exposure prevented overexposure and underexposure resulting in data that can provide more meaningful quantitation. Saturation is indicated in red.
Resources
For Research Use Only. Not for use in diagnostic procedures.