Improved Quantitation with Digital Imaging

2 IMAGING WESTERN BLOTS 2.1 Leaving the Darkroom – Moving to Digital Imaging for Better Quantitation  Digital imaging hardware and the associated quantification software are becoming ever more popular, surpassing film-based methods for gathering data. Compared to film, digital imaging offers a wider dynamic range, more accurate quantitation, and the ability to conduct multiplex imaging, all of which work together to greatly streamline workflows. Here we discuss the key benefits of leaving the darkroom, and moving to digital imaging. Detection of Signal Saturation  When capturing data from your Western blot, through film or a digital imager, you are measuring the intensity of signal collected in a specific area of the blot. When the signal accumulation for a specific band on a Western blot reaches the saturation level, no further signal accumulation is possible. This means that if two bands on your blot are saturated, you have no way of knowing if they have the same signal level, or if their signal level is vastly different.  Using film, detection of saturation is essentially guesswork and it is easy to miss variations in protein levels or to underestimate the amount of protein present. Digital imaging has two advantages. First, software for the quantification of digital imaging can notify the scientist when the acquired image has bands that are saturated. (Figure 2.1 A, B) Second, software for digital imaging can determine the optimal image time that prevents signal saturation of the most intense band. This feature can prevent over-saturation of the signal, making sure the data is within the linear range of the imaging system. A. Film Digital Image Showing saturation B. Sample Western blot Same blot showing saturation Figure 2.1. A wider dynamic range and automatic saturation detection allow for improved quantification. A. The same blot was imaged on both x-ray film and the Azure c600. The Azure c600 detects when saturation occurs and calculates an auto-exposure time to avoid saturated bands. B. The top blot is a sample western blot showing a variety of band intensities. Below is the same blot showing saturated bands, this blot should therefore be exposed for a shorter period of time for more accurate quantification, a process that is easy to do with today’s digital imagers. Wide Dynamic Range  Dynamic range is the ratio of the maximum detectable signal to the background signal (i.e. noise). When comparing two different systems, the one with a wider dynamic range should provide faster workflows and more robust quantitation since both weak and strong signals can be measured during a single exposure. Without a wide dynamic range, strong signals will saturate before weak bands can be detected, requiring multiple exposures to visualize all bands and making quantitation either less reliable or even impossible (Figure 2.2). 16-bit CCD system 12-bit CCD system 1 sec 0.10 R2 = 0.99185 1 10 Log Picograms Protein Log Pixel Intensity Log Pixel Intensity 10 sec 100 0.10 R2 = 0.75929 1 10 Log Picograms Protein 100 Figure 2.2. 16-bit Imaging for a Wide Dynamic Range. A Western blot was imaged on both a 16-bit system and a 12-bit system. While the 10 second exposure appears similar on the different systems, the 12-bit system produces an image that is saturated and not suitable for analysis. High Resolution  Whether you’re looking at a CCD camera–based system or a scanning imager, the resolution will determine the smallest feature size that you will be able to image. For CCD-based imagers, resolution is typically presented in megapixels (MPs), with a larger number of MPs providing a higher resolution. For scanning imagers resolution is provided in microns and indicates the smallest feature size you will be able to visualize. Higher resolution systems can facilitate imaging blots with poorly-resolved protein bands and can facilitate visualizing samples other than blots. For example, some high resolution scanning imagers can image individual cells within a tissue sample or microscope slide. Multiplex Assays  Film and chemiluminescent imaging limit the assay to one signal detected per Western blot. The most recent generation of digital imagers have introduced the possibility of multiplex analysis (Figure 2.3) to assays that were previously only able to analyze one protein at a time. Fluorescent antibodies spanning the visible and near infrared spectrum enable detection of multiple samples of interest within a single assay, making within-experiment controls possible, supporting the development of novel assays, and greatly improving and accelerating workflows. Red channel Green channel Merged image Image Digitization  To include a Western blot image in any publication, digitization of the image is required. With film, scanning of the film is required in order to analyze or quantitate the image. With digital imaging, the resulting image is immediately digitized and ready for quantitative analysis with software.  summarize, digital imagers represent a significant improvement To over traditional film imaging with increased sensitivity, dynamic range and image quality. Furthermore, they offer significant quality of life improvements such as automatic saturation detection and quantification, while also opening the door to the development of new assays through their multiplex capacity. Figure 2.3. Multiplex imaging significantly optimizes workflows and allows for new assay development. Multiplexing on a single blot is a unique method available with digital imagers. Here, a western blot has been probed for both STAT1 (red channel) and phosphorylated STAT1 (green channel) using fluorescently conjugated secondary antibodies and imaged using infrared detection on the Azure c600. 2.2 Choosing a System – CCD Imagers, Scanners, and Hybrid CCD/Scanning Systems  Once a blot has been prepared the last step in the Western blotting workflow is image capture and analysis. Using the right imager and following best practices for data acquisition and analysis is just as important as choosing the right electrophoresis and blotting conditions when you want to generate reliable, high-quality Western blotting data. The choice of imaging system will depend on the type of studies being done, specifically the detection chemistry used and the sensitivity and resolution needed. Different Imaging Systems for Different Performance Needs Today’s digital imagers typically fall into one of two categories—CCD-based imagers and scanning systems. Choosing between the setups comes down to performance needs and whether you need the flexibility to support multiple imaging chemistries and applications. In both types of systems, one of the most important features to look for is dye flexibility. Not every fluorescent dye works with every imaging system, thus it’s important to verify that the system has enough dye flexibility that it will work with the dyes used in your studies. This is especially true when you want to do multiplex fluorescent Western blots. For multiplexing, it is critical to look at systems with multiple excitation sources and multiple detection wavelengths. Additionally, you will want to make sure that the best light sources have been selected. For example, IR dyes are typically excited with lasers, in part because only lasers emit light close to the excitation peak of these types of dyes. Here we discuss the key features to use for imaging system evaluation. CCD Imagers CCD imagers use a CCD camera for signal detection. The blot (or gel, or other sample being imaged) is placed into a chamber and, if necessary, illuminated using a light source. The emitted light is then captured and digitized by the sensor. With this type of system, because the entire sample is imaged at once, the uniformity of sample illumination and sample detection is critical. It is therefore important to consider the imaging field of view (FOV) in CCD platforms, the larger the FOV the more difficult it is to control illumination uniformity. Excitation uniformity is easier to control with a smaller field of view. Imaging chemiluminescent Western blots is most effectively done using a CCD sensor. The CCD sensor samples the entire light spectrum simultaneously and efficiently, supporting the long integration times (from seconds to minutes) needed for sensitive detection. Like film exposures, CCD imaging allows the user to control the exposure time. Azure Biosystems offers CCD imaging systems that combine affordability with performance. Learn more about our cSeries Imagers at azurebiosystems.com/cseriesimaging-systems. Key Features for Evaluation of CCD Imagers High Resolution, High Sensitivity—One feature essential for digital imaging with a CCD camera is pixel binning. The advantages of pixel binning—higher sensitivity and a higher signal-to-noise ratio—are the result of combining neighboring pixels into a single larger pixel, or “super-pixel” (Figure 2.4). The larger size of the super pixel increases sensitivity by increasing the surface area available for photon detection without similarly increasing the noise (note that the lack of increased noise only applies to on-chip binning and is not a feature of binning performed computationally during data analysis). For example, a binning of 2x2 combines four adjacent pixels into a single super-pixel resulting in a four-fold increase in sensitivity to light, while keeping the noise the same as that of a single unbinned pixel. While the increased pixel size can greatly improve the sensitivity of the detector, the larger pixel size reduces the resolution. A binning of 1x1 uses the native pixel size—no pixels are combined—and thus takes advantage of the full resolution of the sensor.  is important to understand that with high levels of binning the final image will have significantly lower It resolution, and that low resolution images may not be suitable for publication. Upscaling, a form of post capture image manipulation, can be used to rescue the lost resolution, but an upscaled image must not be used for quantitative purposes because pixels (data) is artificially added to the image and only raw data should be quantified. 5.4 µ 1X1 Binning 5.4 µ 40 seconds, unbinned 10.8 µ 2X2 Binning 10.8 µ 40 seconds, 2X2 bin Figure 2.4. Pixel binning improves image sensitivity. Pixel binning is a powerful technique for digital imaging that increases sensitivity by combining pixels to make a larger “super-pixel.” The super-pixel has a higher signal-to-noise ratio (SNR) than the unbinned pixel. Wide Aperture for Chemiluminescent Detection—Especially for chemiluminescence, the F stop is an important value to consider. The smaller the F stop, the wider the aperture, and the more light can be let in. Imagers with a small F stop can deliver more light to the sensor, reducing exposure times. Wide Range of Light Sources—While chemiluminescent does not require a light source, fluorescent imaging does require both a specific light source and emission filter. There are two main light sources used in fluorescent imaging: light emitting diodes (LEDs), and lasers. Historically, LEDs have been used because of their wavelength flexibility and low cost. But laser technology has advanced rapidly and now offers similar wavelength flexibility at a competitive price. Lasers are fundamentally different from and have several advantages over LEDs. Because lasers are monochromatic, they are highly effective at exciting fluorophores at a precise wavelength. In a multiplex experiment, precise excitation is critically important for specific excitation to avoid bleed through. LEDs, which are a broad band light source, can excite many more fluorophores but may also introduce cross excitation / bleed through if proper care is not taken in the experimental design. Scanning Systems Scanning systems use laser light for sample excitation and either a photomultiplier tube (PMT) or avalanche photodiode (APD) to detect the emission signal. The sample is typically placed on a bed area and light excites the sample from the bottom. The emitted light can then be detected by a PMT or APD. Unlike CCD imagers, which image the blot or other sample all at once, scanning systems illuminate the sample one small section at a time as the sample is scanned. While scanning can take slightly longer than imaging with a camera-based system, the higher intensities possible with pinpoint rather than widely-distributed illumination can lead to greater sensitivity for more demanding studies. In addition, the ability to use PMTs and APDs, which are not compatible with a camera-based imaging approach, expands the range of detection chemistries and offers lower detection limits and overall better performance for visible and near infrared (NIR) fluorescence imaging.  Imaging fluorescent Western blots can also be done using a CCD sensor, but better performance can be obtained in a bed scanning system. Fluorescence imaging with a CCD imager is often less sensitive than chemiluminescence because fluorescence imaging does not enjoy the enzymatic signal amplification afforded by the horseradish peroxidase-coupled antibody. Fluorescence imaging with a laser scanner, however, can match or exceed chemiluminescence sensitivity because PMTs and APDs have an internal gain which serves to amplify the emission signal in a similar manner as the enzymatic amplification. Because CCD sensors cannot amplify the emission signal like a PMT or APD, laser scanners using a PMT and/or APD are the best choice for high sensitivity fluorescence imaging. Key Features for Evaluating of Scanning System Resolution—Scanner  resolution is important for application flexibility. Scanning systems enable fine tune control over the spatial resolution, allowing a user to select a resolution appropriate for a specific experiment. Most Western blots need only 100-200µm resolution, but some imaging application require much higher resolution to capture fine detail. For example, imaging stained tissue sections (IHC) may require 10µm resolution to visualize fine details in the tissue.  Scan Speed—Unlike CCD imagers that image the whole field of view at once, scanning systems scan the image line by line. The larger your scan area, the longer your scan will take. This is why it is important to select an imaging system with a fast scan speed, usually listed in cm/s. The faster the scan speed, the less time imaging your blot will take.  Excitation Sources—Scanner devices used for detecting fluorescence most commonly employ laser light for excitation. A laser is a collimated light source, meaning it produces a column of light such that nearly all the photons are travelling in the same direction/angle and nearly all the photons leaving the light source are effectively delivered to the endpoint (sample of interest/fluorophore). By contrast LEDs, which have high divergence, produce a diffused pattern of excitation light where the light scatters/diffuses in many directions. When exciting a fluorophore, the more energy delivered to the fluorophore the greater the emission signal. Because lasers are a collimated light source and do not diverge like LEDs, lasers are much more effective at delivering excitation energy to the fluorophore and therefore produce a brighter emission signal yielding a lower limit of detection compared to LED excitation. This technological difference is critically important to achieving high quality images and data.  Another factor to consider is simply the number of light sources. The more lasers, the more dye flexibility. Furthermore, more lasers enable the detection of more fluorophores in a single sample – for example, a system with four unique lasers would enable the detection of four fluorophores from a single sample, increasing the multiplexing capacity, application flexibility, and throughput. Detector Technology and Light Collection—Quantum efficiency and spectral response are important factors to review when evaluating scanning systems. It is also important to consider the number of detectors included in the scanning system. There are two main types of detectors used in scanning systems: Avalanche Photodiodes (APDs) and Photomultiplier Tubes (PMTs): PMTs—PMTs belong to a class of vacuum tubes that convert photons into an electric signal.  Photo multipliers have high internal gain, making them ideal for low light applications. The high internal gain is essential to high sensitivity fluorescence imaging. Photomultiplier tubes operate using photoelectric effect and secondary emission. When light is incident on the photocathode, it emits electrons into the vacuum tube. These Secondary Focusing electrons electrode electrons are focused towards the electron multiplier (dynode), which multiplies the signal by secondary emission. The multiplied electrons are converted into an output signal by the anode (Figure 2.5).  PMTs are ideal for low light imaging, where internal gain is required in order to amplify the signal. This is a key reason why they are used for phosphor imaging and other high sensitivity blue light detection ie GFP detection. Primary electron Dynode Anode Photocathode Figure 2.5. How a PMT works. Connector pins  APDs—An APD is a highly sensitive semiconductor electronic device that exploits the photo electric effect to convert light to electricity (Figure 2.6). APDs are similar to PMTs in that they also amplify the emission signal resulting in enhanced sensitivity through signal amplification. However, APDs are quite different from PMTs. The most important difference between APDs and PMTs is their quantum efficiency. APDs have a significantly higher quantum efficiency at longer wavelengths compared to PMTs, and because APDs can capture light more effectively than PMTs at longer wavelengths they are better suited for fluorescence imaging at wavelengths greater than 500nm . Combining the low autofluorescence associated with long wavelength fluorophores and the high quantum efficiency of APDs results in one of the most sensitive fluorescence detection systems. Although, bright blue dyes with a PMT detector can offer same or Figure 2.6. APD. similar performance.  Z-axis Flexibility—While all scanners can scan in the both X and Y direction, not all systems can freely scan in the z- axis. Controlling the z-axis scan plane allows users to easily image a variety of samples. Membranes, gels, culture plates, and slides all have different optimal z-axis focal planes, so it is important to consider a system than can easily change the z-axis for optimal imaging of a variety of samples. Hybrid CCD/Scanning Systems Hybrid CCD/scanning systems are a new class of imaging systems offering the best performance for a wide variety of chemistry and sample types. As discussed above, there are CCD systems which are ideal for chemiluminescence and gel imaging because they enable user control of the exposure time – short exposure for gel imaging and longer exposures for high sensitivity chemiluminescence imaging. However, because CCD detectors do not have a built in signal amplification mechanism they are not ideal for high sensitivity fluorescence imaging. Alternatively, laser scanners using PMTs and APDs can amplify fluorescence signals and provide a highly sensitivity fluorescence imaging system. Laser scanners however, do not allow for long exposures required for high sensitivity chemiluminescence detection, so laser scanners are not ideal for chemiluminescence imaging. Combining the advantages of a CCD imager for gels and chemi imaging with the high performance of laser scanning system using PMT and APD Azure Biosystems offers this first-in-class detectors for high sensitivity fluorescence offers the best of both hybrid CCD/scanning system, the Sapphire worlds. Hybrid CCD/scanning systems enable high performance Biomolecular Imager. Learn more at across multiple imaging modalities and a wide variety of sample azurebiosystems.com/sapphire. types for high application flexibility – this is the ideal imaging system both chemiluminescent and fluorescent imaging. CCD Imager vs Scanner vs Hybrid System: How to Choose How to choose an imaging setup depends on the types of studies being done. For many labs, the performance of a CCD-based system covers most needs, especially for gels and chemiluminescent Western blotting. However, for labs that need high performance (i.e. sensitivity, resolution, dynamic range), to image large samples, and/or to use multiple imaging chemistries (phosphor imaging, chemiluminescence, and visible and NIR fluorescence) a scanning system may be a better choice. 2.3. Imaging Beyond the Western Blot  Azure Biosystems offers CCD imagers and hybrid CCD/scanning systems that provide the value and versatility of imaging more than Western blots. Here are just a few examples of the different things you can image with our cSeries and Sapphire imagers. cSeries Images Virulent Newcastle Virus Vs. “Normal” Newcastle Virus in chicken embryo imaged on Azure c600. Images provided by Ray Izquierdo-Lara, Ana Chumbe, Katherine Calderon, Manolo Fernandez. FARVET SAC, Peru. Mouse imaging. Acquisition was in RGB automatic mode – red channel was imaged at 704 ms and green channel for 703 ms, both at 60 µm resolution. Imaged on Azure cSeries. An agar plate with E. coli expressing GFPmut3 (green) and mCherry (red). The plate was imaged using red and green LEDs. Imaged on Azure cSeries. Yeast colonies expressing GFP imaged in the Blue channel. Imaged on Azure cSeries. Western blot using Cy5 and Cy3 of FtsZ1 and FtsZ2-1 from Arabidopsis thaliana. Plant fluorescent Western. Imaged on Azure cSeries. GFP expressing cell monolayers with different protocols for fixation in different columns. Imaged on Azure cSeries. 2D Fluorescent Gel Imaged in Cy5/Cy3/Cy2. Imaged on Azure cSeries. Native Gel. Imaged on Azure cSeries. TAMRA/GFP. Imaged on Azure cSeries. GFP/mCherry. Imaged on Azure cSeries. Antibody Array. Imaged on Azure cSeries. Sapphire Images In-Gel Fluorescence and 2D Gels. Untreated and treated HeLa lysate were labeled with Cy3 and Cy5 respectively and simultaneously separated using IEF in the first dimension and SDS-PAGE in the second dimension then scanned at 100µm using the 520nm and 658nm lasers of the Sapphire Biomolecular Imager. Rat Brain. Three color composite image of a rat brain scanned at 10µm on the Sapphire Biomolecular Imager. Bee Head. Composite image of a bee head scanned with the 488nm and 785nm lasers of the Sapphire Biomolecular Imager. Mixed Tissue. Composite image of mixed tissue scanned at 20µm with the 658nm and 785nm lasers of the Sapphire Biomolecular Imager. Chicken Liver. Coomassie stained chicken liver scanned at 10µm with the 488nm, 520nm and 658nm lasers of the Sapphire Biomolecular Imager. Pelargonium Stem. Two channel composite image of a Pelargonium Stem scanned at 10µm with the 658nm and 785nm lasers of the Sapphire Biomolecular Imager. Pine Cone. Multichannel scanned image of a pine cone sliver, scanned with 488nm and 785nm lasers on the Sapphire Biomolecular Imager using the demo machine in Germany.