Common Misconceptions about Fluorescence Image Sensors

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Misconceptions about Fluorescence Imaging and Image Sensors

Your Fluorescence Imaging Experts

Modern molecular biology labs increasingly rely on digital imaging systems equipped with fluorescence image sensors. This is because digital fluorescence imaging offers countless advantages over traditional film-based chemiluminescence detection methods. For instance, signals produced via fluorescence are more stable and reliable than those produced via time-sensitive enzymatic reactions. Despite clear benefits, digital imaging systems and fluorescence detectors are sometimes misunderstood, even by the most well-respected researchers. Allow us to briefly describe a few types of fluorescence image sensors before we dispel some common misconceptions about them.

Fluorescence Imaging

Digital fluorescence imaging has become popular in recent years, and for good reasons. For one, digital near-infrared (NIR) fluorescent images are significantly more precise than those produced with film. Plus, signals detected via NIR fluorescence are more proportional to the sample amount and can therefore be quantified more accurately than those produced via chemiluminescent reactions. This allows researchers to precisely pinpoint the concentration of their targets, sometimes even weeks or months after the first exposure.

Common Types of Fluorescence Image Sensors

Today's biomolecular imaging systems feature distinct types of image sensors depending on the application. However, most modern image sensors rely on the photoelectric effect in one way or another to produce digital images from incoming light energy. The photoelectric effect simply refers to the release of electrons when light hits a certain material.

Metals and conductors generally release more electrons than other materials when exposed to light. Semiconductors made from silicon can also harness the photoelectric effect to varying degrees depending on their design. Light-sensitive semiconductor components, also known as photodiodes, form the basis of most image sensors found in modern digital cameras, telescopes, microscopes, and biomolecular imagers. Here's a brief overview of some common types of image sensors used for fluorescence detection.

1. Charge-Coupled Devices (CCDs)

One of the most notable digital image sensors is the charge-coupled device (CCD). Developed in 1969, CCD technology greatly influenced the development of digital cameras, webcams, and even deep space imagers like the Hubble Space Telescope. These sensors employ a photodiode that converts light energy into electrical signals, often producing digital images with precise color and intensity data.

CCD sensors feature a photodiode composed of a slab of silicon divided into a grid of square pixels. When the camera's shutter opens, the incoming light forces electrons to the surface of the silicon where they're trapped within the grid's pixels. Each pixel holds the charge associated with the light exposure, then charges are shifted off the photodiode in series (row by row) before being sent to a capacitor and amplifier. Finally, the sensor converts the charges into digital values that form the basis of a digital image.

CCD image sensors, when cooled, are capable of producing relatively low-noise digital images. As such, they're often used to detect an entire field of low chemiluminescent signals with a single long exposure. However, CCD sensors read out images fairly slowly given their serial shifting of individual pixel charges. They also require more power than other photodiode designs. Some CCDs are suited for NIR fluorescence detection, although any fixed-area sensor (CCDs included) with long exposures limits the resolution of the final images.

Digital images produced by CCD sensors offer greater reliability and wider dynamic range than images captured on film. We employ a low-noise CCD sensor in our Odyssey^® XF and Pearl^® Trilogy Imaging Systems, as well as our C-DiGit^® Blot Scanners and D-DiGit^® Gel Scanners. Our Odyssey M Imager can also feature an optional CCD sensor for chemiluminescence applications. However, we prefer to rely on other photodiode architectures for high-speed, high-resolution NIR fluorescence detection.

2. Avalanche Photodiodes (APDs)

Avalanche photodiodes (APDs) are extremely sensitive, high-speed photodiodes made from semiconductors like silicon. As with CCD sensors, they detect the brightness of incoming light via the photoelectric effect. However, APDs feature a unique design and operate under precise conditions, which makes them ideal for a wide variety of applications including NIR fluorescence imaging.

Invented in 1952, these photodiodes are often found in today's long-range weapons, fiber-optic communication, and positron emission tomography systems. Their claim to fame is the aptly named avalanche effect, which produces immense signal multiplication within the photodiode itself. Due to this internal gain, APDs often provide exceptional sensitivity and low dark current when cooled. In fact, some systems employ these image sensors to reliably detect single photons of light. However, to utilize the avalanche effect to the fullest, APDs require high-voltage input.

APDs are generally fast, sensitive, and highly efficient light-detecting sensors. They may also be deliberately designed for low-light, high-speed, or temperature-controlled applications. What's more, they can also be configured to detect specific wavelengths of light, like those in the visible or NIR regions, with extreme precision. That's why high-performance APDs are the fluorescence image sensors of choice for our Odyssey^® DLx Imaging System.

3. Complementary Metal-Oxide Semiconductors (CMOS)

Complementary metal-oxide semiconductors (CMOS) come in many different configurations. They're found in most integrated circuits such as random-access memory (RAM), microprocessors, and digital transistors. CMOS can also be configured into arrays of active pixel sensors (APS) that operate much like CCDs and other photodiodes.

CMOS image sensors appeared around the same time as the CCD. Their early adoption lagged behind CCDs due to manufacturing constraints, but CMOS APS quickly became practical in the 1990s. Today, most modern smartphones feature compact CMOS-based digital cameras because they require about 100 times less power than conventional CCD sensors. This is largely due to the innovative way charges are read out from the sensor.

Without getting too technical, each pixel on a CMOS image sensor has its own amplifier and capacitor. This allows individual pixel charges to be read in parallel rather than in series, which is much faster, requires less power, and produces significantly lower read noise when compared to CCDs.

4. Scientific Complementary Metal-Oxide Semiconductors (sCMOS)

In 2009, CMOS sensors went scientific. Engineers adapted them to overcome limitations of the original CMOS design, bridging the gap that existed between them and conventional CCD sensors. Today, sCMOS image sensors can be found in various scientific instruments including cutting-edge microscopes and telescopes, as well as high-performance multimodal imagers like our Odyssey^® M Imaging System.

These next-generation CMOS image sensors provide superior resolution, wider dynamic range, and lower read noise than conventional CMOS detectors, even at speeds of over 100 frames per second. Therefore, sCMOS image sensors are often ideal for imaging applications where speed is key. Plus, they can even operate with high efficiency in the NIR region, making them excellent fluorescence detectors for quantitative Western blots, protein gels, cell-based assays, and tissue section imaging.

As with many newer technologies, sCMOS image sensors tend to be more expensive than most CMOS, CCDs, and APDs. Perhaps this is why some instrument manufacturers choose to avoid them entirely. However, when paired with well-designed circuitry and optimized protocols, few CMOS-, CCD-, or APD-based imaging systems can compare to the combined speed, resolution, and dynamic range of sCMOS fluorescence imagers.

LI-COR uses the most innovative sCMOS image sensor as a crucial component in our Odyssey M Imaging System. Our experienced team of engineers integrates this chip into our own electronic system, and they design and build the sensor controls according to our patented imaging technologies. This results in an extremely low-noise fluorescence detection system with better performance than any off-the-shelf camera alone, sCMOS or otherwise. We pair this high-end image sensor and custom circuitry with a novel line-scanning configuration to deliver the best of both worlds for our customers—high-resolution sensitivity like that of APDs at exceptional read speeds only available with (s)CMOS image sensors.

Misconceptions about Fluorescence Imaging and Image Sensors

It's no surprise that fluorescence imaging and digital image sensors are often misunderstood given the recent rise of these revolutionary technologies. The truth is many researchers still prefer to capture their assay data on film. This was the original detection method for Western blotting, after all. In time, we hope more researchers will see the NIR light, but until then, let's address some common misconceptions about photodiodes and fluorescence detectors.

1. Detectors Alone Determine Data Quality

One common misconception about fluorescence imagers is that the quality of data produced depends largely on the type of detector used in the system. While this is true to some degree, the image sensor is simply one component that affects the quality of data generated by any digital imaging system. Other important factors include the sensor temperature, voltage, and scanning configuration, as well as the overall circuit and optical design.

The circuit surrounding and enabling any image sensor is truly the unsung hero of world-class fluorescence detection. Even the most sensitive detector may be drowned by noise produced from poorly designed circuitry. What's more, the researcher's protocol will inevitably influence the quality of their data more than any image sensor ever could.

Keep in mind that light must arrive at the image sensor before it can be detected, and this doesn't happen by accident (or at least it shouldn't). The engineers' lens and filter choices ultimately determine how much light is allowed to focus on the sensor in the first place. This means the optical design of any instrument, like its circuitry, is as important to data quality as the detector itself.

2. Fluorescent Assays Are Limited by Detector Sensitivity

The "sensitivity" of image sensors is a widely debated topic. In fact, using this term at all may draw criticism from some engineers when used interchangeably with terms like signal, pixel size, and quantum efficiency. This is where discussions can get technical, so allow us to simplify the subject for you.

Sensitivity is often defined as the signal-to-noise ratio (SNR) of a given sensor or imaging system. In this case, signal refers to the amount of light (percentage of photons) that can be captured by the sensor and converted into electrons. Signal depends on time, the number of photons present, and the quantum efficiency (QE) of the detector itself. Quantum efficiency simply means how effectively and reliably a given sensor will convert photons of certain wavelengths into electrons, often expressed as a percentage. The higher the QE, the better, but no sensor is perfect. Those with high QE in the target wavelength can produce low limits of detection, but only if the signal is at least 3 times larger than the total noise of the entire electronic system.

There are many different types of noise to consider when evaluating fluorescence detectors, but the SNR of any digital imaging system is a function of countless variables. It depends on the choice of fluorophore and excitation wavelength, optical filtering, signal filtering, autofluorescence, and scatter, as well as the detector and even the biological sample itself.

For example, dark noise is common when detecting samples with low background, such as in chemiluminescent assays. However, dark noise is rarely an issue for samples that produce high fluorescence signals. In fact, few if any fluorescent bioassays are affected by the noise inherent in the fluorescence detector itself because fluorescent signals are often so strong. This issue is much more common when imaging weak chemiluminescent samples.

3. sCMOS Image Sensors Are Limited by Read Noise

Faster image sensors tend to produce more read noise than those with slower readouts. For instance, some of the first CMOS image sensors struggled to overcome read noise and other artifacts produced from their unique design. However, modern sCMOS sensors swiftly overcame this limitation, and today's configurations can even generate less than 1 electron of read noise.

It's worth restating that an advanced sCMOS image sensor is simply one component of any digital imaging system used by researchers. The circuit built around it along with the protocol it's included in cannot be discounted as significant sources of noise and error. No fluorescence detector is without inherent noise, and sCMOS cameras are no exception; however, there are effective ways to reduce or eliminate sources of sCMOS read noise. How engineers deploy these innovative configurations clearly distinguishes industry-leading fluorescence imaging systems from those peddled by would-be competitors.

4. Confocal Imaging Is Necessary for Fluorescence Detection

This is simply not true. Confocal imaging is only useful in high numerical aperture (NA) optical configurations typically found in microscopy. When used in a macro configuration suitable for gel, membrane, tissue section, or small animal imaging, the image sensor also rejects light from out-of-focus objects. In these cases, confocal imaging simply substitutes all-important sensitivity for sharper images, which is a terrible tradeoff for macro imaging. Thus, confocal imaging is truly only noticeable in high magnification settings like those used in microscopy.

Your Fluorescence Imaging Experts

Fluorescence imaging is complex, and there are many factors to consider beyond detector selection. However, it's essential for researchers to have a basic understanding of these concepts, especially as they decide which imaging systems to deploy in their lab. The truth is improper normalization, nonspecific antibodies, and outdated protocols can overshadow data captured by even the most advanced imaging systems, which is why we also provide our customers with best-in-class software, optimized reagents, and unparalleled support.

You don't have to be an optical or electrical engineer to benefit from the world's most capable biomolecular imagers. We're your fluorescence imaging experts, always here to dispel myths and support scientists worldwide. We don't stoop to confusion tactics; rather, we invest in the education and experience needed to encourage researchers and design top-of-the-line instruments. If you have any questions about imaging systems, fluorescence detection, or Western blotting protocols, then please don't hesitate to contact us today. We're always eager to empower you and your team, no matter which detection method you decide to use.