APD, PMT and sCMOS for Fluorescent Detection: What Every Lab Needs to Know

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Fluorescent laser scanners (sometimes called biomolecular imagers) and multimodal fluorescent imaging systems are important tools in molecular biology. Commonly used for generating images of fluorescent samples such as Western blots and fluorescent arrays, these systems are the workhorse of the modern molecular biology lab. Scientists depend heavily on the data obtained from the images produced on these instruments to draw conclusions and advance their research. The quality of such fluorescent data is largely dependent on the type of detector used by the system. It is important for you to understand the differences between common detectors, so the right instrument can be chosen for a lab’s fluorescent detection applications.

In this article, we will discuss three common detector types (sCMOS, PMTs, and APDs) (Table 1) and how they’re used in multimodal fluorescent imaging systems, such as the Azure Imaging Systems, and laser scanners, such as the Sapphire  FL.

Table of contents

Table 1. Comparison of Photodiode vs sCMOS Technology for Fluorescent Imaging

AttributePhotodiodes (PMT and APD)sCMOS
Capture ModesData collection one pixel at a time, improved signal to noise, highest sensitivity for fluorescent applicationsEntire field of view imaged at once, ideal for capturing images of non-static enzymatic reactions like chemiluminescence from HRP
SpeedDependent on the resolution specified, one pixel is captured at a time, imaging time longer than an sCMOSSince the entire field of view is captured at once, imaging times can be seconds to a couple of minutes
ResolutionHigh resolution possible over a large field of viewResolution is determined by the pixels in the detector and the field of view
SensitivityReduced noise due to single point detection and maximum true signal output (noiseless gain)With weak fluorescent signals, significant potential for noise due to dark current, read noise, and photon shot noise
Signal to NoiseLower background across the field of view, leading to higher signal to noise ratiosMore susceptible to noise, resulting in lower signal to noise ratios
Confocal laser scanningPoint scan confocal - Able to adjust the plane of focus and only collect light from the specified plane and reject out-of-plane lightLine scan confocal – Background signal from out of focus plane not rejected

What is a sCMOS detector?

Scientific complementary metal-oxide semiconductor (sCMOS) detectors convert light energy to electricity, which is then converted into an image that can be viewed or analyzed. sCMOS cameras have a set resolution based on the number of pixels in the detector, pixel size, and field of view.

How does an imaging system with an sCMOS detector work?

In regards to imaging, sCMOS detectors take an image of the entire field of view all at once with all the pixels, resulting in a static image at one time point. This is ideal for samples for which signal decays over time, such as chemiluminescent Western blots. The static picture ensures the same enzymatic state is captured across the entire blot.

Advantages of using sCMOS as a detector in imaging systems

The primary advantage of sCMOS technology is speed. Due to the array nature of the sensor, sCMOS detectors can gather the information necessary to produce an image at a much faster rate than point detectors. This makes sCMOS detectors ideal for capturing chemiluminescent images which are heavily dependent on time.

Chemiluminescent signal is transient, so even minor differences in imaging times can produce dramatically different results. In situations where time and speed are crucial parameters, sCMOS detectors outperform their competitors.

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Challenges with sCMOS

One challenge of sCMOS detectors is that the maximum resolution and maximum field of view cannot be changed once the system has been designed. For a given detector, there is a tradeoff between resolution and field of view. To obtain high-resolution images, you need to limit your field of view. To image a large field of view (for example, an arrangement of multiple blots), you either need to take a low resolution image of the maximum field of view, or take multiple high resolution images and “stitch” them together using software. Stitching can create artifacts along the border where two images meet.

Should you use sCMOS for chemiluminescent or fluorescent image detection?

Another consideration when using an sCMOS is the camera sensitivity, which is limited by the inherent noise generated by the camera1. Even when cooled, the electronic and dark noise generated by CMOS is higher than 1 photon, making it difficult to reach single photon detection sensitivity. This is typically not an issue in samples with inherent low background, such as chemiluminescence, but it can contribute to reduced sensitivity when detecting fluorescent signals.

In summary, sCMOS detects are excellent for chemiluminescent imaging, for fluorescent imaging when signals are not extremely weak, and when the field of view is relatively small.

What are photodiode detectors?

A photodiode is a semiconductor device that is designed to convert light into an electrical current or voltage. Photodiodes are point detectors.

Photodiode detectors vs sCMOS detectors

Unlike sCMOS cameras, which image an entire field of view at once, photodiodes generate spatial and intensity information for one pixel at a time. Each pixel is detected without the influence of another, improving the signal to noise ratio for fluorescent imaging. The disadvantage of this is that imaging can take longer than with an sCMOS camera. 

What are two kinds of photodiode detectors?

There are two kinds of photodiode detectors typically used in fluorescent biomolecular imagers: PMTs and APDs. APDs and PMTs are used in combination with a scanning mechanism to move the light path laterally relative to the sample for image capture (Figure 1).

How a PMT/APD Detector works
Figure 1. The pinhole aperture in confocal laser scanning allows the detection of only the light coming from the focal plane, eliminating the out-of-focus light from above and below the plane. This optical sectioning capability enhances the resolution and contrast of the obtained images.

Is using photodiodes ideal for detecting chemiluminescence?

Photodiode technology is not ideal for imaging chemiluminescence because the amount of light produced by the enzyme reaction may decrease throughout the length of capture. Stable signals from fluorophores and fluorescent dyes remain constant over longer image acquisitions and are consequently unaffected by this difference.

Using confocal imaging to detect fluorescence

An advantage of point detection is making use of confocal imaging4. The optical design of confocal laser scanners allows light from only the specified focal plane to be collected and rejects out-of-plane fluorescent background (Figure 1). The user may adjust the focal plane to match the height of the sample, for example if the sample is directly on the glass surface of the imaging platform, or if the sample is raised above the surface on a slide or a plate. The laser scanner will only detect signal from the selected plane.

Marina Species Polychaete Worm imaged using Azure Sapphire
Fluorescent image of a polychaete (bristle) worm. Imaged using APD and PMT detectors on the Sapphire.

What is the difference between PMTs and APDs?

How PMTs detect light

PMT stands for photomultiplier tube. A PMT is a highly sensitive detector that is used to measure the intensity of light. They are particularly valuable in situations where very low levels of light need to be detected and amplified. Specifically, PMTs can internally amplify signal (a quality known as “gain”) without noise2. This is crucial for extremely sensitive, low light applications, such as phosphor imaging. PMTs are commonly optimized for the visible spectral range.

How APDs detect light

Avalanche photodiodes (APDs) are also highly sensitivity light detectors. They are typically optimized for detection in the visible to near infrared range, making them optimal for the detection of fluorophores used in Western blotting and many other biological assays.

Similarities between PMTs and APDs

Like PMTs, APDs also provide internal, noiseless gain, enabling them to detect a signal photon2

PMTs and APDs vs. sCMOS

Unlike sCMOS systems, systems with PMTs and APDs offer a wide range of resolutions, unrestricted by the field of view. Image resolution is controlled through the software, and typically can cover a wide range, for example, from 1000 microns to 5 microns. The resolution determines the size of the “point” from which the PMT or APD collects data.

In a sCMOS system, the focal plane is static, so the resulting image will include out of focus fluorescent background along with true signal. This produces an image with higher background and lower contrast. A type of confocal imaging (line-scanned dual-axis confocal) can be performed by a sCMOS system, but there will be significant signal from the background outside the focal plane3 so background is still higher and contrast lower than a point system.

Benefits to using confocal imaging

In addition to its benefits for imaging samples that have different heights off the glass surface, confocal imaging is also useful for imaging of the spatial arrangement and localization of specific targets within a sample in 3D. This makes confocal imaging great for imaging model organisms, like in vivo mice. Shown below are two mice imaged using the Sapphire FL to measure tumor progression of 4T1 cells.

Experimental mice were injected with 4T1 tumor cells expressing RFP, while controls were subjected to RFP-negative 4T1 cells. The mice were imaged at multiple timepoints to monitor tumor growth and progression over time. Tumors were measured at Day 4 and Day 11 post-injection and showed substantial growth over this period. Fluorescent signal from the RFP+ tumors also increased over this time and was captured in vivo using the Sapphire FL
Fluorescently labeled (RFP+) tumors are visible in in vivo images of whole mice captured on the Sapphire FL at 100 µm resolution using the 532 Standard Optical Module with intensity setting 6. The focus height was set to 0.00 mm.

This new application note describes the in vivo imaging of tumors in living mice using the Sapphire FL, including tumor measurement and tracking tumor growth over time.

Imaging systems with both APD and PMT detectors

The Azure Sapphire FL Biomolecular Imager utilizes both APD and PMT detectors depending on the wavelengths of the associated optical modules, ensuring maximum sensitivity and quality. Specifically, its Optical Modules excite below 500 nm and rely on PMT technology. The Optical Modules above 500 nm use APD detectors. As an imaging system, the Sapphire FL supports wavelengths between 375 – 900 nm.

Supporting wavelengths between 375 – 900 nm

The Sapphire FL was designed to be the flexible choice in detection chemistry and samples imaging. It brings precise quantitation of nucleic acids and proteins.
Scientist changing optical modules on the new Azure Sapphire FL
The new Azure Sapphire FL Biomolecular Imager is capable of high-resolution imaging and wide depth of field enable many sample types, including arrays, microarrays, Western blots, tissue slides, and small animals.

While speed of image capture is worth considering when imaging biological samples, it is hardly the most crucial feature of such an imaging system. Much more critical to the data quality are the specificity, sensitivity, and clarity of the images generated.

With the Sapphire FL, you can be sure that each of your fluorescent images will provide the best possible data due largely to its reliance on APD and PMT detectors.

SOURCES

  1. Crisp R. Digital camera design, part 5: Basic noise considerations for CMOS image sensors. EDN Network. Dec 9, 2020. Accessed Oct 3, 2023. https://www.edn.com/digital-camera-design-part-5-basic-noise-considerations-for-cmos-image-sensors/#genecy-interstitial-ad.  
  2. Light detectors (PMTs and APDs): detecting and diagnosing light. FindLight Blog. Updated Sept 19, 2020. Accessed Oct 3, 2023. https://www.findlight.net/blog/light-detectors-detecting-and-diagnosing-light/.  
  3. Wang D, Chen Y, Wang Y, Liu JTC. Comparison of line-scanned and point-scanned dual-axis confocal microscope performance. Opt Lett. 2013;38(24):5280-5283. 
  4. Paddock SW. Principles and practices of laser scanning confocal microscopy. Mol Biotechnol. 2000;16(2):127-149.

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