Optics and photonics technologies are enabling modern-day medical products for both diagnostic testing and treatment. In diagnostics, one of the most ubiquitous and impactful tools is the polymerase chain reaction (PCR) instrument, which plays a pivotal role in diagnosing infectious diseases across various end applications and has played a central role in understanding the spread of SARS-Cov-2, the coronavirus that causes COVID-19.

One of the primary optical methods for rapid diagnostic testing is quantitative polymerase chain reaction (qPCR), which utilizes fluorescence for detection. Here, disease molecules of interest are labeled with fluorescent dyes using antibody chemistry, illuminated with specific wavelengths of light (excitation/absorption wavelength), and then emit red-shifted fluorescence light (emission wavelength). PCR instruments rely on carefully designed optics, optical filters, and photodetectors to enable accurate and reliable testing. The strength of the emission wavelength allows the PCR screening system to determine whether the molecules of interest are present and subsequently indicate a positive or negative sample. Figure 1 shows the layout of a typical qPCR diagnostic system with the subsystems consisting of the illumination source, illumination optics, excitation filters, collection optics, emission filters, and detection optics.

Given the bandwidth of the excitation wavelengths for the tagged disease molecules and potentially low emission signal, high signal-to-noise (SNR) detection in the system is critical to test success. Thus, the success of qPCR as an accurate and reliable diagnostic test method of low sample volumes is related to the design and selection of the light source that matches the fluorophores used in the test assay and yields high signal-to-noise detection.

Therefore, instrument developers must consider the illumination system design, including light source selection and delivery optics design, to achieve this goal. LEDs are an increasingly popular choice, over lamps and lasers, for the illumination source due to their cost and widespread availability, making them easily accessible. However, due to LED’s unique properties, specific design considerations are required compared to other light sources. These design considerations evolve for multi-channel qPCR instruments, which can run multiple tests using multiple fluorophore labels from the same sample. It is necessary to design the complete optical path to minimize signal crosstalk between detection channels.

Table 1. A comparison of the pros and cons of the three commonly used illumination sources in fluorescence imaging.

Illumination Source Selection

When starting a PCR instrument design, a team chooses a test assay fluorescent dye based on several biological factors, and then the next step is to select the optimal illumination source. Two factors that drive the illumination source selection are its peak emission wavelength and the output power. These two factors should overlap with the peak absorption wavelength of the fluorophore and the amount of energy required to excite it efficiently, creating a high number of fluorescence emission photons for detection. Therefore, to achieve optimal fluorescence and subsequently SNR, it is best to choose a light source with an emission wavelength that matches the peak absorption wavelength of the fluorophore or is close to it.

The second consideration for source selection is the intensity required to excite the fluorophore. The source should have high coupling efficiency to the delivery optics to deliver the optimum irradiance to the sample. Careful consideration is required to optimize the illumination optics for high light collection and direction to the sample, typically a microfluidic cartridge or plastic vial. Table 1 compares the pros and cons of the three commonly used illumination sources in fluorescence imaging. From the data presented in Table 1, it is possible to conclude that LEDs and lasers have few advantages over arc lamps.

LEDs as illumination sources for qPCR diagnostics are a great choice. They are available over a wide range of wavelengths and are readily available at absorption wavelengths of commonly used fluorophores. In addition, LEDs last longer than lasers, are less expensive, have fewer eye safety considerations, and are compact, making cell phone-sized qPCR devices possible.

Delivery Optics Design

The purpose of illumination optics is to collect the light from the source and transfer it to the sample plane. The transfer of energy from source to sample is governed by an optical principle known as the Lagrange invariant, given by the following equation:

Fig. 2 - A graphical representation of the Lagrange invariant.

The law states that the collected cone of light from the source is conserved throughout the optical pathway. In this equation, h is the size of the emitter/spot, and nsin is the numerical aperture. Figure 2 shows a graphical representation of the Lagrange invariant.

All real light sources have finite extent and divergence. A challenge with LEDs, compared to lasers, is the relatively high light output divergence and larger emission area, which results in low collection efficiency. However, the difficulties with divergence are readily solvable with the right optical design and selection of illumination optics. The illumination optics design needs to capture the full extent of the LED area and cone angle to ensure the maximum amount of the source radiation to the sample plane. This capture generally is accomplished by using aspheric condenser lenses or TIR lenses.

Fig. 3 - Two common illumination architectures are used in qPCR instruments. Figure 1 shows the illumination and excitation paths separated with a dichroic beam splitter. This figure illustrates the architecture preferred for applications that require multiple target detection channels (i.e., up to 4–6 channels), since this type of system layout is not dependent on dichroic filters.

Figures 1 and 3 show two common illumination architectures used in qPCR instruments. In Figure 1, the illumination and excitation paths are separated with a dichroic beam splitter. This architecture is generally used when it is necessary to have a tightly focused spot at the sample and in instruments with one or two detection channels to reduce the complexity of the dichroic filter. However, for applications that require multiple target detection channels (i.e., up to 4 to 6 channels), the architecture shown in Figure 3 is preferred since this type of system layout is not dependent on dichroic filters. The primary drawback of the second system configuration is illumination uniformity across the sample. Additionally, in some cases, the second configuration requires further optimization to minimize stray light, especially if the instrument developer chooses to forgo collimation optics in front of the LED to save on the cost of goods. Designing the optical path to minimize the effects of stray light from contaminating the detection signal and hence the accuracy of the test should also be considered.

Finally, the thermal effect impact from the LED thermal cycler module is an important design consideration. The temperature change can cause the emission wavelength of the LED to shift, leading to nonoptimal excitation of the fluorophore and consecutive nonoptimal emission of red-shift fluorescence. Overall, “wandering” LED emission wavelengths due to thermal effects lead to reduced fluorescence signal and impact the test assay’s reliability and accuracy.


Developing the right product for the use case and achieving the needed performance requires a comprehensive understanding of the complete system requirements and how to balance design parameters to achieve the desired result. For fluorescence qPCR diagnostics, this starts with illumination source selection and optimizing the optical pathway to ensure that the test results from the fully commercialized product can be trusted time and again. Taking a step further to select an LED light source can save money and time due to the ease of access with specific wavelengths and powers, as long as the team has the skills necessary to complete an efficient and optimized illumination optics design.

This article was written by Rekha Doshi, Principal Optical Engineer, and Madilyn Beckman, Business Development Manager at Gray Optics, Portland, ME. For more information, visit here .