By Hongbai Lao, Iridian Spectral Technologies

Engineers creating polymerase chain reaction (PCR) instrumentation face unique challenges in both qualitative detection of nucleic acid sequences (using end-point analysis) and quantitative detection of nucleic acid sequences using real-time analysis. Quantitative PCR (qPCR) instruments that operate in real time require the most favorable signal-to-noise ratio possible, combined with the utmost sensitivity.

Given this demand for high sensitivity, the center wavelength (CWL) and bandwidth of excitation and emission bandpass filters used to configure each channel of a qPCR instrument must be optimized based on that instrument’s system configuration, as well as how many channels it needs to support. Specifically, the CWL and bandwidth for each filter need to maximize both the excitation and emission signals while minimizing cross-talk between signals occupying the same channel, as well as quashing interference with adjacent signals. 

The Sum of Its Parts

Just as each qPCR instrument is the sum of its channels working harmoniously, the optical filters guiding each channel are best considered as a group, versus individual components. As with other fluorescence applications, the excitation bandpass filter, the emission bandpass filter, and the dichroic filter on each channel perform distinct roles that are intertwined: the excitation filter transmits the desired illumination wavelengths and blocks unwanted signals. The emission filter allows only specific wavelengths associated with the desired fluorophore through to the detector, and the dichroic filter is used to steer the different excitation and emission wavelength ranges on to different beam paths. The performance of each filter affects, and is affected by, its associates.

As with many applications, quality qPCR instrumentation is served best by components that maximize performance at an acceptable price point. Since each channel on a qPCR instrument can be so close to its neighbors in wavelength, technological challenges arise, particularly as more channels are added. Costs to resolve these issues can quickly grow out of hand — particularly when considering most bandpass and dichroic filters for these instruments require a custom design, integrating certain fluorochromes or wavelengths.

Overlap between the excitation signal and the emission signal will interfere with detection of the emission signal. Thus, it is vital to use bandpass filters operating with a very steep edge, resulting in better signal isolation due to high transmission on the passband and deep blocking outside the passband. Standard, commercial off-the-shelf filters might be adequate in some of these applications but, in our experience, more than 90% of optical filters in qPCR instruments require some form of customization to fulfill their intended price/performance balance.

For this reason, early engagement with a vendor is critical. It prevents a qPCR instrument maker from choosing a part that may be inappropriate for the project, or miscalculating a part’s price/performance balance. Ideally, early engagement with a vendor secures valuable input before an  instrument’s design is complete, resulting in the best technical and commercial optical filter (or series of filters) being applied based on the customer’s requirement/ instrument configuration. Typically, wavelength range and CWL comprise the most common tweaks applied to
qPCR filters. However, bandwidth, transmission level, and refraction level remain important parameters to optimize.

How Iridian Can Help

It’s easy to say instrument makers need robust, reliable solutions that are also cost effective. However, the reality is design and component costs, quality considerations, on-time delivery, and simply locating a high-performance filter partner that works to understand and implement your vision all present challenges. The size of each challenge depends on the individual project: one instrument maker may be struggling to meet the technical demands associated with its product, while another may have difficulty locating a vendor whose supply chain can scale from prototype to volume. 

From a technical capability standpoint, Iridian ultra-performance filters are designed specifically for LED-based, real-time PCR applications. Our standard filter solutions are compatible with common excitation sources and fluorophores, but we specialize in working with customers to create and implement customized filter solutions that maximize their instrument’s effectiveness. 

For example, Iridian’s ultra PCR fluorescence filter series is designed to provide a high signal-to-noise ratio of the probes contained in real-time PCR samples when illuminated with an LED light source. Customization of the specific spectral characteristics or physical filter size can be achieved by Iridian’s engineers with a turnaround time of about one week for custom biomedical filter designs. 

Each of these custom filters — excitation, emission, and dichroic — provides more signal, with less background, to fluorescence instrument users and OEMs. They provide high transmission within the passband and deep blocking outside the passband: Iridian’s excitation and emission filters feature steep edge transition (< 0.5% from 50% transmission to blocking OD5), whereas our dichroic filter features < 1% slope from 10% to 90% transmission point. 

Perhaps most important, Iridian PCR filter solutions can accompany your project from “good idea” to “good product.” These filters are scalable from initial prototype through to high-volume manufacture, with no compromise in quality. 

An ISO 9001-certified vendor, Iridian’s facilities and processes also stand up to the medical industry’s regulatory scrutiny. We operate a stable, high-yield, and high-volume filter production process, ensuring a reliable supply chain for our customers. Further, Iridian’s meticulous testing and process control ensure consistent filter performance from filter to filter and batch to batch, plus full traceability for all products.

Conclusions

Iridian Spectral Technologies has built its expertise across a diverse set of applications, ranging from fiber optic communications and optical spectroscopy to 3D entertainment and aerospace instrumentation. This deep experience has given us insight into the regulatory, technical, and logistical challenges faced daily by our customers.

Nonetheless, qPCR instrument-related challenges can be among the most daunting, burdened by tough deadlines, budgets that offer little wiggle room, and expectations for flawless functionality. A combination of Iridian’s excitation, emission, and dichroic filters serving each channel of a qPCR instrument assuages many of these concerns, providing the utmost sensitivity while preserving a desirable signal-to-noise ratio.

For more information, visit www.iridian.ca and follow @IridianSpectral on Twitter.