Engineers creating polymerase chain reaction (PCR) instrumentation face unique challenges in both the quantitative detection of nucleic acid sequences using real-time analysis and the qualitative detection of nucleic acid sequences using end-point analysis.
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Quantitative PCR (qPCR) instruments that operate in real-time need high sensitivity and the most favorable signal-to-noise ratio possible.
The bandwidth of excitation, center wavelength (CWL) and emission bandpass filters utilized to configure each channel of a qPCR instrument must all be optimized based on that instrument’s system configuration, in addition to how many channels it needs to support given this demand for high sensitivity.
The CWL and bandwidth for each filter must maximize both the emission and excitation signals while minimizing cross-talk between signals using the same channel, in addition to quashing interference with adjacent signals.
The sum of its parts
The optical filters guiding each channel are best thought of as a group, not as individual components, just as each qPCR instrument is the sum of its channels working together.
The emission bandpass filter, the excitation bandpass filter and the dichroic filter on each channel perform distinct roles which are intertwined, as with other fluorescence applications. The excitation filter blocks unwanted signals and transmits the desired illumination wavelengths.
The dichroic filter is used to steer the different excitation and emission wavelength ranges onto different beam paths and the emission filter only permits specific wavelengths which are associated with the desired fluorophore through to the detector. The performance of each filter is influenced by, and influences, the other.
Quality qPCR instrumentation is served best by components that maximize performance at an acceptable price point, as with most applications. Technological challenges arise, as each channel on a qPCR instrument can be so close to its neighbors in wavelength, particularly as more channels are added.
Costs to resolve these issues can get out of hand quickly, especially when considering that the majority of bandpass and dichroic filters for these instruments need a custom design, integrating certain wavelengths of fluorochromes.
Any overlap between the emission signal and the excitation signal will interfere with the detection of the emission signal. So, it is crucial to utilize bandpass filters operating with a very steep edge, leading to better signal isolation because of deep blocking outside the passband and high transmission on the passband.
In some of these applications, standard, commercial off-the-shelf filters may be enough, but to fulfill their intended price/performance balance, over 90% of optical filters in qPCR instruments need some type of customization.
It is for this reason that early engagement with a vendor is vital. It stops a qPCR instrument maker from selecting a part that may be inappropriate for the project or miscalculating a part’s price/performance balance.
Early engagement with a vendor ideally secures valuable input prior to an instrument’s design being complete, leading to the best technical and commercial optical filter (or series of filters) being applied based on the customer’s requirement/instrument configuration.
Usually, wavelength range and CWL comprise the most common tweaks applied to
qPCR filters. Yet, transmission level, bandwidth and refraction level remain critical parameters to optimize.
How Iridian can help
Quality considerations, design and component costs, on-time delivery and locating a high-performance filter partner who works to understand and implement your vision all present challenges.
The size of each challenge is dependent on the individual project: one may have difficulty locating a vendor whose supply chain can scale from prototype to volume; one instrument maker could be struggling to meet the technical demands associated with its product.
From a technical capability standpoint, Iridian ultra-performance filters are designed for real-time, LED-based, PCR applications. Iridian’s standard filter solutions are compatible with common excitation sources and fluorophores, but they specialize in working with customers to create and implement customized filter solutions to maximize their instrument’s effectiveness.
For instance, Iridian’s ultra PCR fluorescence filter series is designed to supply a high signal-to-noise ratio of the probes contained in real-time PCR samples when illuminated with an LED light source.
Iridian’s engineers can achieve customization of the specific spectral characteristics or physical filter size with a turnaround time of about one week for custom biomedical filter designs. Each of these custom filters — excitation, emission and dichroic — supplies more signal, with less background, to fluorescence instrument users and OEMs.
They supply 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 their dichroic filter features <1% slope from 10% to 90% transmission point.
Iridian PCR filter solutions can also accompany your project from “good idea” to “good product.” These filters are scalable from the initial prototype through to high-volume manufacture, with no compromise in quality.
Iridian’s facilities and processes also stand up to the medical industry’s regulatory scrutiny, an ISO 9001-certified vendor. They operate a high-yield, stable and high-volume filter production process, to provide a reliable supply chain for their customers.
Iridian’s meticulous process control and testing also 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 over a wide range of applications, ranging from 3D entertainment and aerospace instrumentation to fiber optic communications and optical spectroscopy. This deep experience has provided insight into the technical, regulatory and logistical challenges faced daily by customers.
Nonetheless, qPCR instrument-related challenges can be among the most daunting, burdened by budgets that provide little wiggle room, tough deadlines and expectations for flawless functionality.
A combination of Iridian’s emission, excitation and dichroic filters serving each channel of a qPCR instrument assuages a number of these concerns, supplying the utmost sensitivity while preserving a desirable signal-to-noise ratio.
About Iridian Spectral Technologies Ltd.
Iridian Spectral Technologies Ltd. has maintained itself as a world leader in custom optical filter solutions since its founding in 1998 and we pride ourselves on supporting our customers optical products across a wide range of geographic and market areas. We have at our core the belief that if our customers succeed we succeed as well.
Our team at Iridan uses decades of filter design and manufacturing experience to provide support throughout a product’s life cycle; from initial prototyping to high volume manufacturing and our automated production facility is able to ensure that the most cost effective optical solutions are found for any application.
Iridian’s highly advanced, proprietary thin-film design deposition and manufacturing technology delivers durable high performance filter optics for use in telecommunications and data centers, Raman and fluorescence spectroscopy, mid IR applications such as gas sensors, as well as astronomy and aerospace.
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