Volpi has developed proprietary frameworks and leveraged its expertise in dPCR to address the technical challenges of finding target molecules quickly in a sample. The goal of dPCR is to accurately count the number of partitions that contain the target molecules, but non-uniform target amplification and fluorescence detection system issues can complicate this process. By utilizing the proprietary fluorescence design framework, Volpi reduces prototyping iterations and accelerates the development of assays.
Finding target molecules quickly in a sample is a challenging task. There are many technical challenges and pitfalls on the way to quickly getting a consistently reliable result in dPCR testing.
Volpi has approached and resolved these challenges by deploying ingenuity and proprietary frameworks.
In dPCR, the sample containing the nucleic acid to be detected (target) is diluted and divided into thousands to millions of partitions. Each partition contains one or no target. The goal is to accurately count how many partitions are positive (presence of target) and negative (absence of target) to derive the sample’s absolute number of target molecules. To detect the presence or absence of target molecules in each partition, the target is amplified using routine PCR to a level that can be detected using fluorescence.
That sounds rather trivial: positive partitions will appear bright and negative partitions will be dark, right?
Well, there are many reasons why a negative partition can give a brighter signal than a positive partition. These partitions that cannot be easily distinguished are what we call “rain.” The rain is particularly a problem at low concentrations, so it can be like trying to find a needle in a haystack.
Source: https://definetherain.org.uk/
The root causes for this rain can originate from:
1. Non-uniform partitions’ volume (e.g., microfluidic plate fabrication)
2. Non-uniform target amplification and non-specific amplification (biochemistry)
3. The fluorescence detection system
In planar imaging methods, non-uniform fluorescence detection sensitivity and field curvature can make a positive partition in a corner dimmer than a negative partition in the center. The bright signal from a partition can be spilled over to a neighboring negative partition and significantly increase its signal (e.g., point spread function of the imaging optics). In multiplexed assays, spectral crosstalk can significantly increase the signal of negative partitions and contribute to rain. These examples clearly illustrate the critical role of the optics design in minimizing rain.
Light gray particles appear positive before spectral crosstalk compensation and negative after compensation. A small spectral shift of a few nm of the filters or dye spectra is sufficient to induce such a dramatic effect, especially for highly multiplexed solutions. Volpi’s proprietary design framework enables it to hit the requirements for low spectral crosstalk at the first design iteration.
It’s not enough to find the needle in the haystack; we want to find it as fast as possible. For planar imaging methods where entire plates are scanned, the Volpi approach is to analyze the time budget and optimize along three directions: i) maximizing the field of view to reduce the total number of images to take, ii) minimizing the exposure time and iii) reducing the dead time (the time between images). The first success factor is understanding what an adequate image requires in terms of resolution and signal-to-noise ratio for the algorithm to find the partitions and accurately measure their fluorescence signals. A second factor is an expertise in the cost-efficient design of imaging systems with high light throughput. A third success factor is a fast and accurate mechatronics. Volpi develops and manufactures calibration artifacts to help ensure that the performances remain constant over the instrument’s lifetime. Fluorescence Calibration Tools
Example of Volpi’s bespoke control PCBAs with firmware for driving stages.
What sets Volpi apart?
Volpi is more than “a partner for the optics.” We deliver working plug-and-play solutions – a complete imaging system that includes optics, mechatronics and software.
Our solution is plug and play because the critical-to-assay functions and interfaces are thoroughly tested at our plants before being shipped to our customers.
Click here to learn more about our plug-and-play, 6-color fluorescence imaging solution for QIACuity.
Click here to learn more about our plug-and-play, 6-color plate reader solution for Stilla.
Quality – we speak your language and can translate your needs into valid technical requirements to ensure we develop the right solution for you. With our proven expertise in dPCR, we can develop and manufacture plug-and-play systems that will find the needle in the haystack, and do so quickly. We know how to develop, build and test to guarantee reliable results.
Timeline – we have developed a set of reusable assets to leverage to reduce your time to market. We start from a strong base with our Light Engine, Imaging Systems, Stage, Driver, Control Electronics and Firmware solutions. Volpi can provide early, minimal viable solutions to your team to accelerate the development. of your assays. With our fluorescence design framework that includes proprietary characterization instrumentation and models, we reduce the total iterations during prototyping and meet the targeted performances much faster than alternative solutions.
Cost – we are an experienced system integrator that uses system engineering principles to identify the best system architecture to deliver the highest cost-to-performance solution.
Alexandre Larmagnac, Ph.D.
Head of Applications and Support