MERFISH Nucleic Acid Amplification With Saturatable Signal Control
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Solution Overview
Problem
Existing multiplexed single-molecule RNA imaging methods face challenges such as low photon counts, signal overlap due to light scattering and sample autofluorescence, inefficient signal amplification, and difficulty in extending amplification to multiple molecular signals without introducing brightness variation or increasing signal size, which limits imaging throughput and accuracy.
Innovation Solution
A method involving the use of primary and secondary amplifier nucleic acids that bind to nucleic acid probes, with a saturatable binding capacity, to create codewords based on fluorescence distribution, allowing for efficient and controlled signal amplification, minimizing brightness variation and spot size, and enabling error correction to identify correct targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If signal amplification is applied to increase photon counts, then imaging throughput and detection sensitivity are improved, but signal brightness variation and physical extent of signal increase, causing overlap between adjacent molecules
Solution Approach 1:
The amplification system is segmented into multiple discrete components: primary amplifier nucleic acids that bind to probes, secondary amplifier nucleic acids that bind to primary amplifiers, and fluorescent markers that bind to secondary amplifiers. This segmentation creates a modular, controlled amplification cascade that limits signal spread and maintains spatial resolution while achieving sufficient photon counts for detection.
Solution Approach 2:
Primary amplifier nucleic acids serve as intermediaries between the target probe and the fluorescent signal. These intermediaries provide a controlled binding interface that limits the physical extent of signal amplification while enabling sufficient signal accumulation through the binding of multiple secondary amplifiers and fluorescent markers.
2Adaptability or versatility
If amplification methods are extended to multiple molecular signals, then multiplexing capability is improved, but complexity of the amplification system increases
Solution Approach 1:
The amplification system uses universal primary and secondary amplifier nucleic acids that can bind to multiple different probe types through complementary base pairing. This universality allows the same amplification machinery to amplify signals from multiple different molecular targets simultaneously, enabling multiplexing without requiring separate amplification systems for each target.
Solution Approach 2:
Different molecular signals are distinguished by changing the nucleic acid sequence parameters of the probes and their corresponding amplifiers. By varying the sequence complementarity between probes and amplifiers, the system can selectively amplify multiple different targets using the same fundamental amplification mechanism, maintaining system simplicity while achieving multiplexing.
3Illumination intensity
If more amplifier nucleic acids are used to increase signal intensity, then photon counts are improved, but the time required for sample preparation increases
Solution Approach 1:
The amplification process uses periodic binding cycles where primary amplifiers bind to probes, then secondary amplifiers bind to primary amplifiers, and finally fluorescent markers bind to secondary amplifiers. This periodic, staged binding approach allows efficient accumulation of signal intensity through multiple binding events rather than requiring prolonged continuous incubation, reducing overall preparation time while achieving high photon counts.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enhances imaging throughput by ensuring uniform signal brightness and spot size, improving target identification and resolution, particularly in complex biological samples, while allowing rapid and efficient amplification of multiple molecular signals.
Implementation Method 1
exposing the sample to nucleic acid probes, exposing the nucleic acid probes to primary amplifier nucleic acids able to bind to the nucleic acid probes
Implementation Method 2
determining a distribution of the nucleic acid probes within the sample using fluorescence
Data Source
AI summary
The present invention generally relates to systems and methods for imaging or determining nucleic acids in cells or other samples. In some cases, the transcriptome of a cell may be determined. Certain embodiments are generally directed to determining nucleic acids and other targets in a sample at relatively high resolutions. For instance, nucleic acid probes may be applied to sample, and binding of the nucleic acid probes to a target may be amplified using primary and secondary amplifier nucleic acids. In some cases, there is a maximum number of amplifier nucleic acids that can be bound to a target, e.g., the binding is saturatable, and cannot grow indefinitely, even in the presence of abundant reagents. This may be advantageous, for example, for controlling the brightness of each binding event, controlling the size of the amplified regions (e.g., during imaging), and/or for limiting the degree of amplification noise (i.e. the final variation in amplified signal from molecule to molecule), etc. In addition, in some embodiments, the primary and/or secondary amplifier nucleic acids may be formed from only 3 of the 4 naturally-occurring nucleotides, which may result in less secondary structure, faster binding rates, etc. These properties can in some cases facilitate the rapid design of multiple orthogonal amplification sequences, allowing the extension of such an approach to many distinct molecular targets.