Microfluidic Cycle Multiplexing for In Situ Imaging
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Solution Overview
Problem
Current in situ imaging techniques for biological samples by cycle multiplexing face limitations in high-throughput, sensitivity, and precision due to long protocol durations, tissue degradation, and localization errors, especially in diagnostic applications, where manual handling and repeated mounting/demounting of samples lead to reproducibility issues and sample damage from fluorophore exposure.
Innovation Solution
A method utilizing a microfluidic device with a sample support for continuous, controlled flow of imaging probes within a microfluidic chamber, avoiding sample demounting and using an imaging buffer with antioxidants to prevent cross-linking and enhance reagent elution, allowing for rapid, sensitive, and precise multi-molecular read-outs without degrading sample integrity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If manual handling and repeated mounting/demounting of samples is used in cycle multiplexing, then flexibility in reagent application is improved, but sample integrity deteriorates and analysis time increases
Solution Approach 1:
The patent combines multiple functions (sample mounting, reagent application, imaging, and washing) into a single integrated microfluidic device. The sample remains continuously mounted on the sample support throughout all cycling operations, eliminating repeated demounting and remounting operations while maintaining full operational flexibility through fluidic control.
Solution Approach 2:
The microfluidic device performs multiple functions within a single system: it mounts the sample, delivers various reagents (primary antibodies, secondary antibodies, fluorophores, washing buffers), controls imaging cycles, and removes materials. This multi-functional integration maintains operational flexibility while preserving sample integrity through continuous mounting.
2Measurement precision
If prolonged protocol durations are used to achieve complete staining and imaging cycles, then measurement precision is improved, but tissue degradation increases
Solution Approach 1:
The system maintains continuous useful action through automated cyclic operations where reagent application, washing, and imaging occur in seamless succession without manual intervention delays. The microfluidic device continuously flows reagents through the sample, eliminating idle time between steps while ensuring complete staining and imaging for accurate quantification.
Solution Approach 2:
The patent employs periodic cyclic action with automated repetition of staining and imaging cycles. Each cycle includes systematic steps (reagent application, incubation, washing, imaging) that repeat multiple times with different reagents. This periodic automation reduces total protocol duration by eliminating manual handling time while maintaining measurement precision through complete cyclic operations.
3Measurement precision
If fluorophores are applied at high labeling densities to increase signal output, then detection sensitivity is improved, but self-quenching behavior increases
Solution Approach 1:
The system applies preliminary blocking treatment before fluorophore application to prevent non-specific binding and optimize fluorophore distribution. This preliminary action ensures that fluorophores bind specifically to target antigens rather than accumulating in non-specific locations, maintaining high signal output without self-quenching even at high labeling densities.
Solution Approach 2:
The patent employs dynamic control of fluorophore application through multiple cyclic steps with varying conditions. Instead of a single high-density application, the system uses sequential cycles with controlled fluorophore concentrations and incubation times, optimizing signal output while preventing self-quenching through dynamic parameter adjustment.
4Adaptability or versatility
If spectral multiplexing with multiple parallel stains is used to increase molecular readouts, then multiplexing capability is improved, but device performance deteriorates due to crosstalk
Solution Approach 1:
The patent segments the multiplexing process into distinct temporal cycles, each dedicated to a specific stain application and imaging sequence. Instead of attempting to capture all spectral information simultaneously (which causes crosstalk), the system separates measurements in time by sequentially applying different primary antibodies, secondary antibodies, and fluorophores across multiple cycles, achieving high multiplexing capability with clear signal separation.
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
This approach significantly reduces analysis time, maintains sample integrity, and increases the number of labeling cycles while preventing photo-induced alterations, enabling high-throughput, reliable, and precise molecular profiling with improved reproducibility and sensitivity.
Implementation Method 1
a microfluidic device comprising a microfluidic chamber, at least one fluid inlet at one end of said microfluidic chamber and at least one fluid outlet at another end of said microfluidic chamber configured to conduct a fluid supplied from a fluid feeding system under pressure through the microfluidic chamber for advective transport of fluidic substances and reagents inside said microfluidic chamber
Implementation Method 2
use of an imaging buffer with antioxidants to prevent cross-linking and enhance reagent elution, allowing for rapid, sensitive, and precise multi-molecular read-outs without degrading sample integrity
Data Source
AI summary
The invention relates to a method for in situ imaging of samples by cycle multiplexing that enables imaging of various molecular targets through multi-molecular read-outs on the same sample in a rapid, highly sensitive and reliable manner. The invention is further related to imaging buffers preventing the degradation of the sample and of the imaging reagents, which are particularly useful in a method of in situ imaging of samples by cycle multiplexing according to the invention.