FRET-Oligonucleotide Kinetics for Multiplex Fluorescence Microscopy
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Solution Overview
Problem
Standard fluorescence microscopy is limited by spectral overlap, allowing only four to five distinct fluorophores to be imaged simultaneously due to overlapping excitation/emission wavelengths, and techniques like DNA-PAINT suffer from high background fluorescence and slow image acquisition.
Innovation Solution
The method employs FRET-oligonucleotides that emit fluorescence only when hybridized, allowing for unique fluorescent kinetic profiles to be generated, enabling multiplexed imaging without spectral overlap constraints and reducing background fluorescence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If standard fluorescent microscopy uses multiple fluorophores to image different channels, then more components can be visualized simultaneously, but spectral overlap limits the number of distinct channels to four to five
Solution Approach 1:
The patent changes the fundamental parameter of fluorescence detection from relying on spectral wavelength differentiation to relying on temporal kinetics differentiation. By using FRET-oligonucleotides that exhibit transient binding behavior with distinct kinetic profiles (different on/off rates), the system can distinguish between multiple targets without requiring spectrally distinct fluorophores, thereby overcoming the spectral overlap limitation
Solution Approach 2:
The patent introduces FRET-oligonucleotides as intermediary molecules that mediate between the target analytes and the detection system. These oligonucleotides contain fluorophores and quenchers that undergo FRET interactions only during transient binding events, converting the detection mechanism from direct fluorophore emission to FRET-mediated transient signal generation, which enables multiplexing through kinetic differentiation rather than spectral separation
2Measurement precision
If DNA-PAINT technique uses fluorescently labelled imager probes to achieve super-resolution, then resolution below diffraction limit is achieved, but high background fluorescence from unbound probes occurs
Solution Approach 1:
The patent converts the potentially harmful continuous fluorescence emission into a beneficial transient signal by designing FRET-oligonucleotides where the fluorophore is permanently quenched until transient binding occurs. The binding event itself triggers the beneficial signal (fluorescence turn-on), while the unbound state produces no background, thus converting the DNA-PAINT background problem into a signal-on-only detection scheme
Solution Approach 2:
The patent extracts the harmful background fluorescence component from the detection system by using FRET quenching mechanisms. The quencher is integrated into the FRET-oligonucleotide structure, actively removing (taking out) the harmful continuous emission that plagues DNA-PAINT, allowing only the beneficial transient binding signals to reach the detector
3Object-generated harmful factors
If concentration of imager probes is reduced to lower background in DNA-PAINT, then background fluorescence decreases, but image acquisition speed becomes slow
Solution Approach 1:
The patent inverts the conventional DNA-PAINT approach by using signal-on transient binding instead of signal-off continuous emission. Rather than trying to reduce background from continuous emission, the system design ensures no background exists and only transient binding events generate signals, eliminating the need to balance concentration against background and enabling fast acquisition without background compromise
4Device complexity
If SMLM is limited to maximum of three laser wavelengths for excitation, then equipment complexity is reduced, but ability to image more than three modalities is prevented
Solution Approach 1:
The patent makes the single excitation wavelength universal by designing FRET-oligonucleotides that all respond to the same excitation wavelength but produce distinguishable kinetic responses. Each oligonucleotide variant maintains the same fluorophore-quencher architecture and excitation characteristics, allowing a single laser to excite all targets while kinetic analysis distinguishes between them, thus achieving multi-modality imaging without multi-wavelength equipment
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 achieves high-resolution, low-background fluorescence microscopy capable of multiplexing up to 25 distinct channels, enhancing imaging speed and information density.
Implementation Method 1
The method employs FRET-oligonucleotides that emit fluorescence only when hybridized
Implementation Method 2
Fluorescence microscopy is a staple component of life science and pharmaceutical research
Implementation Method 3
use fluorescently tagged DNA probes that reversibly hybridise to complementary docking strands
Data Source
AI summary
Provided is a method for multiplexed fluorescence microscopy comprising contacting a fixed sample with a set of binding agent-T-oligonucleotide conjugates to allow the binding agents to bind to any binding partners present in the sample, wherein the set comprises a plurality of binding agents having different specificities and the sequence of the T-oligonucleotide is unique to the binding agent to which it is conjugated, contacting the sample and any bound binding agents resulting from step a with a FRET-oligonucleotide, illuminating the sample with a wavelength to cause excitation of the FRET-oligonucleotide's emitter molecule, and observing the fluorescent kinetic profile of the sample at the FRET-oligonucleotide emitter molecule's emission wavelength at one or more pixels over time, wherein the FRET-oligonucleotide can hybridise to multiple T-oligonucleotides in the set, to form multiple pairs, and wherein the dissociation and reassociation between each different pair generates a fluorescent kinetic profile that is unique within that set to that pair. Also provided are associated kits, sets of binding agent T-oligonucleotide conjugates and corresponding FRET-oligonucleotides, and methods of designing such sets.