Single-Molecule Fluorescence Counting With Pulsed Photoactivation
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Solution Overview
Problem
Existing single-molecule fluorescence-based methods for quantifying biomarkers suffer from significant errors due to overcounting or undercounting, particularly from photoblinking events and ambiguous fluorescence signals from closely spaced fluorophores, and lack accurate spatial distribution information.
Innovation Solution
A method involving photoactivatable fluorophores labeled with controlled labeling strategies, using pulsed activation and enhanced photobleaching to distinguish individual fluorophores, and employing specific fluorophore types and intensities to minimize errors and enhance counting accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescence microscopy is used to detect biomarkers, then the overall fluorescence intensity can be measured, but the method is relatively insensitive and gives only a broad estimate of biomarker amount, being unsuitable for low copy numbers
Solution Approach 1:
The patent segments the detection process into multiple imaging cycles with pulsed activation, where fluorophores are activated in controlled bursts rather than continuously. This segmentation allows individual fluorophores to be resolved and counted even at low copy numbers, transforming the undifferentiated fluorescence signal into discrete countable events.
Solution Approach 2:
The patent employs periodic pulsed activation of fluorophores instead of continuous illumination. By activating fluorophores in periodic pulses and measuring fluorescence during each pulse, the method achieves single-molecule sensitivity while maintaining quantitative accuracy, resolving the contradiction between sensitivity and precision.
2Measurement precision
If step-wise photobleaching is used to identify copy number, then individual fluorophores can be resolved, but the method is only suitable where the number of fluorophores is relatively small
Solution Approach 1:
The patent uses periodic pulsed activation to repeatedly bring fluorophores into the active state for detection. This allows the system to track individual fluorophores through multiple activation cycles until they photobleach, providing accurate copy number determination across a wide range of fluorophore numbers rather than being limited to small numbers only.
Solution Approach 2:
The patent implements feedback by monitoring fluorescence intensity changes across multiple imaging cycles and using this information to determine when fluorophores have photobleached. This feedback mechanism enables accurate counting of total fluorophore numbers by tracking the step-wise decrease in signal, adapting to various fluorophore quantities.
3Measurement precision
If photoactivated localisation microscopy (PALM) is used to identify fluorophore positions, then exquisite accuracy in position identification is achieved, but the technique can be relatively slow to implement and presents challenges in achieving accurate quantitative information
Solution Approach 1:
The patent employs periodic pulsed activation with optimized pulse durations and intervals that balance position accuracy with measurement speed. By using shorter, more intense activation pulses rather than prolonged activation, the method achieves sufficient localisation precision while reducing total measurement time, thereby improving productivity without sacrificing essential accuracy.
Solution Approach 2:
The patent optimizes parameters such as activation light intensity, pulse duration, and imaging frame rate to achieve the best compromise between position accuracy and quantification speed. By adjusting these parameters based on the specific experimental requirements, the method can adapt to achieve both high precision and fast throughput.
4Measurement precision
If photoblinking is allowed with gap frames to compensate for overcounting, then some accuracy is improved, but undercounting risk increases when closely-spaced fluorophores are mistakenly identified as photoblinking events
Solution Approach 1:
The patent uses periodic pulsed activation with controlled timing to distinguish between genuine photoblinking events and closely-spaced fluorophores. By activating fluorophores in synchronized pulses and analyzing the temporal pattern of fluorescence appearance and disappearance, the method can reliably differentiate between a single fluorophore blinking and multiple fluorophores activating at different times, thereby maintaining both accuracy and reliability.
Solution Approach 2:
The patent implements feedback mechanisms that monitor fluorescence patterns across multiple cycles and use this information to adjust counting decisions. By analyzing the temporal and spatial correlation of fluorescence events, the system can identify and correct for photoblinking artifacts while avoiding misinterpretation of closely-spaced fluorophores, thus maintaining high reliability.
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
The method provides precise quantitation of biomarker numbers and spatial distribution with reduced ambiguity and increased speed by distinguishing photoblinking events and promoting rapid photobleaching, enhancing the accuracy and efficiency of single-molecule fluorescence microscopy.
Implementation Method 1
a sparse subset of fluorophores is photoactivated from a dark state to an active state using a light source
Implementation Method 2
the activated fluorophores are made to fluoresce using a readout laser beam
Implementation Method 3
the technique relies on being able to resolve individual downward steps in the detected signal intensity as individual fluorophores are photobleached
Data Source
AI summary
The present application discloses single molecule fluorescence methods for quantitating the presence of target molecules in a biological sample. The method involves labelling target molecules of the biological sample with a probe, the probe comprising at least one photoactivatable fluorophore and then imaging the target molecules. The imaging involves carrying out multiple imaging cycles, each imaging cycle having an activation step, an excitation step and a photobleaching step. The application also discloses probes suitable for use in quantitative single molecule fluorescence microscopy assays, as well as diagnostic methods based on quantitating the presence of target molecules.