3D Single Molecule Tracking via Confocal Spatial Filtering

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Solution Overview

Problem

Current methods for tracking single molecules in three dimensions are limited by their inability to effectively observe molecules beyond a certain distance from an interface or require extensive time to read out CCD chips, leading to noise and difficulty in tracking faint fluorescent particles.

Innovation Solution

An apparatus using a light source, objective lens, dichroic element, spatial filters, and single photon-counting avalanche photodiodes to create a confocal optical detection volume, allowing for precise tracking of molecules in three dimensions by monitoring light-based responses and adjusting the position of the molecule within the optical probe volume.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If TIR excitation is used for 3D tracking, then measurement precision is improved, but the observation range is limited to within about 100 nm of an interface

Engineering Contradiction:

Improvez-position measurement precisionVSAvoidobservation range

Core Design Contradiction:

Measurement precisionVSAdaptability or versatility

Solution Approach 1:

The patent changes the excitation method from TIR to confocal excitation, and changes the detection method from intensity variation to photon counting with spatial filtering. This allows extended observation range beyond 100 nm while maintaining precision through the confocal detection volume and single-photon sensitivity

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent introduces spatial filters (pinholes) as intermediaries to define a confocal detection volume. This mediator enables precise 3D localization by blocking out-of-focus photons while allowing in-focus photons to reach the detector, extending the usable range beyond TIR limitations

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If two separate image planes are created to extend depth of focus, then observation range is improved, but reading out the entire CCD chip takes substantial time and introduces noise

Engineering Contradiction:

Improvedepth of focus rangeVSAvoidimage readout time

Core Design Contradiction:

Adaptability or versatilityVSLoss of time

Solution Approach 1:

The patent extracts only the relevant signal from the entire image plane by using spatial filters to define a small confocal detection volume. Instead of reading out the entire CCD chip, only photons within the confocal volume reach the detector, dramatically reducing readout time and noise while maintaining extended depth of focus capability

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent replaces the mechanical/electronic readout of entire CCD chips with optical spatial filtering and single-photon detection. This substitution eliminates the time-consuming charge sweeping process and enables rapid tracking of molecules over extended z-ranges

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Speed

If rapid sweeping of excitation laser is used to track molecules, then tracking speed is improved, but the duty cycle is low and makes tracking faint particles difficult

Engineering Contradiction:

Improvetracking speedVSAvoidtracking reliability for faint particles

Core Design Contradiction:

SpeedVSReliability

Solution Approach 1:

The patent replaces the mechanical sweeping motion of the excitation laser with a stationary confocal excitation volume. Combined with single-photon counting detection, this eliminates the duty cycle problem and enables reliable tracking of faint fluorescent particles while maintaining high tracking speed through electronic detection

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

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

Enables precise and extended tracking of single molecules in three dimensions, increasing observation time and precision, and allowing the study of complex biological processes like intracellular dynamics and protein trafficking without the limitations of previous methods.

Implementation Method 1

a light source for emitting a light beam capable of inducing a measurable light-based response from a chosen molecule or particle in the sample

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Implementation Method 2

at least one spatial filter at the at least one image plane for selecting a portion of the light based response and defining an optical probe volume for that portion of the light based response

Methodology Applied

Scientific EffectOptical sectioning: Filter (optical)

Implementation Method 3

a dichroic element for directing the light beam from the light source to the objective lens

Methodology Applied

Scientific EffectDichroic filtering: Dichroic Filter

Data Source

PatentUS7498551B2Apparatus and method for tracking a molecule or particle in three dimensions

Publication Date: 2009.03.03 TRIAD NATIONAL SECURITY LLC

AI summary

An apparatus and method were used to track the movement of fluorescent particles in three dimensions. Control software was used with the apparatus to implement a tracking algorithm for tracking the motion of the individual particles in glycerol/water mixtures. Monte Carlo simulations suggest that the tracking algorithms in combination with the apparatus may be used for tracking the motion of single fluorescent or fluorescently labeled biomolecules in three dimensions.