BonFIRE Spectroscopy for Single-Molecule Bioimaging
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Solution Overview
Problem
Current infrared bioimaging techniques face limitations such as coarse spatial resolution, strong water background interference, and low detectability of dilute samples, restricting their sensitivity and applicability for bioimaging, especially for detecting specific biomolecules like proteins and lipids.
Innovation Solution
The method of bond-selective fluorescence-detected infrared-excited (BonFIRE) spectroscopy, which involves generating coherent IR and NIR lasers in a counter-propagating configuration, irradiating a dye molecule, and detecting fluorescence to extract bond-selective IR absorption maxima, offering enhanced sensitivity, resolution, and multiplexity for bioimaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If direct IR imaging is used, then chemical specificity is improved, but spatial resolution deteriorates due to long-wavelength excitation
Solution Approach 1:
The patent introduces a fluorescent probe as an intermediary that converts IR excitation into visible fluorescence detection. The probe absorbs IR photons and emits fluorescence at shorter wavelengths, which can be detected with conventional optical microscopy systems, thereby achieving both chemical specificity and improved spatial resolution
Solution Approach 2:
The patent replaces direct optical detection of IR radiation with a two-step process involving IR absorption followed by fluorescence emission detection. This substitution allows the use ofๆ็ optical detection systems with higher resolution capabilities instead of direct IR detection methods
2Measurement precision
If conventional IR microscopy is used, then chemical contrast is improved, but detectability of dilute samples deteriorates
Solution Approach 1:
The fluorescent probe serves as a mediator that amplifies the detection signal. When the probe absorbs an IR photon, the resulting fluorescence emission provides a detectable signal even at low concentrations, overcoming the weak signal problem of direct IR detection for dilute samples
Solution Approach 2:
The patent exploits the change in optical properties of the fluorescent probe upon IR excitation. The probe transitions from absorbing IR radiation to emitting visible fluorescence, creating a detectable signal change that enables sensitive detection of dilute biomolecules
3Measurement precision
If water background is strong in IR imaging, then chemical specificity is improved, but signal-to-background ratio deteriorates
Solution Approach 1:
The fluorescent probe acts as an intermediary that converts the weak IR signal into a stronger visible fluorescence signal. This amplification overcomes the strong water background absorption in the IR region, as the detection occurs at wavelengths where water has minimal absorption
Solution Approach 2:
The patent substitutes direct IR detection with fluorescence detection at shorter wavelengths. This substitution moves the detection to a spectral region where water background interference is minimal, thereby improving the signal-to-background ratio while maintaining chemical specificity
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
BonFIRE spectroscopy achieves superior sensitivity and resolution, enabling the detection of dilute biomolecules with improved signal-to-background ratios and signal-to-noise ratios, facilitating more accurate bioimaging and tracking of cellular dynamics.
Implementation Method 1
irradiating the dye molecule with the IR laser and the NIR laser; and detecting a fluorescence from the dye molecule
Implementation Method 2
detecting a fluorescence from the dye molecule
Implementation Method 3
generating an IR laser and a NIR laser, wherein the IR laser and the NIR laser are coherent; aligning the IR laser and the NIR laser in a counter-propagating configuration on the sample
Data Source
AI summary
Disclosed herein include methods and related systems of bond-selective fluorescence-detected infrared-excited (BonFIRE) spectroscopy. BonFIRE employs two-photon excitation in the mid-IR and near-IR to upconvert vibrational excitations to electronic states for fluorescence detection, thus encoding vibrational information into fluorescence. The method comprises providing a sample comprising a dye molecule having an UV-vis absorption maximum; generating an IR laser and a NIR laser, wherein the IR laser and the NIR laser are coherent; aligning the IR laser and the NIR laser in a counter-propagating configuration on the sample; irradiating the dye molecule with the IR laser and the NIR laser; and detecting a fluorescence from the dye molecule which can be used to extract a bond-selective IR absorption maximum of the dye molecule or form an image of the sample from the fluorescence with single-molecule sensitivity. The WF-BonFIRE technique significantly increases the imaging speed for single-molecule samples and for biological samples.