MD-WISE Microscopy Multiplexed Infrared Encoding
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Solution Overview
Problem
Current multiplexed photoluminescence (PL) imaging techniques face limitations due to the overlap of broad emission spectra from chromophores in visible and adjacent spectral regions, restricting the number of color channels available for multiplexed imaging, and existing nonlinear optical methods are hindered by low sensitivity and require tight focusing, which is not compatible with widefield imaging.
Innovation Solution
The use of femtosecond mid-infrared and visible pulses in a multiplexed widefield imaging method, known as MD-WISE microscopy, which exploits distinct responses of chromophores to IR frequencies and temporal delays between pulses to encode and distinguish PL signals, enabling expansion of visible spectral colors into multiple dimensions and simultaneous tracking of multiple chromophores within a single field of view.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional photoluminescence imaging is used with visible spectral regions, then single-molecule level sensitivity and sub-diffraction spatial resolution are achieved, but the number of color channels available for multiplexed imaging is limited due to broad emission spectra overlap
Solution Approach 1:
The patent extends multiplexed imaging from the traditional single spectral dimension to multiple dimensions by combining mid-infrared excitation frequencies with visible emission detection. This allows chromophores to be distinguished not only by their emission color but also by their characteristic infrared absorption frequencies, effectively creating a multi-dimensional identification space that resolves the spectral overlap limitation
2Measurement precision
If nonlinear optical methods such as stimulated Raman excited fluorescence microscopy are used, then multiplexed PL imaging with single-molecule level sensitivity is achieved, but tight focusing is required which is not compatible with widefield imaging
Solution Approach 1:
The patent segments the excitation process into two distinct steps: first, mid-infrared pulses excite vibrational modes through linear absorption (which has large cross-sections and does not require tight focusing), and second, visible pulses excite electronic transitions to produce fluorescence. This segmentation allows the system to benefit from the large absorption cross-sections of linear IR absorption while maintaining the sensitivity of nonlinear optical processes, enabling widefield imaging geometry
3Loss of information
If infrared photothermal microscopy is used, then vibrational information is detected, but the detection limits are less sensitive than PL-based techniques
Solution Approach 1:
The patent uses fluorescence emission as an intermediary to detect vibrational information. Instead of directly detecting the weak photothermal signal from infrared absorption, the method uses IR excitation to modulate the fluorescence signal produced by subsequent visible excitation. This intermediary approach translates the weak vibrational signal into a much stronger fluorescence readout, achieving both vibrational specificity and high sensitivity
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 increases the information extraction rate in PL imaging, allowing for the differentiation of chromophores with nearly identical PL spectra and enabling high-speed chemical and biological imaging by operating in a widefield mode, which is faster and less damaging than confocal microscopy.
Implementation Method 1
The first type is exciting molecular vibrations through the linear absorption of a mid-IR photon that further modulate the electronic absorptions
Implementation Method 2
spatially focusing femtosecond mid-IR and visible pulses delayed by a controlled temporal delay onto a sample comprising at least one chromophore to excite spontaneous emitted PL signals from the at least one chromophore
Implementation Method 3
The second IR-visible nonlinear interaction is based on the strong electric field of femtosecond mid-IR pulses, which can reach the order of megavolts per centimeter (MV/cm) due to the high peak power and the relatively long wavelength. Such electric fields can ionize excitons in semiconductor materials, affecting the PL intensity of quantum dot (QD) emitters
Implementation Method 4
the visible pulse promotes the molecules to an electronic excited level
Implementation Method 5
excite spontaneous emitted PL signals from the at least one chromophore
Data Source
AI summary
A multiplexed widefield imaging method employs spatially focusing femtosecond infrared (IR) pulses and visible pulses delayed by a controlled temporal delay onto a sample stained with one or more chromophore to excite spontaneous emitted photoluminescence (PL) signals from the chromophores. The resulting PL signals are detected to generate PL images in which intensities of the PL signals are a function of optical frequencies of the IR and visible pulses and the temporal delay.