Acousto-Optic Tunable Filter for Multiplexed Fluorescence Imaging
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Solution Overview
Problem
Conventional fluorescence microscopy is limited in multiplexing capability due to the broad spectral width of fluorescence and struggles with achieving high temporal and spatial resolutions when imaging multiple subcellular targets with spectral overlap, especially in live cells.
Innovation Solution
The use of a single, fixed fluorescence emission detection band combined with frame-synchronized fast scanning of the excitation wavelength via an acousto-optic tunable filter (AOTF) allows for simultaneous imaging of multiple subcellular targets with low crosstalk and high spatiotemporal resolutions, enabling the quantification of different fluorophores through unmixing of excitation spectra.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional bandpass filters are used for fluorescence imaging, then the system is simple and easy to operate, but the multiplexing capability is limited to 3-4 channels due to broad spectral width of fluorescence
Solution Approach 1:
The patent employs dynamically tunable excitation wavelengths via AOTF instead of static bandpass filters. The AOTF can electronically adjust the excitation spectrum in real-time, enabling flexible multiplexing of multiple fluorophores with overlapping emission spectra. This dynamic tuning capability allows the system to accommodate more than 3-4 channels by selectively exciting different fluorophores at specific wavelengths, thereby improving multiplexing capability without requiring complex mechanical filter switching mechanisms.
Solution Approach 2:
The patent changes the excitation wavelength parameter dynamically using an acousto-optic tunable filter (AOTF). By varying the excitation wavelength across multiple channels, the system can distinguish between fluorophores with overlapping emission spectra. This parameter change approach allows for high multiplexing capability (up to 6 channels) while maintaining relatively simple system architecture, as the AOTF integrates wavelength tuning functionality without requiring complex mechanical filter assemblies.
2Adaptability or versatility
If mechanical switching between filter sets is used for multi-target imaging, then the system can accommodate multiple channels, but the temporal resolution is reduced due to slow switching speed
Solution Approach 1:
The patent replaces mechanical filter switching with an electronically controlled AOTF system. The AOTF uses acoustic waves to modulate the refractive index of a crystal, enabling rapid electronic tuning of the excitation wavelength without mechanical movement. This substitution of mechanical switching with an electronic/acoustic mechanism dramatically improves the switching speed and temporal resolution, allowing fast dynamic imaging of multiple targets simultaneously while maintaining multi-channel imaging capability.
3Measurement precision
If point-scanning spectral imaging is used to resolve fluorescence emission spectrum, then spectral information can be obtained, but the temporal resolution is reduced due to sequential scanning of spatial and spectral dimensions
Solution Approach 1:
The patent inverts the conventional spectral imaging approach by scanning the excitation wavelength instead of the emission wavelength. Instead of fixing the excitation wavelength and scanning through emission spectra at each pixel (which requires sequential scanning and reduces temporal resolution), the system fixes the emission detection band and scans through excitation wavelengths. This inversion allows simultaneous acquisition of spectral information across the entire field of view, maintaining high temporal resolution while achieving comprehensive spectral resolution for multiplexed fluorophore imaging.
4Measurement precision
If narrow bandpass filtering is applied to fluorescence emission, then spectral specificity is improved, but the signal intensity is reduced due to inefficient use of scarce signal
Solution Approach 1:
The patent uses dynamically tunable excitation wavelengths via AOTF to match the excitation spectrum of each fluorophore optimally. Instead of using fixed narrow bandpass filters that may not align perfectly with fluorophore excitation peaks, the AOTF can be tuned to the exact excitation wavelength needed, maximizing the excitation efficiency and signal intensity. This dynamic tuning ensures that the available excitation light is used most efficiently, improving signal intensity while maintaining spectral specificity through precise wavelength selection.
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 enables fast, quantitative imaging of up to six subcellular targets with low crosstalk and high temporal resolutions, facilitating the study of complex biological processes such as mitophagy and macromolecular crowding with high sensitivity and spatiotemporal resolution.
Implementation Method 1
frame-synchronized fast scanning of the excitation wavelength from a white lamp via an acousto-optic tunable filter (AOTF)
Implementation Method 2
detect the fluorescence emission spectrum
Data Source
AI summary
Systems and methods are provided for excitation spectral microscopy using frame-synchronized acousto-optic scanning of fluorescent excitation wavelengths. Linear unmixing of the images of targets of components that are individually labeled with different fluorophores can be simultaneously imaged with high temporal resolution and low crosstalk and the local abundance of each fluorophore at each pixel can be quantified. Fluorophore decomposed micrographs of the sample can be obtained by rendering the abundance in each pixel as an image.