Multi-color Fluorescence Imaging via Spectral Segmentation
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Solution Overview
Problem
Current super-resolution fluorescence microscopy techniques, such as single-molecule switching (SMS) microscopy, have limited multi-color imaging capabilities, preventing precise correlation of multiple targets due to limited spatial resolution and inefficient use of fluorescence signals.
Innovation Solution
A method for fluorescence imaging that involves filtering and directing fluorescence signals from different fluorophores with distinct emission spectra to separate cameras, allowing for simultaneous multi-color imaging by salvaging otherwise discarded fluorescence and minimizing cross-talk between signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescence filtering is used to separate emission spectra, then spectral separation is achieved, but cross-talk between fluorophores increases and imaging precision deteriorates
Solution Approach 1:
The emission spectrum is segmented into multiple spectral regions (blue edge, green edge, yellow edge, red edge) with distinct filtering applied to each region. This segmentation allows precise separation of fluorophore signals by capturing different portions of their emission spectra, thereby reducing cross-talk while maintaining high imaging precision.
Solution Approach 2:
Asymmetric filtering is applied where the first fluorophore captures the blue and green edges of its emission spectrum while the second fluorophore captures the yellow and red edges. This asymmetric allocation of spectral regions optimizes signal separation and minimizes overlap between different fluorophores, reducing cross-talk effectively.
2Adaptability or versatility
If multiple fluorophores with overlapping emission spectra are imaged simultaneously, then multi-color imaging capability is improved, but signal separation becomes difficult and measurement precision deteriorates
Solution Approach 1:
The method transitions from temporal or spatial separation to spectral dimension separation by capturing multiple spectral regions of fluorophores simultaneously. By utilizing the spectral dimension (different wavelengths), the system can distinguish and separate multiple fluorophores with overlapping emission spectra, enabling versatile multi-color imaging while maintaining precise signal separation.
3Use of energy by moving object
If fluorescence signals are captured across the full emission spectrum, then signal intensity is maximized, but chromatic aberrations increase and imaging precision deteriorates
Solution Approach 1:
Different regions of the emission spectrum are assigned to different fluorophores based on their local spectral characteristics. Each fluorophore is optimized to capture specific spectral regions (blue/green edges for one, yellow/red edges for another), ensuring that each detector receives the optimal portion of the spectrum for its assigned fluorophore. This local optimization maximizes signal intensity for each channel while minimizing chromatic aberrations.
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 multi-color imaging with reduced cross-talk and chromatic aberrations, allowing for the simultaneous detection of multiple fluorescent signals with high spatial resolution, even when dyes have closely overlapping emission spectra, and reduces phototoxicity in live cell imaging.
Implementation Method 1
a first dichroic mirror that reflects fluorescent signals having a wavelength shorter than a transition wavelength of the first dichroic mirror and transmits fluorescent signals having a wavelength longer than the transition wavelength of the first dichroic mirror
Implementation Method 2
an emission filter that transmits the first fluorescent signal and blocks transmission of the excitation beam
Implementation Method 3
a first fluorophore and a second fluorophore with different emission spectra
Data Source
AI summary
Methods and systems for fluorescence imaging are described herein. The method can include: receiving a fluorescence signal including an excitation signal, a first emission signal for a first fluorophore having a first emission spectra, and a second emission signal for a second fluorophore having a second emission spectra; filtering the fluorescence signal to: isolate a first channel encompassing at least one of: a bandwidth of at least 1 nm within which an emission intensity of the first emission spectra is at least twice an emission intensity of the second emission spectra, a bandwidth having a rising edge of the first emission spectra; and at least 10% by height of a rising edge of the first emission spectra, and produce a channel including the fluorescence signal less the first channel; and directing the first channel and the resulting channel to different regions of one or more cameras for collecting fluorescence emissions.