Fluorescence Image Acquisition Using Frequency-Domain Signal Separation
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Solution Overview
Problem
Multi-wavelength fluorescence microscopy is limited by overlapping spectra of fluorescent materials, requiring narrow optical filters and increased excitation light intensity, which can lead to phototoxicity and signal loss.
Innovation Solution
A fluorescence image acquisition apparatus and method that modulates optical signals at the same frequency with different time delays, using a controller to separate fluorescence images and phase images based on a four-bucket scheme, allowing for the extraction of phase information and separation of fluorescence signals by wavelength without additional filters.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If optical filters with narrow pass bandwidths are used to remove overlapping fluorescence signals, then the ability to distinguish fluorescent materials is improved, but the loss of useful fluorescence signals increases
Solution Approach 1:
The patent applies periodic modulation to the excitation light source at different frequencies for different wavelengths. By modulating the excitation light periodically and detecting the modulated fluorescence signals, the system can distinguish overlapping signals without using narrow optical filters, thus avoiding signal loss while maintaining measurement precision.
Solution Approach 2:
The patent changes the frequency parameter of the excitation light source to differentiate between fluorescent materials with overlapping spectra. By assigning different modulation frequencies to different excitation wavelengths, the system can resolve signal overlap through frequency domain separation rather than spatial filtering, eliminating the need for narrow bandwidth filters.
2Strength
If the intensity of the excitation light source is increased to increase the strength of the fluorescence signal, then the fluorescence signal strength is improved, but phototoxicity to the living sample increases
Solution Approach 1:
The patent uses periodic modulation of the excitation light source at different frequencies, allowing the system to extract fluorescence signals through frequency-domain detection. This approach enables signal detection without increasing overall light intensity, as the modulation technique enhances signal discrimination rather than signal strength, thereby reducing phototoxicity while maintaining adequate signal levels.
Solution Approach 2:
The patent replaces the mechanical approach of increasing light intensity to improve signal strength with an electronic/detector-based approach using frequency modulation and demodulation. By substituting intensity-based signal enhancement with frequency-based signal discrimination, the system achieves strong fluorescence detection without the harmful effects of high-intensity illumination.
3Adaptability or versatility
If multiple fluorescent materials with different wavelengths are used to observe intracellular components, then the ability to monitor molecular interactions is improved, but the spectral overlap between fluorescent materials increases
Solution Approach 1:
The patent applies periodic modulation at different frequencies to excitation light sources of different wavelengths. This allows simultaneous excitation of multiple fluorescent materials with overlapping spectra, and the modulated fluorescence signals are detected and separated in the frequency domain, preserving the ability to monitor multiple molecular interactions without spectral interference.
Solution Approach 2:
The patent transitions from spatial/wavelength-based signal separation to frequency-domain separation by modulating excitation light at different frequencies. This dimensional change from the spectral domain to the temporal frequency domain enables the simultaneous use of multiple fluorescent materials with overlapping spectra, as each can be distinguished by its unique modulation frequency rather than by wavelength alone.
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 effectively separates multi-wavelength fluorescence images, reducing background noise and increasing the signal-to-noise ratio and contrast ratio, while minimizing phototoxicity and signal loss.
Implementation Method 1
a light source configured to generate, at different time delays, a plurality of optical signals that are modulated at the same frequency, an illuminator configured to control paths of the plurality of modulated optical signals so that the plurality of modulated optical signals are illuminated onto a sample including a plurality of fluorescent materials
Implementation Method 2
a photodetector configured to detect a plurality of fluorescence signals that are emitted from the plurality of fluorescent materials, respectively
Data Source
AI summary
Provided is a fluorescence image acquisition apparatus for acquiring fluorescence images and phase images using optical signals that are modulated at the same frequency and that have different time delays. The fluorescence image acquisition apparatus may include a light source configured to generate, at different time delays, a plurality of optical signals that are modulated at the same frequency, an illuminator configured to control paths of the plurality of modulated optical signals so that the plurality of modulated optical signals are illuminated onto a sample including a plurality of fluorescent materials, a photodetector configured to detect a plurality of fluorescence signals that are emitted from the plurality of fluorescent materials, respectively, and a controller configured to acquire a plurality of fluorescence images and a plurality of phase images from the plurality of detected fluorescence signals.