Fluorescence Inhibiting Light Beam Modulation for Microscopy
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Solution Overview
Problem
Current high-resolution scanning fluorescence light microscopy techniques often result in extensive bleaching of sensitive fluorescent dyes due to high light intensities, limiting their suitability for imaging structures marked with these dyes.
Innovation Solution
A method and apparatus that modulate the wave fronts of a fluorescence inhibiting light beam to create a minimum intensity at the focal point of the fluorescence excitation light beam, allowing for the detection and allocation of individually emitted photons, thereby reducing bleaching and increasing the yield of fluorescence light.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If high light intensities are used to maximize spatial resolution in fluorescence microscopy, then measurement precision is improved, but the fluorescent dye becomes extensively bleached before reaching the measurement area
Solution Approach 1:
The patent applies preliminary action by using a fluorescence inhibiting light beam to suppress fluorescence emission in advance in all areas except the measurement area. This prevents the fluorescent dye from being excited and bleached before the excitation light reaches the focal point, thereby preserving dye integrity while maintaining high spatial resolution.
Solution Approach 2:
The patent implements preliminary anti-action by introducing a fluorescence inhibiting light beam that counteracts the potential harmful effect of premature fluorescence excitation. This inhibiting beam creates a dark state in the dye molecules before they encounter the excitation light, preventing unwanted bleaching while allowing high-intensity excitation at the focal point for high-resolution imaging.
2Productivity
If scanning speed is increased to improve productivity, then the rate of fluorescence light detection decreases due to reduced exposure time
Solution Approach 1:
The patent applies continuity of useful action by using a continuous wave fluorescence inhibiting light beam that continuously suppresses fluorescence emission throughout the scanning process. This continuous inhibition ensures that fluorescence is only emitted from the measurement area regardless of scanning speed, allowing high-speed scanning without loss of signal yield.
Solution Approach 2:
The patent utilizes parameter changes by modulating the intensity and temporal profile of the fluorescence inhibiting light beam to match the scanning speed. By dynamically adjusting the inhibition parameters, the system maintains optimal fluorescence suppression while accommodating varying scanning speeds, thus preserving both productivity and signal yield.
3Measurement precision
If the scanning range is repeatedly scanned to improve measurement precision, then the fluorescent dye becomes bleached due to cumulative light exposure
Solution Approach 1:
The patent applies preliminary action by establishing a protective inhibiting light field before each scanning pass. This pre-established inhibition prevents cumulative bleaching by ensuring that fluorescent dye molecules are not excited during non-measurement areas of repeated scans, allowing multiple scans for improved precision without proportional dye loss.
Solution Approach 2:
The patent converts the potentially harmful effect of repeated scanning (cumulative bleaching) into a benefit by using the fluorescence inhibiting light beam to selectively suppress emission outside the measurement area. The repeated scanning necessary for high precision imaging no longer causes proportional dye loss because the inhibiting beam protects the dye in non-measurement areas during each scan repetition.
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 high-resolution imaging of structures with highly susceptible fluorescent dyes by minimizing bleaching and maximizing the yield of fluorescence light, allowing for the generation of detailed images with reduced stress on the dyes, even at increased scanning speeds.
Implementation Method 1
fluorescence excitation light beam of fluorescence excitation light... fluorescence light emitted out of the scanning range
Implementation Method 2
fluorescence inhibiting light beam of fluorescence inhibiting light whose wave fronts are modulated such that a fluorescence inhibiting light intensity distribution comprises a minimum at the focal point
Implementation Method 3
an objective lens focusing the fluorescence excitation light beam and the fluorescence inhibiting light beam
Data Source
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AI summary
In a method for imaging a structure (33) marked with a fluorescent dye in a sample, the sample is repeatedly scanned in a scanning range (28) with a light intensity distribution localised around a focal point (29) of a focused fluorescence excitation light beam. The light intensity distribution further comprises a focused fluorescence inhibiting light beam (7) whose wave fronts are modulated so that a fluorescence inhibiting light intensity distribution comprises a minimum at the focal point (29) of the fluorescence excitation light beam (4). The scanning conditions are coordinated in such a way that the fluorescence light is emitted out of the scanning range (28) as individually detectable photons. When these photons are detected, the location (32) of the focal point (28) at the respective point in time is allocated to them. An image of the structure (33) is composed of the locations (32) to which the detected photons have been allocated during several repetitions of scanning the scanning range (28).