STED Microscope Spatial Offset Detection via Fluorescence Timing
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Solution Overview
Problem
Current STED microscopy methods face challenges in achieving precise superposition of excitation and de-excitation light distributions, leading to reduced image brightness and unwanted secondary maxima due to misalignment, and are inflexible with respect to wavelengths and fluorophores, with existing solutions being complex, slow, or not accounting for the sample's refractive index variations.
Innovation Solution
A fluorescence scanning microscope that uses an excitation light source and a de-excitation light source to generate light distributions superimposed at illumination target points, with a detector evaluating fluorescence photons by arrival times to generate two sample images, allowing for the determination of spatial offset between light distribution maxima and minima, enabling precise alignment without the need for calibration slides or phase masks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Shape
If a phase mask is used to influence the de-excitation light distribution, then the light distribution can be shaped, but the phase mask must be optimized for specific wavelengths and reduces flexibility with respect to usable wavelengths and fluorophores
Solution Approach 1:
The patent employs a spatial light modulator (SLM) that can be dynamically adjusted to change the phase and amplitude distribution of the de-excitation light. This dynamic adjustment capability allows the system to adapt to different wavelengths and fluorophores without requiring physical reconfiguration, resolving the contradiction between achieving precise light distribution shaping and maintaining wavelength flexibility.
Solution Approach 2:
The system changes the optical parameters (phase, amplitude, wavelength) of the de-excitation light through the SLM to achieve the desired light distribution. By programmably adjusting these parameters rather than using fixed phase masks, the system maintains versatility across different wavelengths and fluorophore combinations while still achieving precise spatial control.
2Measurement precision
If calibration slides or positioning elements are used to align light distributions, then alignment can be achieved, but the process is slow and complex
Solution Approach 1:
The system performs self-alignment by automatically detecting the relative position of excitation and de-excitation light distributions and adjusting the SLM to compensate for any misalignment. This eliminates the need for external calibration slides or manual positioning, achieving precise alignment rapidly through automated feedback and adjustment.
Solution Approach 2:
The patent implements a feedback mechanism where the system monitors the actual light distributions and adjusts the SLM parameters accordingly to maintain optimal superposition. This closed-loop control enables rapid and precise alignment without requiring time-consuming calibration procedures or manual intervention.
3Ease of operation
If the zero point of de-excitation light distribution does not coincide with the maximum of excitation light distribution, then misalignment occurs, but this reduces image brightness and emphasizes secondary maxima
Solution Approach 1:
The system automatically compensates for misalignment by detecting the relative position of the excitation maximum and de-excitation zero point, then adjusting the SLM to realign them. This self-correcting mechanism ensures consistent high-quality images without requiring manual alignment operations, resolving the contradiction between operational simplicity and image quality reliability.
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 allows for automatic and adaptive superposition of light distributions, improving image quality by minimizing misalignment effects and accommodating sample refractive index variations, thus enhancing imaging resolution and flexibility with existing STED configurations.
Implementation Method 1
The excitation light is designed to excite fluorophores present in the sample to the spontaneous emission of fluorescent light
Implementation Method 2
the de-excitation light, the wavelength of which is different from the wavelength of the excitation light, is used to de-excite fluorophores excited by the excitation light in the course of a stimulated emission of fluorescent light
Data Source
AI summary
A fluorescence scanning microscope includes excitation and de-excitation light sources, which are designed to generate an excitation and a de-excitation light distribution, respectively. An illumination unit combines the light distributions to form a light distribution scanning over multiple illumination target points of a sample in such a way that an intensity maximum of the excitation light distribution and an intensity minimum of the de-excitation light distribution are spatially superimposed on one another. A detector detects fluorescence photons emitted from the respective illumination target point as a function of their arrival times. A processor evaluates the fluorescence photons with respect to the arrival times, generates a first pixel and a second pixel based thereon, assembles the first and second pixels to form first and second sample images, respectively, and, by means of the two sample images, determines a spatial offset between the intensity maximum and the intensity minimum.