RESOLFT Microscopy Illumination Using Long-Pulse Excitation
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Solution Overview
Problem
RESOLFT microscopy faces challenges in achieving high resolution while maintaining cost-effectiveness due to the need for expensive short-pulse excitation light sources and the suboptimal resolution provided by continuous wave STED methods.
Innovation Solution
Irradiating excitation light in pulses longer than 150 picoseconds, up to a few nanoseconds, and using inexpensive laser sources, which allows for improved signal detection and resolution without the need for expensive short-pulse excitation light sources, and allowing stimulation light to be delivered only after the excitation pulse to enhance photon detection from the center of the excitation distribution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If short-pulse excitation light sources are used, then resolution is improved, but cost increases
Solution Approach 1:
The patent changes the pulse duration parameter from short pulses (typically <100 ps) to longer pulses (150 ps to a few nanoseconds). This parameter change allows the use of less expensive laser sources while maintaining the ability to achieve super-resolution through the RESOLFT mechanism, thus resolving the contradiction between resolution and cost.
2Ease of manufacture
If continuous wave STED methods are used, then cost is reduced, but resolution deteriorates
Solution Approach 1:
The patent employs periodic pulsed action for both excitation and stimulation light delivery. The excitation light is delivered in pulses longer than 150 ps, and the stimulation light is delivered in subsequent pulses. This periodic pulsed operation enables the system to achieve high resolution like short-pulse methods while using cost-effective continuous wave or pulsed laser sources, thus resolving the contradiction between cost and resolution.
3Ease of manufacture
If excitation pulse duration is increased, then cost-effective laser sources can be used, but signal detection becomes suboptimal
Solution Approach 1:
The patent uses a gating mechanism that detects photons only during a specific time window after the excitation pulse. This preliminary timing action ensures that only photons from the center of the excitation distribution (where stimulation light was delivered) are detected, filtering out background signal. This resolves the contradiction by maintaining cost-effective long-pulse operation while optimizing signal detection through temporal gating.
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 simplifies the light source requirements, increases the number of detectable photons, and improves resolution by ensuring that only photons from the center of the excitation distribution are detected, while being cost-effective and reliable.
Implementation Method 1
the capability of distinguishing between closely spaced features is about 200 nm. This is due to the wave nature of light. For example, in conventional light microscopes, the resolution limit is mainly determined by the wavelength of the light used and the numerical aperture.
Implementation Method 2
STED (Stimulated Emission Depletion) microscopy, for example, belongs to RESOLFT microscopy. In that method, a fluorescent dye can change back and forth between an electronic ground state and an excited state and fluoresce in the process. In the dark state, the dye is permanently maintained in its ground state through stimulated emission.
Data Source
AI summary
A method for illumination and detection in RESOLFT microscopy using a pulsed or continuous light source for excitation light and switching light is characterized in that the excitation light (4) is irradiated in pulses and in that the pulse of the excitation light (4) is longer than 150 picoseconds, preferably up to a few hundred picoseconds, and even up to a few nanoseconds. A corresponding apparatus uses the method according to the present invention.