Phase Mask Fluorescence Microscope Localized Radiation Minimum
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Solution Overview
Problem
Current microscopy methods face challenges in achieving high-resolution imaging beyond the Abbe diffraction limit and efficiently localizing individual molecules with precise accuracy, particularly in optimizing local photon statistics and simultaneous detection of multiple molecules.
Innovation Solution
The implementation of phase masks and structured illumination techniques, such as spiral phase masks and Bessel beams, along with spectral splitting and achromatic beam separation, to generate customized point spread functions (PSFs) that enhance localization precision and allow simultaneous detection of multiple molecules by optimizing photon statistics and background suppression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional wide-field illumination is used for molecule activation, then the activation process is simple and statistical, but the localization precision is limited by photon statistics and background noise
Solution Approach 1:
The patent applies local quality by using structured illumination patterns (such as lines or dots) that selectively activate molecules in specific spatial regions rather than uniform wide-field activation. This localized activation approach improves signal-to-background ratio and localization precision by concentrating excitation energy where needed while reducing overall background fluorescence.
Solution Approach 2:
The patent employs periodic action through time-dependent illumination patterns that switch between different activation states. By using time-resolved illumination sequences with structured patterns, the system can modulate molecule activation in a periodic manner, enabling precise temporal control over which molecules are activated and detected, thereby improving localization accuracy.
2Productivity
If multiple molecules are detected simultaneously with a single detector, then detection efficiency increases, but distinguishing and localizing individual molecules becomes more difficult
Solution Approach 1:
The patent applies segmentation by spatially separating the detection process into multiple independent detection regions or channels. By dividing the field of view into distinct zones with structured illumination, each detector element or detection channel can independently analyze and localize molecules in its specific region, maintaining high detection speed while preserving the ability to distinguish individual molecules through spatial segmentation.
3Manufacturing precision
If the point spread function (PSF) is conventional, then the system is simple to implement, but the resolution is limited by the Abbe diffraction limit
Solution Approach 1:
The patent applies parameter changes by modifying the optical parameters of the PSF through structured illumination patterns. By changing the spatial frequency content, intensity distribution, and temporal characteristics of the illumination, the system transforms the conventional diffraction-limited PSF into a structured PSF with enhanced resolution capabilities, effectively overcoming the Abbe limit through parameter optimization.
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
These techniques enable improved molecular localization accuracy and faster image acquisition while maintaining high resolution, effectively overcoming the limitations of conventional microscopy in resolving molecular positions and correlations within samples.
Implementation Method 1
a phase mask is provided in the beam path, preferably the detection, which generates a light distribution (PSF) with at least partially limited, local radiation minimum in the detector plane
Implementation Method 2
Preferred phase masks that can be used are spiral phase masks, half-space phase masks and other masks with a continuous or discontinuous change in phase delay
Implementation Method 3
a phase mask or an axicon is provided in the beam path for generating a Bessel distribution (PSF) in the detector plane
Implementation Method 4
These can consist of phase and / or amplitude gratings or means for splitting and superimposing coherent light, so that the structuring is created by interference in the object plane
Implementation Method 5
means for the spectral splitting of the sample light, such as a grating and / or prism, are provided in the detection beam path
Implementation Method 6
means for the spectral splitting of the sample light, such as a grating and / or prism, are provided in the detection beam path
Implementation Method 7
a receiver array is formed from position-sensitive receivers (psd), the position and positional accuracy of the light distribution being read out for each element of the array
Implementation Method 8
A corresponding fluorescence microscope has an excitation light source and a switching light source, which apply corresponding signals to the sample
Implementation Method 9
The excitation radiation is then applied to the sample and the fluorescent sample is imaged, the fluorescent radiation then only being able to originate from the activated subset
Data Source
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AI summary
Disclosed is an apparatus, especially a microscope, characterized by a diffraction-limited resolution volume, comprising multiple dye molecules (UF) that can be switched between different states, at least one of which is fluorescent. The fluorescence is focused using an objective (O) and is imaged onto a spatially resolving detector. In at least one portion of the sample, the UF have a distribution density that is greater than the inverse of the diffraction-limited resolution volume. Said apparatus further comprises one or more light sources for emitting a switching radiation in order to switch a first subset of the UF in the sample, and for emitting an excitation radiation in order to excite the first subset of UF. A phase mask which generates a light distribution (PSF) having an at least partially limited local minimum radiation on the detector plane is provided in the beam path, preferably in the detection beam path. Alternatively, an axicon is provided for generating a Bessel distribution (PSF) on the detector plane, a means for structuring the illumination distribution is provided in the illumination beam path, means for spectrally splitting the sample light are provided in the detection beam path, a receiver array composed of position-sensitive receivers (psd) is provided for detection purposes, means causing a color-dependent light distribution (PSF) on the detector plane are provided in the detection beam path, or an achromatic beam splitter is arranged in or near the pupil.