Light Sheet Microscopy for Isotropic 3D Resolution
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Solution Overview
Problem
Current microscopy methods, such as PALM and SPIM, face challenges in achieving high-resolution 3D imaging due to limitations in axial resolution, out-of-focus autofluorescence, and the need for nonlinear photoactivation, which slows down data acquisition and damages samples.
Innovation Solution
A combination of PALM with TIRF technology and MultiView method, where photoactivation occurs within the focal plane using a light sheet, minimizing out-of-focus autofluorescence and enabling isotropic optical resolution in x, y, z, without nonlinear photoactivation, and utilizing structured illumination to enhance resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If statistical photoactivation of molecules is used in PALM method, then high-resolution image can be generated, but large amounts of data must be processed and measurement is slowed down
Solution Approach 1:
The patent applies periodic structured illumination to activate fluorophores in a controlled pattern rather than statistically. By using light sheets with periodic intensity distributions, the system deterministically activates specific molecular groups in sequence, reducing the total number of activation cycles needed while maintaining super-resolution capability and increasing acquisition speed.
Solution Approach 2:
The patent changes the illumination parameter from wide-field statistical activation to structured light sheet patterns. By modifying the spatial distribution of activation light intensity, the system achieves deterministic molecular activation with fewer cycles, thereby improving both resolution and acquisition rate simultaneously.
2Quantity of substance
If wide-field illumination is used for photoactivation, then molecules can be activated, but autofluorescence light is excited in the entire focus cone which reduces contrast
Solution Approach 1:
The patent applies local quality by using structured light sheets that concentrate activation light only in the focal plane where imaging occurs. This localized illumination activates molecules precisely where needed while leaving other regions unaffected, thereby eliminating out-of-focus autofluorescence generation and improving image contrast.
Solution Approach 2:
The patent extracts the harmful out-of-focus autofluorescence component by replacing wide-field illumination with structured light sheets. This removes the source of background interference while preserving the necessary molecular activation in the focal plane, achieving high contrast imaging.
3Measurement precision
If nonlinear photoactivation with high intensities is used, then photoactivation can be achieved, but dye damage or sample damage can occur
Solution Approach 1:
The patent changes the illumination intensity parameter from high-intensity nonlinear activation to low-intensity linear structured illumination. By using light sheets with optimized intensity distributions, the system achieves precise molecular activation without exceeding damage thresholds, preserving both dye and sample integrity.
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 significantly increases the image acquisition rate, reduces the number of images needed, and allows for high-resolution 3D imaging without sample damage, while avoiding out-of-focus autofluorescence and improving axial resolution.
Implementation Method 1
fluorophores in the sample are usually excited with laser light in the form of a light sheet
Implementation Method 2
The periodically structured light sheet excites the fluorescence at the locations of high intensity. Structuring is used to suppress stray light from extra-focal planes and to increase resolution through structured illumination
Data Source
Figure 1a~1b
Figure 2
Figure 3
AI summary
Apparatus, in particular a microscope, and method for high spatial resolution imaging of a structure of a sample characterized by a diffraction-limited resolution volume with a plurality of dye molecules (UF) which can be switched between different states, with at least one state being fluorescent, the fluorescence being collected by an objective (O) and imaged on a spatially resolving detector using an optical system, the UF having a distribution density in at least part of the sample which is greater than the inverse of the diffraction-limited resolution volume; 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 the UF, with at least one of the light sources being arranged such that it transilluminates the sample and the UF in the sample being switched and/or excited to fluoresce at least in one direction which is approximately perpendicular to the optical axis and in particular in the focus of the objective (O), the switching advantageously being a photoactivation or photodeactivation of the UF and provision being made of the light source for switching and/or the light source for exciting, a focusing arrangement for generating a line-like illumination region extending in the direction of the illumination, at least in one direction, at least approximately perpendicular to the optical axis of the objective.