Light-pad Microscope Spatial Resolution via Light-Sheet Illumination
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Solution Overview
Problem
Current methods for analyzing diffusion processes of biomolecules in complex cellular environments are limited by the inability to provide spatially resolved information, leading to challenges in visualizing diffusion processes and other protein parameters across entire cells or organisms.
Innovation Solution
The development of a light-pad microscope that uses a diffraction-limited light-sheet for illumination, allowing for full spatial control over fluorescence excitation and avoiding out-of-focus exposure, combined with a detection system that captures spatially resolved FCS data.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If confocal laser scanning microscopes are used for FCS measurements, then diffraction limited imaging with ultra sensitive photon counting is achieved, but sequential data acquisition limits spatial resolution and total fluorescence photon yield is reduced due to out-of-focus illumination
Solution Approach 1:
The patent transitions from sequential point-by-point scanning (1D time sequence) to parallel plane illumination (2D spatial plane), where the entire illumination plane is exposed simultaneously rather than scanning through points sequentially. This dimensional change enables both high spatial resolution across the entire field of view and efficient parallel data acquisition
Solution Approach 2:
The illumination is segmented into a structured light sheet that illuminates only the focal plane of interest, dividing the illumination space into in-focus and out-of-focus regions. This segmentation eliminates out-of-focus illumination while maintaining diffraction-limited spatial resolution in the segmented focal plane
2Measurement precision
If confocal laser scanning microscopes perform sequential point measurements, then specific positions can be analyzed, but spatially resolved information across entire cells cannot be generated
Solution Approach 1:
The patent merges the advantages of confocal microscopy (diffraction-limited spatial resolution) with light-sheet illumination (parallel plane exposure), combining the precision of point measurement with the coverage of plane illumination to achieve spatially resolved FCS data across entire cells simultaneously
Solution Approach 2:
The system expands from 1D sequential point scanning to 2D parallel plane illumination, capturing spatially resolved information across the entire illumination plane simultaneously, thereby recovering the spatial information that was lost in sequential scanning
3Loss of energy
If light-sheet illumination is used, then out-of-focus exposure is avoided and fluorescence excitation efficiency is improved, but device complexity increases compared to confocal systems
Solution Approach 1:
The patent adapts the light-sheet illumination approach for multi-purpose use in FCS measurements, combining it with standard confocal detection optics. This universal approach allows the system to achieve both improved excitation efficiency and spatially resolved data using integrated components from both light-sheet and confocal microscopy
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 the generation of spatially resolved maps of protein concentrations and mobilities, providing detailed insights into diffusion processes and protein interactions within cells, while also increasing the efficiency of FCS measurements compared to traditional confocal methods.
Implementation Method 1
diffraction-limited light-sheet for illumination
Implementation Method 2
illumination objective lens focusing the illumination light onto a sample
Implementation Method 3
detection objective lens detecting the light at a perpendicular orientation to the illuminating light path
Implementation Method 4
fluorescence excitation and avoiding out-of-focus exposure
Data Source
Figure 1A~1F
Figure 2
Figure 3a~3c
AI summary
The present disclosure teaches a microscope (1) having an illumination light path (20) for illuminating a sample or object (18) and a viewing light path (40,50) for viewing the sample. The microscope comprises an illumination light path focussing arrangement in the illumination light path, the illumination light path focussing arrangement defining a substantially two-dimensional sample or object illumination region (22) extending along an illumination direction of the illumination light path and transversely thereto. The microscope further comprises an illumination region-confining device (27) in the illumination light path for selectively illuminating a portion (10) of the substantially two-dimensional object illumination region, wherein the portion of the substantially two-dimensional object illumination region is confined at least in the illumination direction and/or in the direction transversely thereto.