Image Scanning Microscopy for Spectral Fluorophore Separation
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Solution Overview
Problem
Existing image scanning microscopes are limited in their ability to accurately determine the spatial distribution of concentrations of different fluorophore species in a sample.
Innovation Solution
An image scanning microscope and method that utilize a spectral encoding element to modulate detection light based on wavelength, combined with an array detector and control unit to detect photon arrival times, enabling the reconstruction of a high-resolution image and determination of fluorophore species concentrations through fingerprints, including temporal emission behavior and spectral excitation behavior.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a multi-element photodetector array is used in image scanning microscopy, then spatial resolution and signal-to-noise ratio are improved, but the capability to distinguish different fluorophore species is limited
Solution Approach 1:
The detection array is segmented into multiple independently controllable detector elements, each capable of detecting photons from different fluorophore species. This segmentation allows simultaneous detection of multiple fluorophore types while maintaining high spatial resolution through the multi-element array architecture.
Solution Approach 2:
Different regions of the detection array are assigned different detection characteristics or weighting factors to optimize detection of specific fluorophore species. This local quality differentiation enables the system to distinguish between fluorophore types based on their spatial distribution patterns and detection characteristics.
2Device complexity
If conventional confocal microscopy with a single point detector is used, then device complexity is reduced, but spatial resolution and signal-to-noise ratio are limited
Solution Approach 1:
Multiple detector elements are merged into a single integrated detection array that functions as a unified detection system. This merging allows the system to achieve high spatial resolution through parallel detection while managing complexity through integrated control and processing of the detector array.
Solution Approach 2:
The detection array is designed with multi-functionality to perform both high-resolution imaging and fluorophore species differentiation simultaneously. The universal detection system can handle multiple detection tasks through appropriate algorithmic processing, reducing the need for separate specialized detectors.
3Loss of information
If multiple fluorophore species are detected simultaneously, then information content is increased, but measurement precision for spatial distribution determination is reduced
Solution Approach 1:
The detection system incorporates temporal and spectral dimensions in addition to spatial detection. By detecting photons at different times and wavelengths through the multi-element array, the system creates additional measurement dimensions that enable precise separation and quantification of multiple fluorophore species while maintaining spatial resolution.
Solution Approach 2:
Real-time feedback processing is implemented to continuously adjust detection parameters and algorithms based on the detected signal characteristics. This feedback mechanism optimizes the measurement precision for each fluorophore species by adapting to their specific emission patterns and environmental conditions, preventing information loss while maintaining accuracy.
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
Enables robust distinction of multiple fluorophore species with high spatial resolution and signal-to-noise ratio, allowing for accurate reconstruction of their spatial distribution.
Implementation Method 1
a spectral encoding element configured to change the spatial distribution of the intensity of the detection light based on the wavelength of the detection light
Implementation Method 2
an objective lens directed at a sample space and configured to direct the excitation light into the sample space and to receive the detection light from the sample space
Implementation Method 3
at least one array detector configured to detect the spatial distribution of the intensity of the detection light
Data Source
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AI summary
An image scanning microscope (100) comprises an excitation unit (108) configured to generate excitation light (118) according to at least one excitation modality, and an objective lens (102) directed at a sample space (106) and configured to direct the excitation light (118) into the sample space (106) and to receive the detection light (126) from the sample space (106). A scanning unit (110) of the image scanning microscope (100) is arranged along a beam path between the excitation unit (108) and the objective lens (102) and configured to selectively direct the excitation light (118) into different regions of the sample space (106) via the objective lens (102). The image scanning microscope (100) also comprises a detection arrangement (112) comprising at least one spectral encoding element (128) configured to change the spatial distribution of the intensity of the detection light (126) based on the wavelength of the detection light (126) and at least one array detector (130a, 130b) configured to detect the spatial distribution of the intensity of the detection light (126). A main beam splitter (114) of the image scanning microscope (100) is configured to direct the excitation light (118) into the objective lens (102) via the scanning unit (110), and to direct the detection light (126) into the detection arrangement (112). The image scanning microscope (100) further comprises a control unit (116) configured to control the excitation unit (108) to set the excitation modality, and to determine a spatial distribution of a concentration of at least two different fluorophore species in a sample (104) arranged in the sample space (106) based on the detected spatial distribution of the intensity of the detection light (126) and based on the excitation modality and/or at least one photon arrival time detected by at least one time resolved detector element of the detection arrangement (112).