Microscope Illumination Pattern Manipulation for Parallelized Cell Imaging
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Solution Overview
Problem
Current microscopy techniques, such as confocal laser scanning microscopes and programmable array microscopes, face challenges in achieving high image refresh rates with minimal light exposure for living cell imaging, as they either suffer from slow scanning, high light intensities, significant signal losses, or limitations in image field size and objective selection.
Innovation Solution
A microscope design where the manipulation device is placed upstream of the main beam splitter, allowing for individual pixel activation in the detector based on selected illumination patterns, using a spatial light modulator or rotatable stop disk, and a Single Photon Avalanche Diode array for sensitive and rapid detection, enabling confocal limiting resolution and flexible experimentation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If confocal laser scanning microscope is used to block out-of-focus light, then optical sectioning is achieved, but image acquisition becomes very slow due to sequential scanning
Solution Approach 1:
The patent segments the detection process by dividing the detector into multiple pixel groups that can be independently activated. Each pixel group corresponds to a specific region in the sample plane, allowing parallel detection of fluorescence from different areas simultaneously, thus achieving fast confocal imaging without sequential scanning
Solution Approach 2:
The patent introduces a spatial dimension to the detection process by using a pixel array detector where different pixels detect light from different lateral positions simultaneously. This transforms the sequential one-dimensional scanning approach into a parallel two-dimensional detection approach, dramatically increasing acquisition speed while maintaining optical sectioning
2Measurement precision
If high light intensities are used in confocal laser scanning to achieve acceptable signal-to-noise ratio, then detection sensitivity is improved, but phototoxic effects on living cells increase
Solution Approach 1:
The patent segments the illumination and detection process into multiple independent pixel groups. By activating only the necessary pixel groups corresponding to the region of interest, the total light exposure to the sample is significantly reduced compared to illuminating the entire field, thereby maintaining signal-to-noise ratio while minimizing phototoxic effects
Solution Approach 2:
The patent applies local quality by enabling selective activation of specific pixel groups based on the illumination pattern. This allows concentrated detection sensitivity in regions where fluorescence is actually present while leaving other regions unexposed, thus improving signal-to-noise ratio locally without increasing overall phototoxicity
3Productivity
If DMD array is used as excitation and detection pinhole matrix in programmable array microscope, then parallelization is achieved, but detection efficiency decreases due to signal losses
Solution Approach 1:
The patent extracts the detection function from the illumination path by using a separate pixel array detector. The detector directly receives fluorescence from the sample through the beam splitter without requiring the DMD array to perform both illumination and detection functions, thereby eliminating signal losses associated with DMD reflection and diffraction
Solution Approach 2:
The patent makes the pixel array detector universal by having it serve both as the detection element and as the basis for defining detection regions. The same pixel array that detects fluorescence also determines which areas are monitored, eliminating the need for a separate DMD detection matrix and reducing overall system complexity and signal loss
4Object-affected harmful factors
If wide-field microscope is used to minimize light exposure, then phototoxic effects are reduced, but out-of-focus light superimposes on the signal reducing image quality
Solution Approach 1:
The patent segments the detection process into multiple pixel groups that can be independently controlled. By activating only the pixel groups corresponding to the in-focus plane while keeping other pixel groups inactive, the system achieves optical sectioning similar to confocal microscopy but with much lower light exposure since only necessary regions are illuminated and detected
Solution Approach 2:
The patent applies partial action by illuminating and detecting only the specific regions of interest in the sample plane rather than the entire field. This selective partial illumination and detection maintains wide-field microscopy's low light exposure advantage while achieving confocal-like optical sectioning by excluding out-of-focus regions from detection
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 allows for extremely parallelized scanning with confocal resolution, reduced light exposure, and increased frame rates, making it suitable for living cell imaging while minimizing phototoxic effects and optimizing image quality.
Implementation Method 1
a light source for providing illumination light
Implementation Method 2
a main beam splitter for splitting illumination light and fluorescent light
Implementation Method 3
a detector having a multiplicity of pixels for detecting fluorescent light emitted by the sample
Implementation Method 4
Single Photon Avalanche Diode array for sensitive and rapid detection
Data Source
AI summary
A microscope and method of microscopy having a light source for providing illumination light, a controllable manipulation device for generating in a variable manner an illumination pattern of the illumination light to be selected, an illumination beam path with a microscope lens for guiding the illumination pattern to a sample to be examined, a detector having a plurality of pixels for examining the fluorescent light emitted by the sample, a detection beam path for guiding the fluorescent light emitted by the sample to the detector, a main beam splitter for splitting illumination light and fluorescent light, a control and evaluation unit for controlling the manipulation device and for evaluating the data measured by the detector. The manipulation device is arranged in the illumination beam path upstream from the main beam splitter such that the pixel of the detector can be individually activated using the control and evaluation unit and in read out patterns to be selected.