Cytometric Analysis Using Continuous Microscope Scanning
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Solution Overview
Problem
Current cytometric analysis methods for cell samples face challenges in reducing mechanical loads and light exposure, which can cause phototoxicity and vibrations, especially when analyzing adherent cells in microtiter plates, leading to impaired cell viability and incomplete data acquisition.
Innovation Solution
A method involving continuous movement of cell samples relative to a microscope's optical system, alternating between transmission and fluorescence modes, allowing for brief illumination times and reduced mechanical stress, enabling the recording and analysis of images in both modes to quantify fluorescence intensities while minimizing damage to cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If the sample carrier is moved incrementally with jerky movements to record images, then complete image coverage is achieved, but acceleration forces and vibrations cause cell culture changes and surface waves that impede transmission image recording
Solution Approach 1:
Instead of moving the sample carrier incrementally with stopping at each position, the patent inverts the approach by moving the microscope optics continuously over the stationary sample carrier. This allows smooth, continuous scanning without jerky movements, eliminating acceleration forces and vibrations while achieving complete image coverage through the scanning motion of the optical system.
Solution Approach 2:
The patent replaces the mechanical sample carrier movement system with a scanning optical system. By using galvanometer mirrors or piezoelectric actuators to deflect the light path and scan the microscope objective over the sample, the system achieves precise positioning without physical movement of the heavy sample carrier, thereby eliminating mechanical vibrations and acceleration forces.
2Manufacturing precision
If the entire optical system is moved relative to the sample carrier to prevent oscillations, then image recording stability is improved, but the heavy weight of microscope components makes quick movements difficult
Solution Approach 1:
The patent replaces the heavy mechanical optical system movement with a stationary optical system and uses electronic scanning through galvanometer mirrors or piezoelectric actuators. This substitution allows rapid positioning and scanning without the inertia and mechanical constraints of moving the entire optical assembly, achieving both stability and speed.
Solution Approach 2:
Instead of moving the optical system in three dimensions (x, y, z) which is mechanically complex and slow, the patent uses a stationary optical system with scanning mirrors that achieve the same effect by deflecting light paths in two dimensions (x, y) through angular movements, adding a dimensional aspect to the positioning approach.
3Measurement precision
If cells are covered with liquid during examination, then fluorescence quantification is enabled, but surface waves are generated that impede transmission image recording
Solution Approach 1:
The patent uses pulsed or periodic illumination instead of continuous lighting, synchronizing the light source activation with the image capture timing. This periodic action allows fluorescence excitation only when needed for measurement, reducing overall light exposure and minimizing liquid surface wave generation while maintaining sufficient signal for quantification.
Solution Approach 2:
The patent applies partial illumination by selectively illuminating only the regions of interest or using just enough light intensity to achieve the required measurement precision. This reduces the overall energy input that causes liquid surface waves, while still obtaining sufficient fluorescence signal for accurate quantification through optimized detection sensitivity.
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 reduces phototoxicity and mechanical stress, allowing for more accurate and non-invasive fluorescence quantification, enabling repeated analysis of the same sample over time and providing detailed sub-cellular information with reduced exposure times, suitable for live cell analysis.
Implementation Method 1
at least one or more images of a sub-region of the cell samples are recorded in the transmission mode
Implementation Method 2
The fluorescent dyes in the cells are excited by the laser beam, as a result of which the cells scatter laser light. In so doing, the light scattering and the emitted fluorescent light are detected.
Implementation Method 3
the cells scatter laser light. In so doing, the light scattering and the emitted fluorescent light are detected.
Data Source
AI summary
The present application relates to a method for the cytometric analysis of multiple cell samples by a microscope for examining multiple cell samples under a microscope, wherein the microscope can be or is operated, selectively and/or alternatingly, in a transmission mode and/or in a fluorescence mode, and wherein at least one cell sample has at least one fluorescence marker. The method includes; moving the cell samples continuously in one plane relative to an optical system of the microscope having at least one microscope camera, wherein, during the movement of the cell samples, at least one or more images of a sub-region of the cell samples are recorded in the transmission mode or in the fluorescence mode and at least one or more images of the same sub-region of the cell samples are recorded in the fluorescence mode by at least one microscope camera.