Beam-Homogenized Microscope for Uniform Multi-Well TIRF Imaging
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Solution Overview
Problem
Existing fluorescence microscopy techniques, including TIRF microscopes, are inadequate for studying living, electrically active cells due to limitations such as the need for a prism that occludes culture media and prevents physical access, leading to unsatisfactory optical measurement of cellular activity.
Innovation Solution
A microscope with a beam homogenizer system that illuminates multi-well plates from beneath, using near-total-internal-reflection (TIR) to uniformly illuminate the bottom of wells, allowing for spatially-patterned illumination and simultaneous imaging of multiple wells with reduced autofluorescence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If a laser beam is used for illumination, then the illumination intensity is high and the coherence length is long, but the beam exhibits strong spatial coherence which causes speckle noise and reduces image quality
Solution Approach 1:
The patent segments the laser beam into multiple independent beams using a beam splitter, which divides the original coherent beam into several separate paths. This segmentation reduces the spatial coherence of the illumination while maintaining high intensity, thereby eliminating speckle noise and improving image quality without sacrificing illumination strength.
2Manufacturing precision
If multiple independent light sources are used to reduce speckle noise, then the spatial coherence is reduced, but the illumination intensity decreases and the coherence length becomes too short
Solution Approach 1:
The patent combines multiple independent light sources (LEDs) into a single illumination system that maintains high intensity. The beam splitter divides this combined illumination into multiple independent beams that illuminate different regions of the specimen, reducing spatial coherence and speckle noise while preserving the high illumination intensity and long coherence length needed for high-quality imaging.
3Manufacturing precision
If a beam splitter is introduced to divide the laser beam, then the spatial coherence is reduced improving image quality, but the device complexity increases
Solution Approach 1:
The beam splitter is designed to serve multiple functions simultaneously: it divides the laser beam into multiple independent beams for speckle reduction, maintains the optical path length for coherence preservation, and enables flexible illumination of multiple specimen regions. This multi-functionality reduces the need for additional separate components, thereby limiting the increase in device complexity while achieving significant improvements in image quality.
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 clear imaging of living cells with high signal-to-noise ratio and uniform illumination, facilitating optogenetic analysis and high-throughput recording of electrical activity in cells like neurons.
Implementation Method 1
A beam homogenizer divides the laser beam into a number of independent beams that collectively and uniformly illuminate the entire field of view
Implementation Method 2
A spatial light modulator performs optical sectioning to eliminate out-of-focus light
Implementation Method 3
Fluorescence microscopy is widely used in the life sciences because it is non-invasive and can be used to image living specimens
Data Source
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AI summary
The inventions provide microscopes for imaging samples within wells of multi-well plates. Microscopes of the disclosure include a beam homogenizer system that shapes a beam from a light source into a shape specific to the bottom of a well of a multi-well plate. In particular, microscopes of the disclosure can illuminate wells for imaging by passing light through a prism that is beneath the sample. The light enters the prism from the side and as refracted into the well at a steep angle such that the light only illuminates about a bottom ten microns of the well. The beam homogenizer shapes the light from the light source so that, instead of hitting the prism as a spot with an irregular shape, the light enters the prism in a substantially rectangular pattern with homogeneous optical power level over the pattern.