Multi-Well Plate Microscope with Beam-Homogenized TIR Illumination

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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 in sample access and autofluorescence, which overwhelm the signal from the sample.

Innovation Solution

A microscope design that illuminates samples within multi-well plates using a beam homogenizer to shape light into a uniform rectangular pattern, allowing near-total-internal-reflection (TIR) illumination of the sample bottom, coupled with a digital micromirror device (DMD) for spatially patterned illumination, and optical systems to minimize autofluorescence.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Object-affected harmful factors

If TIRF microscopy is used to reduce background fluorescence, then autofluorescence is reduced, but sample access is severely limited and culture media cannot be used

Engineering Contradiction:

ImproveautofluorescenceVSAvoidsample access

Core Design Contradiction:

Object-affected harmful factorsVSEase of operation

Solution Approach 1:

Instead of placing the prism above the sample (conventional TIRF), the prism is positioned below the multi-well plate to illuminate samples from the bottom. This inversion allows culture media and physical access from above while maintaining TIRF's advantage of reducing background fluorescence through total internal reflection at the glass-bottom well interface

Inventive Principle:

Principle #13The other way round (Inversion)

2Object-affected harmful factors

If conventional TIRF prism configuration is used, then background fluorescence is reduced, but optical power distribution is non-uniform across the sample

Engineering Contradiction:

Improvebackground fluorescenceVSAvoidoptical power uniformity

Core Design Contradiction:

Object-affected harmful factorsVSIllumination intensity

Solution Approach 1:

A beam homogenizer is introduced between the light source and the prism to create a uniform optical power distribution across the illumination area. This homogenizer ensures that different regions of the sample receive equal illumination intensity, solving the non-uniformity problem while maintaining the TIRF configuration for background reduction

Inventive Principle:

Principle #3Local quality

3Area of stationary object

If light illuminates the entire well, then complete sample coverage is achieved, but off-target light causes increased autofluorescence

Engineering Contradiction:

Improveillumination coverageVSAvoidoff-target autofluorescence

Core Design Contradiction:

Area of stationary objectVSObject-affected harmful factors

Solution Approach 1:

The illumination is configured to target only the bottom portion of the well (approximately bottom 10 microns) where cells are located, rather than illuminating the entire well volume. This selective illumination parameter change reduces off-target excitation of culture media and other components that would produce autofluorescence, while maintaining complete coverage of the sample at the well bottom

Inventive Principle:

Principle #35Parameter changes

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 spatial control, facilitating optogenetic analysis of electrical activity in cells like neurons, while minimizing autofluorescence and allowing physical access to the sample.

Implementation Method 1

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

Methodology Applied

Scientific EffectBeam homogenization:

Implementation Method 2

The light enters the prism from the side and is refracted into the well at an angle such that the light only illuminates about a bottom ten microns of the well

Methodology Applied

Scientific EffectRefraction: Refraction

Implementation Method 3

The light is restricted to the lower portion of the well by sending the beam into the prism at angle that promotes near-total-internal-reflection (TIR) within a glass bottom of the well

Methodology Applied

Scientific EffectTotal internal reflection: Total Internal Reflection

Implementation Method 4

The cells can include fluorescent reporter proteins that emit light in response to cellular electrical activity

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS12422660B2Microscope for imaging samples within wells of multi-well plates hold by stage and beam homogenizer for homogenizing beam

Publication Date: 2025.09.23 QUIVER HOLDINGS INC

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.