Serial Multichannel Microscopy Without Destaining Steps

Solution Overview

Problem

Current methods for repetitive fluorescent labelling and imaging in biological specimens require a destaining step between imaging steps, which is time-consuming and stressful for the specimens, and are prone to acquisition bleaching due to variable fluorochrome stability and binding constants, leading to misleading differential images.

Innovation Solution

A method involving repetitive fluorescent labelling and imaging without destaining steps, where images from different cycles are subtracted to account for acquisition bleaching, using degradation functions to adjust for fluorochrome decay, and spacing out reagents to enhance precision.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If destaining steps are used between imaging steps, then fluorescent labelling can be removed, but the process time increases considerably and specimen stress increases

Engineering Contradiction:

Improvefluorescent labelling removalVSAvoidprocess time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent extracts the harmful destaining step from the imaging process by using image subtraction to remove the need for chemical or radiation-based destaining. The fluorescent label is not physically removed but mathematically eliminated through computational methods, thereby saving time and reducing specimen stress while maintaining labelling reliability.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent replaces the mechanical/chemical destaining process with an information-processing approach. Instead of using oxygenation reagents or radiation to destroy fluorescent labels, the system uses digital image subtraction to eliminate the need for physical destaining, substituting a computational mechanism for a chemical/radiation-based one.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Reliability

If destaining steps are used between imaging steps, then fluorescent labelling can be removed, but specimen stress increases

Engineering Contradiction:

Improvefluorescent labelling removalVSAvoidspecimen stress

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent extracts the harmful destaining step from the imaging process by using image subtraction to remove the need for chemical or radiation-based destaining. The fluorescent label is not physically removed but mathematically eliminated through computational methods, thereby saving time and reducing specimen stress while maintaining labelling reliability.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent replaces the mechanical/chemical destaining process with an information-processing approach. Instead of using oxygenation reagents or radiation to destroy fluorescent labels, the system uses digital image subtraction to eliminate the need for physical destaining, substituting a computational mechanism for a chemical/radiation-based one.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Adaptability or versatility

If image subtraction is used to detect different target moieties, then multiple informations can be generated, but acquisition bleaching causes decay of fluorescence emission over time

Engineering Contradiction:

Improvemultiple information detectionVSAvoidfluorescence emission stability

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent applies preliminary action by measuring the fluorescence intensity of a reference sample before the imaging experiment. This reference measurement is used to calculate a correction factor that compensates for acquisition bleaching during the experiment, allowing accurate detection of multiple target moieties without the reliability issues caused by fluorescence decay.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent implements feedback by continuously monitoring fluorescence intensity changes and using the reference sample data to dynamically adjust the image subtraction calculations. The correction factor based on reference measurements provides feedback that compensates for bleaching effects, ensuring reliable detection throughout the experiment.

Inventive Principle:

Principle #23Feedback

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 precise, time-efficient imaging without destaining, minimizing specimen stress and improving image precision by accounting for fluorochrome decay, enabling miniaturization and parallel processing of multiple samples.

Implementation Method 1

a first conjugate comprising a first antigen recognizing moiety Y and a first fluorescent moiety X, thereby binding at least a part of the first conjugate to the target moieties recognized by the first antigen recognizing moiety Y

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Implementation Method 2

the intensity of the first image is reduced by a degradation function and then subtracted from the at least one second image

Methodology Applied

Scientific EffectAcquisition bleaching:

Data Source

PatentUS12601683B2Serial multichannel microscopy

Publication Date: 2026.04.14 MILTENYI BIOTEC BV & CO KG

AI summary

The invention is directed to a much faster way of combining multiple colour channels in fluorescence microscopy for complex fast diagnostic and research purposes. The advantage is that the procedure and the microscope setting can be simplified and miniaturised to an extend that the construction of such automatic systems become much easier and by omitting the erasing step the speed of analysis is faster and moved to the processing of the images by image processing systems, nowadays working at almost real time.