# US 12,601,683 B2 · Serial multichannel microscopy

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- Number: US 12,601,683 B2
- Document ID: US12601683B2
- Status: Granted
- Kind: B2
- Application number: 17/768,276
- Filed: 2020-11-11
- Published: 2024-04-25
- Granted: 2026-04-14
- Earliest claimed: 2019-11-11
- Assignees: Miltenyi Biotec GmbH
- Applicants: Miltenyi Biotec B.V. & Co. KG
- Inventors: Werner Muller
- Web page: https://patentgrep.com/patent/US12601683B2

## CPC

- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 33/58: involving labelled substances (G01N33/53 takes precedence)
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G01N 33/582: {with fluorescent label}
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

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.

## Claims

### Claim 1

A method for detecting target moieties in a sample of biological specimen by:
a) contacting the sample with 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
b) removing the first conjugates not bound to the target moieties from the sample
c) obtaining a first image of the sample thereby detecting the target moieties labelled with the first conjugate; and
d) repeating steps a) to c) with at least one second conjugate comprising a second antigen recognizing moiety Y′ and a second fluorescent moiety X′, thereby obtaining at least one second image and wherein the first and second antigen recognizing moieties Y and Y′ bind to different target moieties characterized in that the intensity of the first image is reduced by a degradation function and then subtracted from the at least one second image.

### Claim 2 (depends on 1)

The method according to claim 1, characterized in that the degradation function is calculated as a decrease in intensity of 5-50%.

### Claim 3 (depends on 1)

The method according to claim 1, characterized in that steps a) to c) are repeated in at least 2 cycles, wherein in each cycle an image is obtained and wherein after each cycle the image of the previous cycle is subtracted from the image of the current cycle.

### Claim 4 (depends on 1)

The method according to claim 1, characterized in that steps a) to c) are repeated in at least 2 cycles, wherein in each cycle an image is obtained and wherein after the last cycle, the image of each cycle is subtracted from the image of the respective previous cycle.

### Claim 5 (depends on 1)

The method according to claim 1, characterized in that the images are obtained as pixel graphic images and the subtraction of the images is obtained by a subtracting algorithm of the pixels.

### Claim 6 (depends on 1)

The method according to claim 1 characterized in that the conjugates not bonded to the target moieties are removed from the sample by washing.

### Claim 7 (depends on 1)

The method according to claim 1 characterized in that after the last cycle is performed, the conjugates bonded to the target moieties are removed from the target moieties at least in part by radiation, oxidative or enzymatic treatment.

### Claim 8 (depends on 1)

The method according to claim 1 characterized in that the fluorescent moiety X is selected from the group consisting of xanthene dyes, rhodamine dyes, coumarine dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyl oxazole dyes, pyromethene dyes, acridine dyes, oxadiazole dyes, carbopyronine dyes, benzpyrylium dyes, fluorene dyes, fluorescent oligomers or fluorescent polymers.

### Claim 9 (depends on 1)

The method according to claim 1, characterized in that the fluorescent moieties of the conjugates decay by exposure to radiation.

### Claim 10 (depends on 1)

The method according to claim 1, characterized in that images of the first and second conjugates not bound to the respective target moieties are used to calibrate the decay by exposure to radiation.

### Claim 11 (depends on 1)

The method according to claim 1, characterized in that the two fluorescent moieties X and X′ provided have absorption maxima differing by at least 10 nm.