Microscopic imaging method using a correction factor

Patent metadata

Field	Value
Publication number	US-11238575-B2
Application number	US-201916445802-A
Country	US
Kind code	B2
Filing date	Jun 19, 2019
Priority date	Jun 28, 2018
Publication date	Feb 1, 2022
Grant date	Feb 1, 2022

Abstract

A microscopic imaging method, includes illuminating a specimen with illumination radiation and capturing detection radiation along a detection axis. The detection radiation is caused by the illumination radiation, at a first time as a wide-field signal and at a second time as a composite signal. The composite signal is formed by a superposition of a confocal image and a wide-field image; extracting the confocal image by subtracting the wide-field signal from the composite signal, wherein a correction factor is used. A current correction factor is ascertained for each executed imaging and/or for each imaged specimen (1) and the confocal image is extracted using the respective current correction factor.

First claim

What is claimed is:

1. Microscopic imaging method, comprising:
illuminating a specimen with illumination radiation and capturing detection radiation along a detection axis, said detection radiation having been caused by the illumination radiation, at a first time as a wide-field signal and at a second time as a composite signal,
forming said composite signal by a superposition of a confocal signal and a wide-field signal,
extracting the confocal signal by subtracting the wide-field signal from the composite signal, wherein a correction factor is used,
ascertaining a current correction factor for each executed imaging and/or for each imaged specimen and
extracting the confocal signal using the respective current correction factor,

wherein, for the purposes of ascertaining the current correction factor in a correction plane chosen along the detection axis at a distance from a surface of the specimen, at least one correction wide-field image and one correction composite image are captured in each case, the image data thereof are ascertained and a current correction factor is ascertained on the basis of the ascertained image data; wherein the distance of the correction plane is chosen to be so large that no structures of the surface pass through the correction plane.

2. Method according to claim 1 , wherein the distance of the correction plane is chosen from a range of four to six full widths at half maximum of the pointspread function of the objective lens used to capture the detection radiation.

3. Method according to claim 1 , wherein at least one correction wide-field image and one correction composite image are captured in each case in a correction plane in front of and behind a current focal plane.

4. Method according to claim 1 , wherein the current correction factor is ascertained from a mean brightnesses of a correction wide-field image and a correction composite image.

5. Method according to claim 1 , wherein a current correction factor is ascertained for each pixel pair of the correction images.

Assignees

• Zeiss Carl Microscopy Gmbh

Inventors

• Drescher Viktor
• Langholz Nils

Classifications

• H04N23/56

  provided with illuminating means · CPC title

• H04N23/951

  by using two or more images to influence resolution, frame rate or aspect ratio · CPC title

• H04N5/3205

  using subtraction imaging techniques · CPC title

• G06T5/50 Primary

  using two or more images, e.g. averaging or subtraction · CPC title

• G02B21/008 Primary

  Details of detection or image processing, including general computer control · CPC title