Method of locally imaging a structure in a sample at high spatial resolution in order to detect reactions of an object of interest to altered environmental conditions

Patent metadata

Field	Value
Publication number	US-2019011367-A1
Application number	US-201816123280-A
Country	US
Kind code	A1
Filing date	Sep 6, 2018
Priority date	Mar 7, 2016
Publication date	Jan 10, 2019
Grant date	—

Abstract

For high spatial resolution imaging a structure marked with luminescence markers, light that has an effect on the emission of luminescence light by the luminescence markers is directed onto a sample with an intensity distribution having a central zero point. Scan areas of the sample are scanned with the zero point. Luminescence light emitted out of a local area including the zero point is registered and assigned to the respective location of the zero point in the sample. Several copies of an object of interest are arranged in the scan areas and subjected to varying surrounding conditions. The individual scan areas are scanned with the respective zero point at least two times at two different stages of reactions to the varying surrounding conditions. Dimensions of the scan areas are limited such that they are not larger than 75% of a distance of intensity maxima delimiting the zero point.

First claim

We claim:

1 . A method of high resolution imaging a structure in a sample, the structure being marked with luminescence markers, the method comprising
directing light that has an effect on the emission of luminescence light by the luminescence markers onto the sample with an intensity distribution which has a zero point and intensity maxima neighboring the zero point in at least one direction and having a distance in the at least one direction; scanning scan areas with the zero point, the scan areas being parts of the sample; while scanning the scan areas, registering luminescence light emitted out of a local area including the zero point in the sample; assigning the registered luminescence light to a respective location of the zero point in the sample; and limiting dimensions of the scan areas, in the at least one direction in which the intensity maxima are neighboring the zero point in the sample, to not more than 75% of the distance of the intensity maxima in the at least one direction, wherein each of a plurality of copies of an object of interest is arranged such that it overlaps with one of the scan areas, and wherein the plurality of copies of the object of interest are subjected to varying surrounding conditions to measure reactions of the object of interest to the varying surrounding conditions, wherein the individual scan areas are scanned with the respective zero point at least two times at two different stages of the reactions to the varying surrounding conditions.

2 . The method of claim 1 , wherein the two times at which the scan areas are scanned with the respective zero point are selected from
a time prior to varying the surrounding conditions, a first time during varying the surrounding conditions, a second time during varying the surrounding conditions arranged at an interval of time to the first time, and a time after varying the surrounding conditions.

3 . The method of claim 1 , wherein different sets of the plurality of copies of the object of interest in the sample are subjected to different varying surrounding conditions.

4 . The method of claim 1 , wherein the surrounding conditions are varied by adding a chemical substance.

5 . The method of claim 1 , wherein the plurality of copies of the object of interest are arranged in a pattern defined with regard to fixed points of the sample, and wherein the scan areas are approached with the zero point in relation to the fixed points of the sample.

6 . The method of claim 1 , wherein at at least one time at which the individual scan areas are scanned with the respective zero point, the zero point is arranged in not more than 3 n or 2 n positions per scan area, wherein n is the number of the spatial dimensions in which the scan areas are scanned.

7 . The method of claim 1 , wherein the object of interest arranged in a plurality of copies in the sample is selected from a group including molecules, proteins, complexes, synapses, membranes, cell components and viruses.

8 . The method of claim 1 , wherein the dimensions of the scan areas in the at least one direction in which the intensity maxima are neighboring the zero point in the sample are not larger than 25% of the distance of the intensity maxima in the at least one direction.

9 . The method of claim 1 , wherein the dimensions of the scan areas in the at least one direction in which the intensity maxima are neighboring the zero point in the sample are not larger than a distance over which an intensity of the light in the at least one direction, starting at the zero point, increases up to 25% of the intensity of the light in the neighboring intensity maxima.

10 . The method of claim 1 , wherein, prior to scanning the scan areas, the structure in the sample is images in another way to determine a position of the scan areas in the sample.

11 . The method of claim 10 , wherein, prior to scanning the scan areas, a larger area of the sample larger than the scan areas is scanned with the zero point at at least one of
an at least 50% lower intensity of the light and an at least 50% higher scanning speed.

12 . The method of claim 10 , wherein a scanner is used for scanning the scan areas, and wherein another scanner is used for scanning the larger area of the sample.

13 . The method of claim 12 , wherein a sample holder is moved relative to an objective lens by which the light is directed onto the sample for scanning the larger area of the sample, and wherein at least one of
an electro-optical scanner, an acousto-optical deflector, a galvo scanner and a galvo mirror
is used for scanning the scan areas in the at least one direction.

14 . The method of claim 1 , wherein the light that has an effect on the emission of luminescence light by the luminescence markers is luminescence enabling light.

15 . The method of claim 14 , wherein the luminescence enabling light enables the emission of luminescence light by the luminescence markers in that it excites the luminescence markers for luminescence or transferred out of a dark state into an excitable state.

16 . The method of claim 1 , wherein the light that has an effect on the emission of luminescence light by the luminescence markers is luminescence inhibiting light whose wavelength is selected such as to inhibit the emission of luminescence light by the luminescence markers outside the zero point.

17 . The method of claim 16 , wherein the luminescence inhibiting light is stimulation light whose wavelength is selected such as to inhibit the emission of luminescence light by the luminescence markers outside the zero point by stimulated emission, wherein the stimulation light is directed onto the sample together with excitation light whose wavelength is selected such as to excite the luminescence markers for emission of luminescence light and which has an intensity distribution comprising a maximum overlapping with the zero point of the luminescence inhibiting light.

18 . The method of claim 14 , wherein, prior to scanning the scan areas with the zero point, additional switch off light is directed onto the sample with such an intensity distribution that the switch off light, in neighboring areas, switches the luminescence markers into an inactive state, wherein the neighboring areas are neighboring the scan areas in the at least one direction in which the intensity maxima are neighboring the zero point in the sample.

19 . The method of claim 18 , wherein the sample is scanned with the scan areas, wherein the scan areas in all positions or in selected positions of the scan areas in the sample are scanned with the zero point.

20 . The method of claim 18 , wherein the intensity distribution of the switch off light comprises a local intensity minimum formed by destructive interference in the scan area.

21 . The method of claim 18 , wherein, prior to or temporarily overlapping with directing the switch off light to the sample, switch on light is directed onto the scan areas that switches the luminescence markers into their active state.

22 . The method of claim 18 , wherein luminescence light emitted by the switchable luminescence markers upon being switched on or off is registered and evaluated.

23 . The method of claim 22 , wherein a result of the step of evaluating is at least one of the following:
whether a respective one of the scan areas delimited by a respective one of the neighboring areas will be scanned with the zero point; wheth

Assignees

• Max Planck Gesellschaft

Inventors

• Hell Stefan W
• Goettfert Fabian
• Westphal Volker

Classifications

• G02B21/008

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

• G02B21/0036

  Scanning details, e.g. scanning stages · CPC title

• G02B21/0072

  details concerning resolution or correction, including general design of CSOM objectives · CPC title

• G02B27/58

  Optics for apodization or superresolution; Optical synthetic aperture systems · CPC title

• G01N21/6428

  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) · CPC title