Locally imaging a structure in a sample at high spatial resolution

Patent metadata

Field	Value
Publication number	US-9891417-B2
Application number	US-201615271888-A
Country	US
Kind code	B2
Filing date	Sep 21, 2016
Priority date	Sep 22, 2015
Publication date	Feb 13, 2018
Grant date	Feb 13, 2018

Abstract

For high spatial resolution imaging a structure in a sample, the structure being marked with luminescence markers, light that has an effect on the emission of luminescence light by the luminescence markers is directed onto the sample with an intensity distribution having a zero point and intensity maxima neighboring the zero point in at least one direction. A scan area which is a part of the sample is 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. Dimensions of the scan area, in at least one direction in which the intensity maxima are neighboring the zero point, are limited such that they are not larger than 75% of a distance of the intensity maxima in the at least one direction.

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 a scan area with the zero point, the scan area being a part of the sample;
while scanning the scan area 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 area, 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.

2. The method of claim 1 , wherein the dimensions of the scan area 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.

3. The method of claim 1 , wherein the dimensions of the scan area 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.

4. The method of claim 1 , wherein the scan area is repeatedly scanned with the zero point.

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

6. The method of claim 5 , wherein, prior to scanning the scan area, a larger partial area of the sample 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.

7. The method of claim 5 , wherein a scanner is used for scanning the scan area, and wherein another scanner is used for scanning the larger area of the sample.

8. The method of claim 5 , 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 area in the at least one direction.

9. The method of claim 1 , wherein the light 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.

10. The method of claim 9 , 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 in the area of the zero point of the luminescence inhibiting light.

11. The method of claim 10 , wherein, prior to scanning the scan area with the zero point, additional switch off light is directed onto the sample with such an intensity distribution that the switch off light, in a neighboring area, switches the luminescence markers into an inactive state, wherein the neighboring area is neighboring the scan area in the at least one direction in which the intensity maxima are neighboring the zero point of the stimulation light in the sample.

12. The method of claim 11 , wherein the sample is scanned with the scan area, wherein the scan area in all positions or in selected positions of the scan area in the sample is scanned with the zero point.

13. The method of claim 11 , wherein the intensity distribution of the switch off light comprises a local intensity minimum formed by destructive interference in the scan area.

14. The method of claim 11 , wherein, prior to or temporarily overlapping with directing the switch off light to the sample, switch on light is directed onto the scan area of the sample that switches the luminescence markers into their active state.

15. The method of claim 14 , wherein luminescence light emitted by the switchable luminescence markers upon being switched on or off is registered and evaluated.

16. The method of claim 15 , wherein a result of the step of evaluating is at least one of the following:
whether the scan area delimited by the neighboring area will be scanned with the zero point;
whether the excitation light will be directed onto the sample in the scan area delimited by the neighboring partial area; and
under which conditions directing the stimulation light onto the sample in each position of the zero point in the scan area delimited by the neighboring area, and registering the luminescence light emitted out of the area of the zero point will be interrupted.

17. The method of claim 1 , wherein the luminescence light emitted out of the area of the zero point is registered with a point sensor whose position with regard to the sample remains unchanged during scanning the scan area.

18. The method of claim 1 , wherein several scan areas of the sample are scanned simultaneously.

19. The method of claim 18 , wherein several copies of an object of interest are arranged in the sample with an overlap with the several scan areas, wherein an image of the object of interest is composed of partial images of the object which are obtained in scanning the several scan areas.

20. A method of high spatial 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 at an intensity distribution which has a zero point and intensity maxima neighboring the zero point in at least one direction;
scanning a scan area with the zero point, the scan area being a part of the sample;
while scanning the scan area registering luminescence light emitted out of a local area including the zero point in the sample; and
assigning the registered luminescence light to a respective location of the zero point in the sample;
wherein the scan area is scanned with the zero point starting at a center point and with increasing distance to the center point in the at least one direction.

21. The method of claim 20 , wherein the scan area is scanned along a spiral course around the center point.

22. A scanning luminescence light microscope comprising
a light source configured to provide light;
a light shaper configured to direct the light onto a sample with an intensity distribution having a zero point and intensity maxima neighboring the zero point;
a scanner configured to scan a scan area with the zero point, the scan area being a partial area of the sample;
a detector configured to register luminescence light emitted out of the area of the zero point; and
a controller programmed with software implementing a method of high resolution

Assignees

• Max Planck Gesellschaft

Inventors

• Hell Stefan W
• Goettfert Fabian
• Westphal Volker

Classifications

• G02B21/0076

  arrangements using fluorescence or luminescence · CPC title

• G02B21/0036 Primary

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

• G02B27/58

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

• G02B21/16

  adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)} · CPC title

• G01N21/6458

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title