Luminescence microscopy

Patent metadata

Field	Value
Publication number	US-9404867-B2
Application number	US-201013518115-A
Country	US
Kind code	B2
Filing date	Oct 22, 2010
Priority date	Dec 22, 2009
Publication date	Aug 2, 2016
Grant date	Aug 2, 2016

Abstract

A luminescence microscopy method includes a sample being used, which comprises a certain substance, wherein the certain substance can be converted repeatedly from a first state, in which it can be excited into emitting luminescence radiation, into a second state, in which it cannot be excited into emitting luminescence radiation. The substance present in the sample can be brought into the first state by irradiating switch radiation. The certain substance can be excited into emitting luminescence radiation by irradiating excitation radiation. The sample emitting luminescence radiation can be displayed. A high-resolution selection of sample regions extending perpendicularly to a sample surface is carried out by irradiating either the switch radiation or the excitation radiation as structured illumination of the sample. A high-resolution selection of the sample surface is carried out by irradiating the switch radiation and/or the excitation radiation as TIRF illumination of the sample.

First claim

What is claimed is:

1. A luminescence microscopy method comprising:
providing a sample having a sample surface and including a specific substance, wherein the specific substance can be repeatedly transformed from a first state in which it can be excited to emit luminescence radiation into a second state in which it cannot be excited to emit luminescence radiation;
transforming the specific substance present in the sample from the second state into the first state by irradiation of switching radiation; and
exciting the specific substance present in the sample to emit luminescence radiation by irradiation of excitation radiation and imaging the sample emitting luminescence radiation;
wherein one or more sample areas extending perpendicular to the sample surface are selected with high resolution by irradiating one of the switching radiation and the excitation radiation as structured sample illumination being structured in a plane parallel to the sample surface,
and wherein the one or more sample areas extending parallel to the sample surface selected with high resolution by irradiating the other one of the switching radiation and the excitation radiation as TIRF illumination of the sample.

2. The method of claim 1 , wherein irradiating the switching beam and irradiating the excitation radiation is repeated while performing at least one of rotating and changing the structure of the structured illumination, and the method further comprising combining the images obtained during the repetitions into a high-resolution image.

3. The method according to claim 2 , wherein the specific substance of the sample is transformed by irradiation of reset radiation into a state that cannot be excited to luminescence, and wherein after the imaging the sample is irradiated with reset radiation each time.

4. A microscope for luminescence microscopy of a sample, the sample including a specific substance, wherein the specific substance can be repeatedly transformed from a first state in which it can be excited to emit luminescence radiation into a second state in which it cannot be excited to emit luminescence radiation, the microscope comprising:
a switching radiation source and a switching beam path provided thereto for irradiating switching radiation onto the sample, in order to transform the specific substance present in the sample from the second state into the first state;
an excitation radiation source and an excitation beam path provided thereto for irradiating excitation radiation onto the sample, in order to excite the emission of luminescence radiation in the sample and to transform the specific substance present in the sample into the second state; and
a detector and a detection beam path provided thereto for imaging the sample emitting the luminescence radiation,
wherein, for the high-resolution selection of sample areas extending perpendicular to a sample surface, one of the switching beam path for irradiating the switching radiation and the excitation beam path for irradiating the excitation radiation effects a structured illumination of the sample, wherein this illumination is structured in a plane parallel to the sample surface,
and wherein, for the high-resolution selection of sample areas extending parallel to the sample surface, the other one of the switching beam path for irradiating the switching radiation and the excitation beam path for irradiating the excitation radiation effects a TIRF illumination of the sample.

5. The microscope of claim 4 , further comprising a device for rotating the structure of the structured illumination relative to the sample.

6. The microscope of claim 5 , further comprising a reset radiation source and a reset radiation path provided thereto for irradiating reset radiation in order to transform the sample into a state that cannot be excited to luminescence.

7. Microscope according to claim 4 , further comprising a reset radiation source and a reset radiation path provided thereto for irradiating reset radiation in order to transform the sample into a state that cannot be excited to luminescence.

8. The method of claim 1 , wherein irradiating the switching beam and irradiating the excitation radiation is repeated while performing shifting of the structured illumination, the method further comprising combining the images obtained during the repetitions into a high-resolution image.

9. The microscope of claim 4 , further comprising a device for shifting the structure of the structured illumination relative to the sample.

10. The method of claim 1 , wherein the structured sample illumination is generated by imaging a diffraction pattern into the sample.

11. The microscope of claim 4 , wherein the one of switching beam path and excitation beam path images a diffraction pattern into the sample to generate the structured sample illumination.

Assignees

• Kempe Michael
• Netz Ralf
• Krampert Gerhard
• Zeiss Carl Microscopy Gmbh

Inventors

• Kempe Michael
• Netz Ralf
• Krampert Gerhard

Classifications

• G01N21/6458 Primary

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

• G02B21/16

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