Method and apparatus for imaging a sample

Patent metadata

Field	Value
Publication number	US-2018266958-A1
Application number	US-201715460517-A
Country	US
Kind code	A1
Filing date	Mar 16, 2017
Priority date	Mar 16, 2017
Publication date	Sep 20, 2018
Grant date	—

Abstract

A method and apparatus for imaging an area of interest (22) of a sample (20) is disclosed. The sample (20) comprises a plurality of fluorophores (25). The method comprises illuminating (320) along an illumination path (40) the area of interest (22) and adjacent areas (27a, 27b) of the sample (20) with radiation at a first wavelength to excite the fluorophores (25), providing (330) along the illumination path (40) an illumination pattern with a minimum intensity in the area of interest to switch off the fluorophores (25) in the adjacent areas, and imaging (340) the area of interest (22) at a detection path, the to detection path being substantially perpendicular to the illumination path (40).

First claim

1 . A method for imaging an area of interest ( 22 ) of a sample ( 20 ), wherein the sample ( 20 ) comprises a plurality of fluorophores ( 25 ), the method comprising:
illuminating ( 320 ) along an illumination path ( 40 ) the area of interest ( 22 ) and adjacent areas ( 27 a , 27 b ) of the sample ( 20 ) with radiation at a first wavelength to excite the fluorophores ( 25 ); providing ( 330 ) along the illumination path ( 40 ) an illumination pattern with a minimum intensity in the area of interest to switch off the fluorophores ( 25 ) in the adjacent to areas; imaging ( 340 ) the area of interest ( 22 ) at a detection path, the detection path being substantially perpendicular to the illumination path ( 40 ).

2 . The method of claim 1 , wherein the providing of the illumination path ( 40 ) comprises providing an illumination beam having two orthogonal phase components.

3 . The method of claim 1 , wherein the illumination of the area of interest ( 22 ) and the adjacent areas ( 27 a , 27 b ) is substantially a two-dimensional plane extending in the direction of the illumination path ( 40 ),

4 . The method of claim 1 , further comprising providing a further illumination pattern having a plurality of minimum intensities in a direction substantially perpendicular to the illumination path ( 40 ) and the detection path.

5 . The method of claim 1 , wherein the first wavelength is about 405 nm.

6 . The method of claim 1 , wherein the second wavelength is about 488 mm

7 . The method of claim 1 , further comprising combing images of a plurality of areas of interest ( 22 ) to form a final image.

8 . An apparatus ( 10 ) for the imaging a sample ( 20 ), wherein the sample ( 20 ) comprises a plurality of fluorophores ( 25 ), the apparatus ( 10 ) comprising:
a first laser ( 30 ) emitting radiation at a first wavelength along an illumination path ( 40 ) and illuminating the sample ( 20 ); a second laser ( 50 ) emitting radiation at a second wavelength along the illumination path ( 40 ) and providing an illumination pattern with a minimum intensity at the sample ( 20 ); an illumination objective lens ( 45 ) in the illumination path ( 40 ); and a detection objective lens ( 60 ) for imaging fluoresced radiation from the sample to ( 20 ) along a detection path ( 70 ), wherein the detection path ( 70 ) is substantially perpendicular to the illumination path ( 40 ).

9 . The apparatus ( 10 ) of claim 8 , further comprising a phase delay device ( 90 ) for creating from the radiation at a second wavelength an illumination beam having two orthogonal phase components.

10 . The apparatus ( 10 ) of claim 9 , wherein the phase delay device is a half-moon plate ( 90 ).

11 . The apparatus ( 10 ) of claim 8 , further comprising an Glann-Thompson prism ( 100 ) in the illumination path ( 40 ).

12 . The apparatus of claim 8 , further comprising a lambda half plate ( 110 ) in the illumination path ( 40 ).

13 . The apparatus of claim 8 , further comprising a cylinder lens ( 120 ) in the illumination path ( 40 ).

14 . The apparatus ( 10 ) of claim 8 , further comprising a unit for providing a further illumination pattern having a plurality of minimum intensities in a direction substantially perpendicular to the illumination path ( 40 ) and the detection path ( 70 ).

15 . The apparatus ( 10 ) of claim 8 , further comprising a read-out laser ( 80 ) for illuminating the sample ( 20 ) at the area of interest ( 22 ) along the illumination path ( 40 ).

16 . The apparatus ( 10 ) of claim 8 , further comprising a recording device ( 130 ) connected to the detection objective lens ( 60 ).

Assignees

• European Molecular Biology Laboratory
• Deutsches Krebsforschungszentrum Stiftung Des Oeffentlichen Rechts

Inventors

• Hufnagel Lars
• Hoyer Patrick

Classifications

• G01N21/6402

  Atomic fluorescence; Laser induced fluorescence · CPC title

• G01N21/6458 Primary

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

• G01N2021/6463

  Optics · CPC title

• G02F1/0126

  Opto-optical modulation, i.e. control of one light beam by another light beam, not otherwise provided for in this subclass · 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