Resolution enhancement for line scanning excitation microscopy systems and methods

• US 10,247,930 B2
• Filed: 09/22/2015
• Issued: 04/02/2019
• Est. Priority Date: 09/24/2014
• Status: Active Grant

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Shroff, Hari, York, Andrew, Giannini, John, Kumar, Abhishek
• Primary Examiner(s): Font, Frank G
• Application Number:
  US15/512,870
• Publication Number:
  US 20170254997A1
• Time in Patent Office: 1,288 Days
• Field of Search: 359385, 359388, 359389
• CPC Class Codes: G02B 21/0032 Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06
• CPC Class Codes: G02B 21/0036 Scanning details, e.g. scanning stages
• CPC Class Codes: G02B 21/0048 scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 26/101 with both horizontal and vertical deflecting means, e.g. raster or XY scanners colour television using laser beams scanning a display screen H04N9/3129
• CPC Class Codes: G02B 27/58 Optics for apodization or superresolution; Optical synthetic aperture systems

Abstract

A resolution enhancement technique for a line scanning confocal microscopy system that generates vertical and horizontal line scanning patterns onto a sample is disclosed. The line scanning confocal microscopy system is capable of producing line scanning patterns through the use of two alternative pathways that generate either the vertical line scanning pattern or horizontal line scanning pattern.

Claims

1. A line scanning microscopy system comprising:

a light source for transmitting a single light beam;

a scanning apparatus arrangement for relaying the single light beam along either a first pathway for generating line scans that form a vertical line-scan pattern or a second pathway for generating line scans that form a horizontal line-scan pattern;

an optic arrangement for relaying the line scans of the vertical line-scan pattern or line scans of the horizontal line-scan pattern to illuminate a sample and generate fluorescent line scan emissions formed in either the vertical line-scan pattern or the horizontal line-scan pattern;

a scaling component for scaling the fluorescent line scan emissions, to either locally contract each of the fluorescent line scan emissions to produce a contracted fluorescent line scan emissions that form a contracted line-scan pattern or expand each of the fluorescent line-scan emissions to produce an expanded fluorescent line scan emissions to form an expanded line-scan pattern; and

a detection apparatus for capturing an image of the expanded line-scan pattern or the contracted line-scan pattern.

2. The microscopy system of claim 1, wherein the contracted fluorescent line scan emissions of the contracted line-scan pattern define a first width that is less than a second width defined by each of the fluorescent line scan emissions of the vertical line-scan pattern or the horizontal line-scan pattern.

3. The microscopy system of claim 1, wherein each of the contracted fluorescent line-scan emissions of the contracted line-scan pattern define a first longitudinal axis and each of the fluorescent line scan emissions of the vertical line-scan pattern or the horizontal line-scan pattern define a second longitudinal axis, and wherein the first longitudinal axis of each of the contracted fluorescent line scan emissions of the contracted line-scan pattern is spaced from an adjacent first longitudinal axis of another scaled fluorescent line scan emissions by a first distance, and wherein the second longitudinal axis of each of the fluorescent line scan emissions of the vertical line-scan pattern or horizontal line-scan pattern is spaced from an adjacent second longitudinal axis of another fluorescent longitudinal axis by a second distance, and wherein the first distance is equal to the second distance.

4. The microscopy system of claim 1, wherein each of the expanded fluorescent line scan emissions of the expanded line-scan pattern define a first width that is equal to a second width defined by each of the fluorescent line scan emissions of the vertical line-scan pattern or the horizontal line-scan pattern.

5. The microscopy system of claim 1, wherein each of the expanded fluorescent line scan emissions of the expanded line-scan pattern define a first longitudinal axis and each of the fluorescent line scan emissions of the vertical line-scan pattern or the horizontal line-scan pattern define a second longitudinal axis, and wherein the first longitudinal axis of each of the expanded fluorescent line scan emissions of the expanded line-scan pattern is spaced from an adjacent first longitudinal axis of another scaled fluorescent line scan emissions by a first distance, and wherein the second longitudinal axis of each of the fluorescent line scan emissions of the vertical line-scan pattern or the horizontal line-scan pattern is spaced from an adjacent second longitudinal axis of another adjacent fluorescent line scan emissions by a second distance, wherein the first distance is greater than the second distance.

6. The microscopy system of claim 1, wherein the scaling component comprises a scanning apparatus for scanning the contracted fluorescent line scan emissions of the contracted line-scan pattern or the expanded fluorescent line scan emission expanded line-scan pattern onto the detection apparatus.

7. The microscopy system of claim 1, wherein the light source comprises a laser.

8. The microscopy system of claim 1, wherein the scanning apparatus arrangement for the first pathway comprises a first scanning apparatus that relays the single light beam to a first mirror that reflects the single light beam through a first pair of lenses to a second mirror for relaying the single light beam to a second scanning apparatus.

9. The microscopy system of claim 1, wherein the scanning apparatus arrangement for the second pathway comprises the first scanning apparatus that relays the single light beam to a third mirror that reflects the single light beam through a second pair of lenses to a fourth mirror for relaying the single light beam to a second scanning apparatus.

10. The microscopy system of claim 1, wherein the scanning apparatus arrangement comprises a first image rotation device along the first pathway and a second image rotation device along the second pathway.

11. A microscopy system comprising:

a first light source that transmits a first single light beam and a second light source that transmits a second single light beam;

a first optic arrangement that combines the first single light beam and the second single light beam to form a combined light beam;

a scanning apparatus arrangement for relaying the combined light beam along either a first pathway for generating line scans that form a vertical line-scan pattern or a second pathway for generating line scans that form a horizontal line-scan pattern;

a second optic arrangement for relaying the line scans of the vertical line-scan pattern or line scans of the horizontal line-scan pattern to illuminate a sample and generate a fluorescent line-scan emissions formed in either the vertical line-scan pattern or the horizontal line-scan pattern;

a scaling component for scaling the fluorescent line scan emissions to either locally contract each of the fluorescent line scan emissions to produce a contracted fluorescent line scan emissions to form a contracted line-scan pattern, or expand each of the fluorescent line-scan emissions to produce an expanded fluorescent line scan emissions to form an expanded line-scan pattern; and

a detection apparatus for capturing an image of the expanded line-scan pattern or the contracted line-scan pattern.

12. The microscopy system of claim 11, wherein the first light source or the second light source comprises a depletion laser source that transmits a stimulated emission depletion beam.

13. The microscopy system of claim 12, further comprising:

a third optic arrangement in operative association with the depletion laser source for providing pulse-stretching, polarization control and shuttering functions.

14. The microscopy system of claim 11, wherein the contracted fluorescent line scan emissions of the contracted line-scan pattern define a first width that is less than a second width defined by each of the fluorescent line scan emissions of the vertical line-scan pattern or the horizontal line-scan pattern.

15. The microscopy system of claim 11, wherein each of the contracted fluorescent line-scan emissions of the contracted line-scan pattern define a first longitudinal axis and each of the fluorescent line scan emissions of the vertical line-scan pattern or the horizontal line-scan pattern define a second longitudinal axis, and wherein the first longitudinal axis of each of the contracted fluorescent line scan emissions of the contracted line-scan pattern is spaced from an adjacent first longitudinal axis of another contracted fluorescent line scan emissions by a first distance, and wherein the second longitudinal axis of each of the fluorescent line scan emissions of the vertical line-scan pattern or horizontal line-scan pattern is spaced from an adjacent second longitudinal axis of another second longitudinal axis by a second distance, and wherein the first distance is equal to the second distance.

16. The microscopy system of claim 11, wherein each of the expanded fluorescent line scan emissions of the expanded line-scan pattern define a first width that is equal to a second width defined by each of the fluorescent line scan emissions of the vertical line-scan pattern or the horizontal line-scan pattern.

17. The microscopy system of claim 11, wherein each of the expanded fluorescent line scan emissions of the expanded line-scan pattern define a first longitudinal axis and each of the fluorescent line scan emissions of the vertical line-scan pattern or the horizontal line-scan pattern define a second longitudinal axis, and wherein the first longitudinal axis of each of the expanded fluorescent line scan emissions of the expanded line-scan pattern is spaced from an adjacent first longitudinal axis of another expanded fluorescent line scan emissions by a first distance, and wherein the second longitudinal axis of each of the fluorescent line scan emissions of the vertical line-scan pattern or the horizontal line-scan pattern is spaced from an adjacent second longitudinal axis of another adjacent fluorescent line scan emissions by a second distance, wherein the first distance is greater than the second distance.

18. A microscopy system comprising:

a light source for transmitting a single light beam;

a scanning apparatus arrangement for relaying the single light beam and generating a light sheet;

an excitation objective for imaging the light sheet onto a sample for generating fluorescence emissions;

a detection objective for relaying the fluorescence emissions through an optic arrangement;

a scaling component for scaling the fluorescence emissions received from the optic arrangement to either locally contract the fluorescence emissions arising from the light sheet to produce fluorescence emissions that are contracted or locally expand the fluorescence emissions arising from the light sheet to produce fluorescence emissions that are expanded; and

a detection apparatus for capturing an image of the expanded or the contracted fluorescence emissions arising from the light sheet.

19. The microscopy system of claim 18, wherein the detection objective is aligned at a perpendicular relation relative to the excitation objective.

20. The microscopy system of claim 18, further comprising:

beam shaping optics positioned between the light source and the scanning apparatus arrangement for shaping the single light beam into a line beam.