MULTI-FOCAL STRUCTURED ILLUMINATION MICROSCOPY SYSTEMS AND METHODS

• US 20160238827A1
• Filed: 02/22/2013
• Published: 08/18/2016
• Est. Priority Date: 02/23/2012
• Status: Active Grant

• Current Assignee: The USA of America As Represented By The Secretary Dept of Health and Human Services
• Original Assignee: The USA of America As Represented By The Secretary Dept of Health and Human Services
• Inventors: Shroff, Hari, York, Andrew
• Granted Patent: US 9,696,534 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: G01N 2021/6478 Special lenses
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 2201/0633 Directed, collimated illumination
• 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 21/004 fixed arrays, e.g. switchable aperture arrays
• CPC Class Codes: G02B 21/0072 details concerning resolution or correction, including general design of CSOM objectives
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence

Abstract

A multi-focal selective illumination microscopy (SIM) system for generating multi-focal patterns of a sample is disclosed. The multi-focal SIM system performs a focusing, scaling and summing operation on each multi-focal pattern in a sequence of multi-focal patterns that completely scan the sample to produce a high resolution composite image.

Claims

1. A microscopy system comprising:

a light source for transmitting a single light beam;

a beam splitter for splitting the single light beam into a plurality of light beams forming a multi-focal pattern;

a scanner for scanning the plurality of light beams that forms the multi-focal pattern onto a sample such that the sample generates a plurality of fluorescent emissions with each multi-focal pattern;

a focusing component defining an aperture configured to block out-of-focus fluorescence emissions of the plurality of fluorescent emissions for each multi-focal pattern and allowing through in-focus fluorescent emissions to pass through the aperture;

a scaling component for scaling down the plurality of in-focus fluorescent emissions for each multi-focal pattern such that each of the plurality of in-focus fluorescent emissions is scaled down to produce a plurality of scaled in-focus fluorescent emissions for each multi-focal pattern; and

a summing component for summing each of the plurality of scaled in-focus fluorescent emissions to produce a plurality of summed, scaled in-focus fluorescent emissions that form a composite image of the plurality of summed, scaled in-focus fluorescent emissions.

2. The microscopy system of claim 1, wherein the beam splitter is a micro-lens array.

3. The microscopy system of claim 1, wherein the focusing component is a pinhole array.

4. The microscopy system of claim 1, wherein the scaling component is a plurality of micro-lens arrays.

5. The microscopy system of claim 1, wherein each multi-focal pattern defines a plurality of respective focal points for each of the plurality of in-focus fluorescent emissions, wherein the scaling component scales the plurality of focal points in a local contraction in which each of the plurality of focal points in the multi-focal pattern maintains the same proportional distance from the other plurality of focal points of the multi-focal pattern as the plurality of fluorescent emissions contract by a predetermined factor.

6. The microscopy system of claim 1, further comprising:

a processing sub-system for deconvolving the composite image.

7. The microscopy system of claim 1, wherein the scanner de-scans the plurality of fluorescent emissions emitted by the sample to the focus component.

8. The microscopy system of claim 1, further comprising:

a dichroic mirror for allowing the plurality of light beams to pass through in one direction and redirecting the plurality of fluorescent emissions from the opposite direction.

9. The microscopy system of claim 1, further comprising:

a first lens for imaging the plurality of light beams through a tube lens and an objective lens to illuminate the sample.

10. The microscopy system of claim 1, wherein the plurality of light beams has a wavelength between 350 nm to 1200 nm.

11. The microscopy system of claim 1, wherein the plurality of fluorescent emissions has a wavelength between 350 nm to 1200 nm.

12. The microscopy system of claim 11, further comprising:

an emission filter for allowing scaled, in-focus fluorescent emissions of about 350 nm to 1200 nm to pass through to a detector.

13. The microscopy system of claim 1, further comprising:

a detector for detecting the plurality of scaled in-focus fluorescent emissions.

14. The microscopy system of claim 12, wherein the scanner re-scans the scaled, in-focus fluorescent emissions to the detector.

15. A microscopy system comprising:

a light source for transmitting a single light beam;

a beam splitter for splitting the single light beam into a plurality of light beams forming a plurality of multi-focal patterns, wherein each of the multi-focal patterns defines a plurality of focal points;

a scanner for scanning the plurality of light beams that forms each of the multi-focal patterns onto a sample such that the sample generates a plurality of multi-focal fluorescent emissions in each of the multi-focal patterns that defines a plurality of fluorescence focal points;

a detector for collecting the plurality of multi-focal fluorescent emissions that results from each of the multi-focal patterns; and

a processing system for processing the plurality of multi-focal fluorescent emissions that results from each of the multi-focal patterns collected from the detector comprising;

a processor in operative communication with a database for storing the plurality of collected multi-focal fluorescent emissions resulting from the multi-focal patterns, wherein the processor removes out-of-focus fluorescent emissions for each of the plurality of fluorescent emission to leave only in-focus fluorescent emissions resulting from each of the plurality of multi-focal patterns, wherein the processor then scales the in-focus fluorescence emissions resulting from each of the plurality of multi-focal patterns in a local contraction operation in which each of the plurality of fluorescent focal points resulting from each of the multi-focal patterns maintains the same distance from another plurality of fluorescent focal points resulting from the multi-focal pattern as the plurality of fluorescent focal points contract to produce scaled, in-focus fluorescent emissions;

wherein the processor sums each scaled, in-focus fluorescence emissions to produce a composite image.

16. The microscopy system of claim 15, further comprising the processor executing a deconvolution operation for processing the plurality of multi-focal patterns with the scaled, in-focus fluorescent emissions.

17. The microscopy system of claim 15, wherein the beam splitter is a digital micromirror device or a swept-field confocal unit, or a spinning-disk, or Nipkow confocal scan head.

18. The microscopy system of claim 15, wherein the processing system executes an automatic lattice detection of each of the plurality of multi-focal patterns to determine the position of one or more illumination spots.

19. The microscopy system of claim 18, wherein the automatic lattice detection determines a plurality of vectors to locate the position of each of the one or more illumination spots.

20. The microscopy system of claim 19, wherein the plurality of vectors comprises lattice vectors, shift vectors, and/or offset vectors.

21. The microscopy system of claim 20, wherein two of the lattice vectors specify a two-dimensional displacement between any two neighboring illumination spots.

22. A method for multi-focal structured illumination microscopy comprising:

generating a single light beam;

splitting the single light beam into a plurality of light beams in which the focal point of each the plurality of light beam forms a plurality of multi-focal illumination patterns,

illuminating a sample with the plurality of light beams forming each of the plurality of multi-focal illumination patterns, wherein the illuminated sample produces a plurality of fluorescence emissions in the plurality of multi-focal patterns;

performing a pinholing operation in which out-of-focus fluorescent emissions from the plurality of fluorescent emissions are blocked and only in-focus fluorescent emissions from the plurality of fluorescent emissions are permitted to pass during the pinholing operation;

scaling each focal point of the plurality of in-focus fluorescent emissions resulting from the plurality of multi-focal patterns by a predetermined factor to produce a plurality of scaled, in-focus fluorescent emissions resulting from the plurality of multi-focal patterns; and

summing each of the plurality of scaled, in-focus fluorescent emissions to produce a plurality of summed, scaled, in-focus fluorescent emissions that form a composite image.

23. The method of claim 22, wherein the plurality of scaled, in-focus fluorescent emissions resulting from each of the plurality of multi-focal patterns undergo a local contraction in which the relative distance between each of the focal points in a multi-focal pattern remains proportional the same.

24. The method of claim 22 further comprising:

scanning the plurality of light beams in the plurality of multi-focal patterns onto the sample.

25. The method of claim 24, wherein scanning the plurality of multi-focal patterns onto the sample is performed by a galvanometer.

26. The method of claim 22, further comprising:

performing a deconvolution operation on the composite image composed of summed, scaled, in-focus fluorescent emissions.

27. A method for multi-focal structured illumination microscopy may include:

generating a single light beam;

splitting the single light beam into a plurality of light beams in which the focal point of each the plurality of light beams forms a plurality of multi-focal patterns,

illuminating a sample with the plurality of light beams forming each of the plurality of multi-focal patterns, wherein the illuminated sample produces a plurality of fluorescent emissions resulting from each the plurality of multi-focal patterns;

performing a pinholing operation in which out-of-focus fluorescent emissions from the plurality of fluorescent emissions are blocked and only in-focus fluorescent emissions from the plurality of fluorescent emissions are permitted to pass during the pinholing operation;

scaling each focal point of the plurality of in-focus fluorescent emissions resulting from the plurality of multi-focal patterns by a predetermined factor to produce a plurality of scaled, in-focus fluorescent emissions resulting from the plurality of multi-focal patterns; and

summing each of the plurality of scaled, in-focus fluorescent emissions to form a composite image.

28. The method of claim 27, further comprising:

performing a deconvolution operation on the composite image resulting from the summed, scaled, in-focus fluorescent emissions.