Pulse-illuminated edge deduction microscopy

Patent metadata

Field	Value
Publication number	US-11221473-B2
Application number	US-201816499952-A
Country	US
Kind code	B2
Filing date	Mar 5, 2018
Priority date	Apr 6, 2017
Publication date	Jan 11, 2022
Grant date	Jan 11, 2022

Abstract

Devices and methods for super-resolution optical microscopy are described. Devices include an optical multiplexer to develop an excitation/illumination optical beam that includes alternating pulses of different profiles. Devices also include a signal processing unit to process a sample response to excitation/illumination beam and to subtract the neighboring pulses of the different profiles from one another on a pulse-to-pulse basis. Devices can be incorporated in existing confocal microscopy designs. As the subtraction effectively reduces the volume of the response signal, the spatial resolution of the systems can be markedly improved as compared to previously known optical microscopy approaches.

First claim

What is claimed is:

1. An optical microscopy device comprising:
a multiplexer, the multiplexer comprising a beam splitter and a beam combiner, a first light path having a first length and a second light path having a second length being defined between the beam splitter and the beam combiner, the first length differing from the second length, the beam combiner being configured for combining a first light beam of the first light path with a second light beam of the second light path to form an excitation/illumination beam, the beam combiner being configured for location in optical communication with a sample, the multiplexer further comprising a phase plate located in one of the first light path and the second light path, the phase plate modifying a profile of either the first light beam or the second light beam; and
a signal processing unit configured for processing a signal generated by the sample in response to the excitation/illumination beam, wherein the signal processing unit comprises data processing software configured to carry out pulse-to-pulse subtraction of neighboring pulse pairs of the signal, the signal including a series of pulses comprising interleaved pulses of different profiles.

2. The optical microscopy device of claim 1 , the signal processing unit further comprising a demultiplexer and an analog to digital converter.

3. The optical microscopy device of claim 2 , the signal processing unit comprising a programmable clock, the programmable clock controlling the demultiplexer.

4. The optical microscopy device of claim 1 , wherein at least one of the first length and the second length is adjustable.

5. The optical microscopy device of claim 1 , wherein the phase plate comprises an optical phase modulation device.

6. The optical microscopy device of claim 5 , wherein the optical phase modulation device comprises at least one of an optical vortex phase plate and spatial light modulator.

7. The optical microscopy device of claim 1 , further comprising a photodetector in electrical communication with the signal processing unit.

8. The optical microscopy device of claim 7 , wherein the photodetector comprises a photomultiplier.

9. The optical microscopy device of claim 8 , wherein the photomultiplier comprises a fast photomultiplier tube.

10. The optical microscopy device of claim 1 , further comprising a confocal microscope in optical communication with the multiplexer and in electronic communication with the signal processing unit.

11. A method for examining a sample comprising:
directing a pulsed source light beam to a beam splitter to form a first pulsed beam traveling on a first light path and a second pulsed beam traveling on a second light path, the first light path having a first length and the second light path having a second length, the first and second lengths differing from one another;
modifying a profile of one of the first pulsed beam and the second pulsed beam to form a modified pulsed beam, the other of the first pulsed beam and the second pulsed beam being unmodified;
combining the modified pulsed beam and the unmodified pulsed beam to form a third pulsed beam that includes pulses of the modified pulsed beam interleaved with pulses of the unmodified pulsed beam;
directing the third pulsed beam at a sample, the sample thereby generating a response pulsed beam;
deducting components of the response pulsed beam from one another to form a processed signal; and
forming an image of the sample by use of the processed signal.

12. The method of claim 11 , further comprising demultiplexing the response pulsed beam to form first and second analog signals and converting the first and second analog signals to digital signals prior to deducting components of the response pulsed beam from one another.

13. The method of claim 11 , wherein the pulsed source light beam defines a profile that focuses to a solid spot.

14. The method of claim 11 , wherein the profile of the modified pulsed beam defines a profile that focuses to a circular profile surrounding a dark center.

15. The method of claim 11 , wherein a difference between the first length and the second length provides a delay in pulses of one of the first pulsed beam or the second pulsed beam such that the pulses of the first pulsed beam and the pulses of the second pulsed beam are temporally shifted relative to one another.

16. The method of claim 15 , wherein the pulses of the first pulsed beam and the pulses of the second pulsed beam are temporally shifted relative to one another by about one-half of a pulse repetition cycle.

17. The method of claim 11 , wherein the response pulsed beam comprises a fluorescent response of the sample.

18. The method of claim 17 wherein the fluorescent response comprises an autofluorescent response of the sample.

19. The method of claim 11 , wherein the sample is a biological sample comprising live or fixed cells.

20. An optical microscopy device comprising:
a multiplexer, the multiplexer comprising a beam splitter and a beam combiner, a first light path having a first length and a second light path having a second length being defined between the beam splitter and the beam combiner, the first length differing from the second length, the beam combiner being configured for combining a first light beam of the first light path with a second light beam of the second light path to form an excitation/illumination beam, the beam combiner being configured for location in optical communication with a sample, the multiplexer further comprising a phase plate located in one of the first light path and the second light path, the phase plate comprising at least one of an optical vortex phase plate and a spatial light modulator, the phase plate modifying a profile of either the first light beam or the second light beam; and
a signal processing unit, wherein the signal processing unit comprises data processing software configured to deduct signal components from one another, the signal components being components of a signal generated by the sample in response to the excitation/illumination beam.

Assignees

• Univ Clemson
• Univ Clemson Res Foundation

Inventors

• Li Yang
• Ye Tong
• Chen Xun

Classifications

• G02B21/0076 Primary

  arrangements using fluorescence or luminescence · CPC title

• G01N21/6458

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

• G02B27/58

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

• G02B21/0072

  details concerning resolution or correction, including general design of CSOM objectives · CPC title

• G02B21/0084

  time-scale detection, e.g. strobed, ultra-fast, heterodyne detection · CPC title