# US 2019/0170993 A1 · Methods, systems, and devices for super resolution solid immersion lens microscopy

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- Number: US 2019/0170993 A1
- Document ID: US20190170993A1
- Status: Published application
- Kind: A1
- Application number: 16/325,289
- Filed: 2017-08-18
- Published: 2019-06-06
- Earliest claimed: 2016-08-19
- Assignees: SuRe Optics, Inc.
- Applicants: SuRe Optics, Inc.
- Inventors: Zong-Long Liau; Frederick J. Leonberger
- Web page: https://patentgrep.com/patent/US20190170993A1

## CPC

- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G02B 21/33: Immersion oils {, or microscope systems or objectives for use with immersion fluids}
- 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)}
- G02B 21/0076: {arrangements using fluorescence or luminescence}
- G02B 21/16: adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)}
- G02B 21/0024: {Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS

## Abstract

Described herein are methods and systems for the optical imaging of a physical specimen of interest that is in contact with, or in close proximity to, the backplane of a high refractive index solid-immersion lens (SIL), wherein the specimen comprises features of interest that act as a local high-refractive index regions. The SIL lens preferably comprises fiducial markers.

## Claims

### Claim 1

A method of imaging a specimen containing features of interest with resolution in the sub-100 nm down to sub-nm range, using an annular solid immersion lens (ASIL), the method comprising the steps of:
(a) directing electromagnetic radiation (emr) to the ASIL, using a laser scanning microscope (LSM), wherein:
(i) the ASIL comprises a first surface comprising a convex region and a second surface that is substantially planar;
(ii) at least a portion of the emr is transmitted through the convex region of the first surface of the ASIL, and propagates inside the ASIL toward the second surface;
(iii) the portion of the emr transmitted through the convex region forms a focus near and/or on the second surface of the ASIL; and
(iv) the second surface of the ASIL is in contact with a sample medium having a refractive index that is lower than the refractive index of the ASIL;
(b) positioning a feature of interest of the specimen within the sample medium in close proximity to and/or in contact with the second surface of the ASIL, wherein the feature of interest has a refractive index that is higher than that of material immediately surrounding the feature of interest, and wherein the feature of interest is positioned within the focus formed by the emr, thereby illuminating the feature;
(c) detecting emr that is either (i) transmitted from inside the ASIL into the sample medium, or (ii) reflected from inside the ASIL back toward the first surface of the ASIL; and
(d) constructing an image of the specimen that resolves specimen features that are less than 100 nm using a LSM.

### Claim at position 2

-3. (canceled)

### Claim 4 (depends on 1)

The method of claim 1, wherein the feature of interest forms a local high-index region in close proximity to the second surface of the ASIL, thereby acting as a source of a spherical wave with a well-defined center.

### Claim at position 4

-10. (canceled)

### Claim 11 (depends on 1)

The method of claim 1, wherein the specimen is a biological specimen.

### Claim at position 6

-20. (canceled)

### Claim 21 (depends on 1)

The method of claim 1, wherein:
the emr is detected using a detector that converts the detected emr into a signal having a value that is a function of an intensity of the detected emr, and the method comprises displaying the value of the signal for visualization.

### Claim 22

(canceled)

### Claim 23 (depends on 1)

The method of claim 1, wherein the ASIL has a refractive index greater than 3.

### Claim 24

(canceled)

### Claim 25

(canceled)

### Claim 26

(canceled)

### Claim 27 (depends on 1)

The method of claim 1, wherein the sample medium is a medium selected from the group consisting of air, water, organic material and a biological media.

### Claim 28

(canceled)

### Claim 29 (depends on 1)

The method of claim 1, wherein the second surface of the ASIL comprises one or more fiducial markings, said fiducial markings having been fabricated by one or more techniques selected from the group consisting of etching, nanoparticle deposition, and ion bombardment.

### Claim at position 16

-41. (canceled)

### Claim 42 (depends on 1)

The method of claim 1, comprising:
directing emr to the ASIL using a laser scanning microscope (LSM); and
constructing the image of the specimen using the LSM.

### Claim 43 (depends on 1)

The method of claim 1, comprising detecting emr that is transmitted through the sample medium and/or specimen, away from the second surface of the ASIL.

### Claim 44

(canceled)

### Claim 45

A method of imaging a specimen containing features of interest with resolution in the sub-100 nm down to sub-nm range, using an annular solid immersion lens (ASIL), the method comprising the steps of:
(a) directing electromagnetic radiation (emr) to the ASIL, wherein:
(i) the ASIL comprises a first surface comprising a convex region, and a second surface that is substantially planar;
(ii) at least a portion of the emr is transmitted through the convex region of the first surface of the ASIL, and propagates inside the ASIL toward the second surface;
(iii) the portion of the emr transmitted through the convex region forms a focus near and/or on the second surface of the ASIL; and
(iii) the second surface of the ASIL is in contact with a sample medium having a refractive index that is lower than the refractive index of the ASIL;
(b) positioning specimen feature of interest of the specimen within the sample medium in close proximity to the second surface of the ASIL, wherein the feature of interest has a refractive index that is higher than that of material immediately surrounding the feature of interest, and wherein the feature of interest is positioned within the focus formed by the emr, thereby illuminating the specimen;
(c) detecting fluorescent light that is emitted by at least one of (i) the feature of interest itself, and (ii) one or more secondary fluorescent objects within the sample medium; and
(d) constructing an image that resolves specimen features that are less than 100 nm.

### Claim 46

(canceled)

### Claim 47 (depends on 45)

The method of claim 45, wherein the secondary fluorescent objects are excited by excitation light that is transmitted into the sample medium.

### Claim at position 23

-49. (canceled)

### Claim 50 (depends on 45)

The method of claim 45, wherein fluorescent light that is emitted by at least one of (i) the feature of interest itself, and (ii) one or more secondary fluorescent objects in close proximity to the feature of interest is detected by a detector following the sample medium.

### Claim 51 (depends on 45)

The method of claim 45, wherein fluorescent light that is emitted by at least one of (i) the feature of interest itself, and (ii) one or more secondary fluorescent objects in close proximity to the feature of interest is transmitted from the sample medium into the ASIL, and detected after it propagates inside the ASIL and is transmitted through the first surface of the ASIL.

### Claim 52

(canceled)

### Claim 53 (depends on 45)

The method of claim 45, wherein the feature of interest forms a local high-index region in close proximity to, and/or in contact with, the second surface of the ASIL, thereby acting as a source of a spherical wave with a well-defined center, including the case wherein the center is a dimensionless point.

### Claim at position 28

54-61. (canceled)

### Claim 62 (depends on 45)

The method of claim 45, wherein the specimen is a biological or bio-molecular specimen.

### Claim at position 30

-71. (canceled)

### Claim 72 (depends on 45)

The method of claim 45, wherein:
the emr is light incident onto the SIL, the emr is detected using a detector that converts the detected emr into a signal having a value that is a function of an intensity of the detected emr, and the method comprises displaying the value of the signal for visualization.

### Claim 73

(canceled)

### Claim 74 (depends on 45)

The method of claim 45, wherein the ASIL has a refractive index greater than 3.

### Claim 75

(canceled)

### Claim 76

(canceled)

### Claim 77

(canceled)

### Claim 78 (depends on 45)

The method of claim 45, wherein the sample medium is a medium selected from the group consisting of air, water, organic material, and a biological media.

### Claim 79

(canceled)

### Claim 80 (depends on 45)

The method of claim 45, wherein the second surface of the SIL comprises one or more fiducial markings, made by etching, nanoparticles deposition, or ion bombardment.

### Claim at position 40

-92. (canceled)

### Claim 93 (depends on 45)

The method of claim 45, comprising:
directing emr to the ASIL using a laser scanning microscope (LSM); and
constructing the image of the specimen using the LSM.

### Claim 94 (depends on 45)

The method of claim 45, comprising detecting fluorescent light that is transmitted through the sample medium and/or specimen, away from the second surface of the ASIL.

### Claim 95

(canceled)

### Claim 96

A system for imaging a specimen containing features of interest with resolution in the sub-100 nm down to sub-nm range using an annular solid immersion lens (ASIL), the system comprising:
a source of electromagnetic radiation (emr);
an ASIL in contact with a sample medium and positioned in close proximity to, and/or in contact with, the specimen, the sample medium having a refractive index that is lower than the refractive index of the ASIL, the ASIL comprising:
a first surface comprising a convex region, and
a second surface that is substantially planar, wherein the second surface of the ASIL is in contact with the sample medium,
such that at least a portion of the emr from the source is transmitted through the convex region of the first surface of the ASIL, and propagates inside the ASIL toward the second surface, the portion of the emr transmitted through the convex region forms a focus near and/or on the second surface of the ASIL, wherein a feature of interest of the specimen is in close proximity to the second surface of the ASIL, wherein the feature of interest has a refractive index that is higher than that of the sample medium, and wherein the feature of interest is positioned within the focus formed by the emr, thereby illuminating the feature of interest; and
a detector for detecting emr that is either (i) transmitted from inside the ASIL into the sample medium, or (ii) reflected from inside the ASIL back toward the first surface of the ASIL.

### Claim at position 45

97-99. (canceled)

### Claim 100 (depends on 97)

The system of claim 97, wherein the source, ASIL, and detector are part of a laser scanning microscope (LSM) and the source is aligned to direct the emr to the ASIL via optics of the LSM.

### Claim 101 (depends on 97)

The system of claim 97, wherein the detector is mounted following the specimen and sample medium.

### Claim at position 48

102-112. (canceled)