AU2020103836A4 - A fiber-end super-resolution nano-fluorescence microscopic illumination probe - Google Patents
A fiber-end super-resolution nano-fluorescence microscopic illumination probe Download PDFInfo
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- AU2020103836A4 AU2020103836A4 AU2020103836A AU2020103836A AU2020103836A4 AU 2020103836 A4 AU2020103836 A4 AU 2020103836A4 AU 2020103836 A AU2020103836 A AU 2020103836A AU 2020103836 A AU2020103836 A AU 2020103836A AU 2020103836 A4 AU2020103836 A4 AU 2020103836A4
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- fluorescence
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B6/00—Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings
- G02B6/24—Coupling light guides
- G02B6/26—Optical coupling means
- G02B6/32—Optical coupling means having lens focusing means positioned between opposed fibre ends
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B6/00—Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings
- G02B6/24—Coupling light guides
- G02B6/26—Optical coupling means
- G02B6/262—Optical details of coupling light into, or out of, or between fibre ends, e.g. special fibre end shapes or associated optical elements
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B6/00—Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings
- G02B6/24—Coupling light guides
- G02B6/36—Mechanical coupling means
- G02B6/3616—Holders, macro size fixtures for mechanically holding or positioning fibres, e.g. on an optical bench
- G02B6/3624—Fibre head, e.g. fibre probe termination
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- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention provides a fiber-end super-resolution nano-fluorescence microscopic illumination
probe: it is characterized by the composition of a coaxial dual-waveguide fiber optical tweezers
system and a cascade of two dielectric microsphere lenses. The coaxial dual-waveguide fiber has
a center core and an annular core, and there are symmetrical truncated cone structures at the fiber
ends. They reflect and focus the annular trapped beam transmitted in the annular core to form a
trap and traps a microsphere lens. A groove is etched in the center of the end face of the fiber and
another microsphere lens is adhered to the groove. Two microsphere lenses are coaxially
distributed with the fiber, and the fluorescence excitation beam transmitted by the center core is
compressed in two stages by the two microsphere lenses to form a super-resolution optical nano
jet that exceeds the diffraction limit, enabling super-resolution fluorescence illumination at the
nanometer scale.
1/5
DRAWINGS
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FIG.1
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Step 1 Step
Step 2 Step 5
3
FIG.2
Description
1/5 DRAWINGS
1-2
FIG.1
1
Step 1 Step
Step 2 Step 5
3
FIG.2
A fiber-end super-resolution nano-fluorescence microscopic illumination probe
[0001] The invention relates to a fiber-end super-resolution nano-fluorescence microscopic
illumination probe, which can compress the fluorescent excitation beam to form a nanometer
scale optical jet that exceeds the diffraction limit, and realize the super-resolution fluorescence
microscopic illumination and detection. This belongs to the nano-optical technology field.
[0002] Modern biomedical research needs to observe the precise location and distribution of
intracellular organelles, viruses, parasites, etc. in three-dimensional cellular space in order to
better understand the processes of human life and the mechanisms of disease generation. On the
other hand, the study of protein science in the post-genomic era requires the elucidation of: the
relationship between protein structure, localization and function and the spatial and temporal
sequence of the interactions between protein and protein; how biomolecules, mainly structural
proteins and RNA and their complexes, form the basic structural system of the cell; and how
important active factors regulate the major life activities of the cell, such as cell proliferation,
cell differentiation, apoptosis and cell signaling. The characteristic scales reflecting the
properties of these systems are on the nanometer scale, far beyond the resolution limits of conventional optical microscopy.
[0003] In the past few decades, people have been trying different methods to overcome the
limitations in optical microscopy due to the Abbe diffraction limit. Several super-resolution
optical imaging technologies, including scanning near-field optical microscopes, stimulated
emission loss microscopes, metamaterial hyperlens microscopes, solid immersion lens
microscopes, and super-oscillation lens microscopes, have been successfully implemented.
Although they have good performance, they are expensive, long preparation time, and
complicated detection methods.
[0004] The optical nanojet technology based on microsphere lens is a technology in which a light
beam is irradiated on a medium microsphere, and the light beam is compressed to a size smaller
than the diffraction limit at one end of the microsphere. Since the beam waist diameter of the
compressed spot is on the order of nanometers, it has a spatial resolution of the order of
nanometers and has a high energy density. Therefore, it has a wide range of applications in
technical fields such as super-resolution imaging (KRIVITSKY, Leonid A., et al. Locomotion of
microspheres for super-resolution imaging. Scientific reports, 2013, 3: 3501.), nano-fluorescence
enhancement (LECLER, Sylvain, et al. Photonic jet driven non-linear optics: example of two
photon fluorescence enhancement by dielectric microspheres. Optics express, 2007, 15.8: 4935
4942.), and Raman scattering enhancement (US2013/0308127A1).
[0005] The type of optical nanojet technology usually uses micrometer-scale standard dielectric
spheres, for optical tweezers systems, this type of dielectric microspheres are just the size range
that can be stably trapped and manipulated. Therefore, combining the two technologies of optical
tweezers and optical nanojet can achieve more abundant application scenarios, especially has a
wide application onto super-resolution fluorescence illumination and imaging.
[0006] The purpose of the invention is to provide a fiber-end super-resolution nano-fluorescence microscopic illumination probe, which can achieve the super-resolution fluorescence illumination and imaging on single molecules.
[0007] The purpose of the invention is achieved as follows:
[0008] A fiber-end super-resolution nano-fluorescence microscopic illumination probe, it is characterized by the composition of a coaxial dual-waveguide fiber optical tweezers system and a cascade of two dielectric microsphere lenses. The coaxial dual-waveguide fiber has a center core and an annular core, and there are symmetrical truncated cone structures at the fiber ends. They reflect and focus the annular trapped beam transmitted in the annular core to form optical tweezers, to trap a microsphere lens. A groove is etched in the center of the end face of the fiber and another microsphere lens is adhered to the groove. Two microsphere lenses are coaxially distributed with the fiber, and the fluorescence excitation beam transmitted by the center core is compressed in two stages by the two microsphere lenses to form a super-resolution optical nano jet that exceeds the diffraction limit, enabling super-resolution fluorescence illumination at the nanometer scale.
[0009] The truncated cone structure is a symmetrical reflective truncated cone structure prepared by precision polishing and processing at the coaxial dual-waveguide fiber end, it can also be an arc-optimized reflective truncated cone structure. The optimized reflective truncated cone structure allows for better focusing of the trapped beam and higher spatial resolution for more stable trapping of smaller microsphere lenses.
[0010] The groove is located at the center of the end face of the fiber, which can be prepared by
femtosecond laser etching, and is sized so that the microsphere lens can be placed just right. The
microsphere lens in the groove is adhered by a low refractive index glue with a diameter greater
than or equal to the diameter of the center core of the coaxial dual-waveguide fiber and less than
the diameter of the truncated cone end face, and its refractive index is greater than that of the low
refractive index glue.
[0011] The light beams transmitted in the annular core of the coaxial dual-waveguide fiber are
reflected and focused by the truncated cone structure, forming optical tweezers that contactlessly
traps a microsphere lens whose refractive index is greater than of the background ambient
refractive index. The refractive index of the trapped microsphere lens should be in the range of
1.33-1.8, taking water as the background ambient.
[0012] The trapped microsphere lens may be an extracellular dielectric microsphere, a dielectric
microsphere placed inside the cell, such as a polystyrene microsphere or a spherical oil droplet,
or a spherical biological dielectric contained within the cell itself, such as fat particles in
adipocytes.
[0013] The present invention has at least the following significant advances and advantages over
the prior arts:
[0014] (1) As an optical fiber is used as the light transmitting medium, it is obvious that the
invention has the great advantage of compact structure and flexible operation compared with the
fluorescent illumination systems of space light.
[0015] (2) A two-stage compression of the fluorescence excitation beam is achieved in the form of a combination of two microsphere lenses to compress the low-energy-density large-diameter beam into a super-resolution high-energy-density nano-jet, which not only ensures the energy density of the fluorescence excitation light transmitted within the fiber is low, but also makes full use of the beam's energy.
[0016] (3) Contactless microsphere lenses trappings enable single molecule fluorescence
illumination in living cells, i.e., generation of optical nano-jets in the interior of living cells
without destroying the cells.
[0017] FIG. 1 is a schematic diagram of the end face structure of a coaxial dual-waveguide fiber.
[0018] FIG. 2 is the fabrication method and steps of a fiber-end super-resolution nano
fluorescence microscopic illumination probe.
[0019] FIG. 3 shows two types of reflective truncated cone structures at the fiber ends, in which,
(a) is a precision-grinded symmetrical truncated cone structure, and (b) is an optimized arc
shaped symmetrical truncated cone structure.
[0020] FIG. 4 is a schematic diagram of the generation of the optical nano-jets for super
resolution probes.
[0021] FIG. 5 is a graph of the simulation results of the contactless trap formed by the short beam, transmitted in the annular core of the coaxial dual-waveguide fiber, reflected and focused when passing the truncated cone structure, which shows the intensity distribution of the electric field modes in the end section of the fiber.
[0022] FIG. 6 shows the magnitude of optical forces received by a polystyrene sphere with refractive index of 1.6 at different positions on the fiber axis. The intersection position of the curve with Fz=O is the stable trapping point of the microsphere.
[0023] FIG. 7 is a graph of the simulation results of the fluorescence excitation beam under the compression of two cascaded microsphere lenses to generate an optical nano-jet.
[0024] FIG. 8 shows the energy distribution in the x-axis direction at the highest value of the energy density of the optical nano-jet in FIG. 7.
[0025] FIG. 9 is a graph of the compression simulation results of fluorescence excitation light in the absence of trapped microspheres, with only the microsphere lens adhered to the groove.
[0026] FIG. 10 is a schematic diagram of the principle of a fiber-end super-resolution nano fluorescence microscopic illumination probe used for the illumination and imaging inside a cell.
[0027] The invention will be described in detail below in conjunction with specific embodiments and drawings.
[0028] Embodiment 1: Preparation method of the probe.
[0029] Firstly, the method of preparing the coaxial dual-waveguide fiber and the probe used in
this invention is described.
[0030] As shown in FIG. 1, the coaxial dual-waveguide fiber 1 of the present invention has an
center core 1-1 and a coaxially distributed annular core 1-2, wherein the annular core 1-2 is used
to transmit a trapped beam, such as a near infrared beam at a wavelength of 980 nm, and the
center core 1-1 is used to transmit a fluorescence excitation beam, such as a green light beam at a
wavelength of 532 nm.
[0031] FIG. 2 is used as an example to describe the preparation method of the fiber-end super
resolution nano-fluorescence microscopic probe proposed by the present invention.
[0032] Step 1: Take a coaxial dual-waveguide fiber 1, remove the 20-30mm coating layer, cut
flat the end face of the fiber, and use a femtosecond laser to etch a1Oum x1Oum x1Oum groove
2 in the middle of the end face of the fiber.
[0033] Step 2: A microsphere lens 3 with a diameter of 1Oum and a refractive index of 1.5 is
placed in the groove 2, and the microsphere lens 3 is cured using glue with a refractive index of
1.4 so that the center of the microsphere lens 3 is on the axis of the fiber.
[0034] Step 3: Grind the end face of the optical fiber to remove excess glue block 4, so that the
end face of the optical fiber is flat.
[0035] Step 4: Grind the fiber end face truncated cone structure 5 to form the fiber end face with a 69 degrees cone base angle of the truncated cone, and precision-polish to make the truncated cone's bevel smooth.
[0036] Step 5: Pass a 980 nm capture beam into the annular core 1-2 to capture a microsphere lens 6 with a diameter of 2um and refractive index of 1.65, and then pass a 532 nm fluorescence excitation light into the center core to generate an optical nano-jet to complete the preparation of the super-resolution fluorescence microscopic illumination probe.
[0037] In step 4, the end faces of the optical fibers are precisely ground to form a symmetrical truncated cone reflective structure 5 as shown in FIG. 3(a), or a more precise and complex grinding method can be used to make the bevel of the truncated cone become arced, as shown in FIG. 3(b), such a curved structure 5-1 can make the focusing effect of the reflected annular beam better and trapping the 1-5um diameter microsphere lenses more stably.
[0038] Embodiment 2: Generation of super-resolution optical nano-jet embodiment.
[0039] The schematic diagram of the optical nano-jet generation of the super-resolution probe is shown in FIG. 4. FIG. 4(a) is a three-dimensional diagram of the fiber end, FIG. 4(b) is an axial section of the fiber, and FIG. 4(c) is a local magnification of the optical nano-jet generation site. The end-face truncated cone structure 5 of the coaxial dual-waveguide fiber 1 can reflect the annular trapped beam (980 nm) 7, focus outside the fiber end, forming a focus-trapping point, which can stably trap the 2um microsphere lens 6. The fluorescence excitation beam (532 nm) 8 transmitted in the center core 1-1 is compressed after passing through the microsphere lens 3 in the groove and the trapped microsphere lens 6 to form a sub-wavelength scale optical nano-jet 9 that can be used for super-resolution fluorescence excitation illumination.
[0040] FIG. 5 is a graph of the simulation results of the contactless trap formed by the short beam, transmitted in the annular core 1-1 of the coaxial dual-waveguide fiber 1, reflected and focused when passing the truncated cone structure, which shows the intensity distribution of the electric field modes in the end section of the fiber. It can be seen that in the case of the truncated cone base angle being at 69 degrees, the distance between the trapping point and the end face of the fiber is about 16um, which means that non-contact microsphere trapping can be achieved. FIG. 6 shows the magnitude of the optical force received by a microsphere with refractive index of 1.65 at different positions on the fiber axis. The intersection of the curve with Fz=0 is the stable trapping point of the microsphere.
[0041] FIG. 7 is a graph of the simulation results of the fluorescence excitation beam under the compression of two cascaded microsphere lenses to generate an optical nano-jet. The microsphere lens in the groove in the figure has a diameter of 12um and a refractive index of 1.5, the glue used has a refractive index of 1.4, the ambient is water with a refractive index of 1.33, and the trapped microsphere lens has a diameter of 2um and a refractive index of 1.65. The local magnification diagram shows that the spatial size of the fluorescence excitation beam is greatly compressed and the energy density is enhanced by the compression of the two microsphere lenses. FIG. 8 shows the energy distribution of the optical nano-jet in the x-axis direction at the highest value of energy density in FIG. 7, and the half-maximal full width of the compressed optical nano-jet FWHM = 0.68X can be obtained, which breaks the diffraction limit.
[0042] As a comparison, FIG. 9 is a graph of the compression simulation results of fluorescence excitation light in the absence of trapped microspheres, with only the microsphere lens adhered to the groove. The obtained compression effect is not satisfactory, the spatial scale of the beam is large and the energy density is low. Therefore, the proposed method achieves a very significant beam compression effect through two cascaded microsphere lenses, which compresses the size of the fluorescence excitation beam to the sub-wavelength level and enables super-resolution fluorescence illumination, especially suitable for single-molecule fluorescence excitation illumination.
[0043] Embodiment 3: A fiber-end super-resolution nano-fluorescence microscopic illumination
probe used for the fluorescence illumination and imaging of intracellular single molecules.
[0044] In order to highlight the significant advancement of the contactless fiber optical tweezers
employed in the present invention, single-molecule fluorescence illumination inside cells is
selected as an example to illustrate. As shown in FIG. 10, the probe prepared from the coaxial
dual-waveguide fiber 1 is manipulated by a micro manipulation table 15 to trap the microsphere
lens 6 placed in the single cell 12 on the carrier stage 14. The microsphere lens 6 can be a
spherical substance that is inherently present in the cell, such as a fat particle in an adipocyte, or
it can be a modified micrometer dielectric sphere placed in the cell by phagocytosis of the cell.
After the trapping beam traps the microsphere lens, the fiber probe is moved to move the
microsphere lens 6 to the position to be measured, e.g., at a DNA molecule 13. Pass the
fluorescence excitation light beam 8 in the center core 1-1, after a two-stage compression of the
two microsphere lenses, the resulting optical nano-jet 9 can achieve super-resolution
fluorescence excitation of DNA molecule 13. The fluorescence is collected by the objective lens
11 and imaged by the camera 10, which enables fluorescence detection and imaging. Thus, the
purpose of super-resolution fluorescence microscopic illumination of single molecules within the
cell is achieved, and the whole process is non-invasive to the cell.
Claims (5)
1. A fiber-end super-resolution nano-fluorescence microscopic illumination probe: it is
characterized by the composition of a coaxial dual-waveguide fiber optical tweezers system and
a cascade of two dielectric microsphere lenses. The coaxial dual-waveguide fiber has a center
core and an annular core, and there are symmetrical truncated cone structures at the fiber ends.
They reflect and focus the annular trapped beam transmitted in the annular core to form optical
tweezers, to trap a microsphere lens. A groove is etched in the center of the end face of the fiber
and another microsphere lens is adhered to the groove. Two microsphere lenses are coaxially
distributed with the fiber, and the fluorescence excitation beam transmitted by the center core is
compressed in two stages by the two microsphere lenses to form a super-resolution optical nano
jet that exceeds the diffraction limit, enabling super-resolution fluorescence illumination at the
nanometer scale.
2. As claimed in claim 1, a fiber-end super-resolution nano-fluorescence microscopic
illumination probe, its characteristic is: the truncated cone structure is a symmetrical reflective
truncated cone structure prepared by precision polishing and processing at the coaxial dual
waveguide fiber end, it can also be an arc-optimized reflective truncated cone structure.
3. As claimed in claim 1, a fiber-end super-resolution nano-fluorescence microscopic
illumination probe, its characteristic is: the microsphere lens in the groove described above is
adhered by a low refractive index glue with a diameter greater than or equal to the diameter of
the center core of the coaxial dual-waveguide fiber and less than the diameter of the truncated
cone end face, and its refractive index is greater than that of the low refractive index glue.
4. As claimed in claim 1, a fiber-end super-resolution nano-fluorescence microscopic
illumination probe, its characteristic is: the light beams transmitted in the annular core of the coaxial dual-waveguide fiber are reflected and focused by the truncated cone structure, forming optical tweezers that contactlessly traps a microsphere lens whose refractive index is greater than of the background ambient refractive index.
5. As claimed in claim 1 and claim 4, a fiber-end super-resolution nano-fluorescence
microscopic illumination probe, its characteristic is: the trapped microsphere lens may be an
extracellular dielectric microsphere, a dielectric microsphere placed inside the cell, or a spherical
biological dielectric contained within the cell itself.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| AU2020103836A AU2020103836A4 (en) | 2020-12-02 | 2020-12-02 | A fiber-end super-resolution nano-fluorescence microscopic illumination probe |
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| Application Number | Priority Date | Filing Date | Title |
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| AU2020103836A AU2020103836A4 (en) | 2020-12-02 | 2020-12-02 | A fiber-end super-resolution nano-fluorescence microscopic illumination probe |
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| Publication Number | Publication Date |
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| AU2020103836A4 true AU2020103836A4 (en) | 2021-02-11 |
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| AU2020103836A Ceased AU2020103836A4 (en) | 2020-12-02 | 2020-12-02 | A fiber-end super-resolution nano-fluorescence microscopic illumination probe |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114296228A (en) * | 2021-12-31 | 2022-04-08 | 南京理工大学智能计算成像研究院有限公司 | High-resolution microscopic imaging method based on mixed illumination mode |
-
2020
- 2020-12-02 AU AU2020103836A patent/AU2020103836A4/en not_active Ceased
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114296228A (en) * | 2021-12-31 | 2022-04-08 | 南京理工大学智能计算成像研究院有限公司 | High-resolution microscopic imaging method based on mixed illumination mode |
| CN114296228B (en) * | 2021-12-31 | 2024-01-23 | 南京理工大学智能计算成像研究院有限公司 | High-resolution microscopic imaging method based on mixed illumination mode |
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