NL2030544B1

NL2030544B1 - Optical microscope based on surface plasmon resonance

Info

Publication number: NL2030544B1
Application number: NL2030544A
Authority: NL
Inventors: Zhang Qingwen; Wang Yi
Original assignee: Univ Wenzhou Medical
Priority date: 2022-01-13
Filing date: 2022-01-13
Publication date: 2023-07-25

Abstract

It showed an optical microscope based on surface plasmon resonance (SPR). The optical microscope comprises a laser, an incident angle adjusting assembly, a beam splitter, an optical microscopic amplification objective lens, and a plasmon resonance sensing chip 5 which are sequentially arranged along a light path, and further comprises an image sensor, wherein the incident angle adjusting assembly comprises a polarizer used for converting incident light into p—polarized light, and the incident light of the laser passes through the incident angle adjusting assembly, the beam splitter 10 and the optical microscopic amplification objective lens and is incident on the plasmon resonance sensing chip at a surface plasmon resonance angle (9). Plasmon surface resonance is excited on the plasmon resonance sensing chip, and reflected light on the plasmon resonance sensing chip passes through the optical l5 microscopic amplification objective lens and the beam splitter and is incident and imaged on the image sensor. (+ Fig. l) 20

Description

P1009/NLpd

OPTICAL MICROSCOPE BASED ON SURFACE PLASMON RESONANCE

TECHNICAL FIELD

The present disclosure relates to an optical microscope sys- tem based on surface plasmon resonance (SPR).

BACKGROUND ART

Optical microscopes have always played an extremely important role in life sciences, biological research, materials science and other fields. Compared with other high-resolution microscopes, the optical microscopes have the unique advantages that, for example, optical microscopes have the advantages of observing living sam- ples dynamically in real time compared with electron microscopes.

By means of the advantage, the optical microscope is incomparable in biological research. However, the optical microscopes have their own disadvantages, the biggest of which is that the resolu- tion of optical microscopes is limited by the limit of optical diffraction. The resolution of a common fluorescence microscope is also 200 nm, and an ultra-high resolution fluorescence microscope can realize the imaging of a structure smaller than 10 nm, however a sample needs to be subjected to fluorescence labeling, the true characteristics of the sample cannot be reflected due to the change of activity and other properties of the sample. Therefore, it is crucial for optical microscopy to improve its resolution and achieve no need of fluorescent labeling.

SUMMARY

To overcome defects and disadvantages in the prior art, an objective of the present disclosure is to provide an optical mi- croscope based on SPR with high resolution and without fluores- cence labeling.

To achieve the objective, the technical solution of the pre- sent disclosure comprises a laser, an incident angle adjusting as- sembly, a beam splitter, an optical microscopic amplification ob- jective lens, and a plasmon resonance sensing chip which are se-

gquentially arranged along a light path, and further comprises an imaging sensor. The incident angle adjusting assembly comprises a polarizer for converting incident light into p-polarized light, the incident light of the laser passes through the incident angle adjusting assembly, the beam splitter and the optical microscopic amplification objective lens and arrives at the SPR sensing chip with a SPR angle (8) for the exciting of SPR. The reflected light on the SPR sensing chip passes through the optical microscopic am- plification objective lens and the beam splitter, and is collected by the image sensor.

In further arrangement, the incident angle adjusting assembly comprises a collimating lens arranged at the front end of a light path of the polarizer and a condensing lens arranged at the rear end of light path of the polarizer, and the incident light is fo- cused on a rear focal plane of the optical microscopic amplifica- tion objective lens after passing through the incident angle ad-

Justing assembly and the beam splitter.

In further arrangement, the incident angle adjusting assembly is entirely placed on the movable platform. The incident angle ad-

Jjusting assembly is entirely driven by a movable platform, thus adjusting an incident angle of incident light irradiating on the plasmon resonance sensing chip to meet a SPR angle (8).

In further arrangement, the optical matching oil is filled between the optical microscopic amplification objective lens and the plasmon resonance sensing chip. The refractive index of the optical matching oil ranges from 1.515-1.780.

In further arrangement, the image sensor is a CCD image sen- sor or a CMOS image sensor.

In further arrangement, the polarizer is a natural birefrin- gent crystal or an artificial polarizer.

In further arrangement, the optical microscopic magnification objective lens demonstrate the magnification times of 40 to 100 and a numerical aperture range of 1.40 to 1.69.

In further arrangement, the beam splitter is one of a prism, a dichroic mirror, a thin film beam splitter, or a half-plate to- tal reflector.

In further arrangement, the plasmon resonance sensing chip is composed of a substrate and a metal layer, wherein the substrate material is one of lanthanum glass, BK7 glass, common glass, or a plastic substrate. A layer of chromium metal of 2 nm+1 is firstly evaporated on the substrate, and then a layer of composite layer made of one or more of gold, silver, copper, aluminum and platinum is evaporated on the chromium substrate with the thickness of 40- 60 nm.

In further arrangement, an optical lens is further arranged on a reflection light path between the beam splitter and the image sensor.

A working principle of the present disclosure is that the SPR microscope could effectively improve the resolution of the optical microscope to be less than 50 nm without fluorescent labeling. SPR is a physical optical phenomenon occurring on an interface between metal and dielectric, which is an electromagnetic surface wave, has the field intensity at the surface, and has the field intensi- ty decaying with an exponential trend in a direction perpendicular to the interface. Due to the fact that SPR needs to meet resonance conditions, surface plasmon waves are obtained by generally cou- pling incident light to the surface of a dielectric layer for ex- citing by using a prism or a grating, which generate resonance when having the energy same as that of evanescent waves generated by total internal reflection of the incident light to sharply re- duce the energy of the reflected light.

In the present disclosure, an optical microscopic amplifica- tion objective lens is in direct contact with a dielectric layer which is connected to the objective lens through matching oil, a large enough incident angle can be provided through a high numeri- cal aperture to achieve vector matching, thus achieving SPR which is particularly sensitive to refractive index change of the die- lectric layer. By utilizing the characteristics, materials with different refractive indexes can be distinguished to form images with different brightness and darkness, thus forming a SPR micro- scope with high resolution, high sensitivity and no fluorescence labeling.

The technical solution provided by the present disclosure can be applied to the following fields:

(1) label-free imaging of nanoparticles with a diameter larg- er than 50 nm, e.qg., interaction imaging between the virus and its surface receptors; (2) real-time detection in the field of biomolecules, e.qg., real-time detection of interaction between the biomolecule and bi- ological cells, for example, real-time detection of biomolecule information on DNA or protein chips; (3) real-time label-free detection of cell molecules, e.qg., label-free detection of interaction of cell molecules and cell collagen fiber formation; (4) real-time detection of interaction between drug macromol- ecules, peptides and the like and cells and tissues.

Compared with the prior art, the present disclosure has the advantages that: the SPR microscope can realize high-resolution, high- sensitivity and high-stability real-time detection of biomolecules without fluorescent labeling.

The SPR microscope can quantitatively detect the content of certain particles in the sample. Such technology can make up the defect that other method can only conduct qualitative detection.

The following further describes the present disclosure in conjunction with the drawings and specific embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a structure diagram of the present disclo- sure.

FIG. 2 illustrates an image of reflected light intensity col- lected from a CCD camera.

FIG. 3 illustrates a gray-scale curve of gold nanoparticles on a CCD camera.

FIG. 4 illustrates a reflected light intensity spectrum de- tected from one pixel point of a CCD camera along with change of the incident angle.

FIG. 5 the SPRi curve illustrates light intensity of reflect- ed light detected from a CCD camera along with change of the inci- dent angle, i.e., a surface plasmon resonance image ().

DETAILED DESCRIPTION OF THE EMBODIMENTS

The following describes the present disclosure detail with reference to the embodiments, the embodiments are only used for further describing the present disclosure, but cannot be construed 5 as a limitation to the scope of protection of the present disclo- sure, some non-essential modifications and adjustments of the pre- sent disclosure may be made by engineers skilled in the art in ac- cordance with the content of the present disclosure.

Please referring to a structure diagram shown in FIG. 1, var- ious components arranged in accordance with the sequence of a light path are a laser 1, an optical fiber 2, a movable platform 3, a collimating lens 4, a polarizer 5, a condensing lens 6, a beam splitter 7, an optical microscopic amplification objective lens 8, matching oil 9, a plasmon resonance sensing chip 10, a to- be-detected sample 11, an optical lens 12, and an image sensor 13.

As an example, SPR microscope was employed for the detection of gold nanoparticles with a diameter of 80 nm (1) The plasmon resonance sensing chip is prepared, BK7 glass is used as a substrate, a layer of chromium metal of 2 nm is firstly evaporated on a glass sheet, a metal layer with a thick- ness of about 47 nm is evaporated on the second layer, and a layer of gold nano-particles having a diameter of 80 nm is self- assembled on the surface of a gold chip. (2) The laser is turned on to adjust the light path, thus a strong signal can be collected by the image sensor (preferably a

CCD camera). (3) Referring to FIG. 1, the movable platform is adjusted to move in an x direction to linearly adjust a position of incident light to the optical microscopic amplification objective lens.

Thus the incident angle of a light beam coming out of an objective lens to the gold chip is changed to obtain SPR angle spectrogram, and obtain the incident angle capable of exciting SPR. (4) As shown in FIG. 2, an image is observed from the CCD camera, wherein a bright spot part is one of the gold nano- particles, the gray-scale curve of the bright spot part is as shown in FIG. 3, and the full width at half maximum (FWHM) of the bright spot part is about 650 nm.

(5) The light intensity reflected from the plasmon resonance sensing chip is detected using the image sensor, it can be found that the light intensity reflected to the camera is the weakest when an angle of the incident light on the plasmon resonance sens- ing chip is at a certain value. At the moment, it is noted that

SPR has occurred, and a part of energy of the incident light has been converted into the energy of the surface plasmon wave. When

FIG. 4 illustrates the change of reflected light intensity as the incident angle of the light changes on one pixel point of the im- age sensor. Visibly, a SPR angle spectrogram can also be detected in a size range of about 160 nm x 160 nm for one pixel. Therefore, it is possible to analyze the interaction between molecules on a small area of one pixel, or even to detect single molecules. (6) By scanning an incident angle of the incident light, it can be detected that the reflected light intensity is changed therewith from the image sensor, thus acquiring the SPR angle spectrogram. As shown in FIG. 5, after the single gold nano- particles of 80 nm is adsorbed to a gold chip, the SPR angle is increased by about 2°. Through the changes of the SPR image and the SPR angle, the attributes of the to-be-detected sample, in- cluding information such as content, refractive index, even quali- ty and the like, can be calculated.

Claims

CONCLUSIONS

Surface plasmon resonance (SPR) optical microscope comprising a laser, an optical fiber, an angle of incidence adjustment assembly, a beam splitter, an objective lens for optical microscopic amplification, and an SPR detection chip arranged sequentially along arranged in a light path, and further comprising an image sensor, the angle of incidence adjustment assembly including a polarizer for converting incident light to p-polarized light, the incident light from the laser generator passing through the assembly for adjusting the angle of incidence, the beam splitter, the objective lens for optical microscopic amplification and arrives at the detection chip with an SPR angle for the existence of the SPR on the detection chip, and the reflected light passes through the objective lens for optical microscopic gain and the beam splitter and is received by the image sensor; wherein the objective lens for optical microscopic amplification has gain values of 40 to 100 and a numerical aperture range of 1.40 to 1.69.

The SPR based optical microscope of claim 1, wherein the angle of incidence adjustment assembly comprises a collimating lens mounted at the front end of the light path of the polarizer and a condensing lens mounted at the rear end of the light path from the polarizer, and wherein the incident light is focused onto a rear focal plane of the objective lens for optical microscopic amplification after passing through the angle of incidence and beam splitter assembly ; wherein the angle of attack assembly is located wholly on a movable platform; wherein optical adjustment oil is filled between the outer end of the optical microscopic amplification objective lens and the inner end face of the detection chip, and wherein the refractive index of the optical adjustment oil ranges from

1,515 to 1,780; wherein the image sensor is a CCD image sensor or a CMOS image sensor; wherein the polarizer is a natural birefringent crystal or artificial polarizer; wherein the beam splitter is one of a prism, a dichroic mirror, a thin film beam splitter, or a half-plate total reflector; wherein the sensing chip is composed of a substrate and a metal layer, wherein the substrate is one of glass or a plastic substrate, wherein a layer of chromium metal of 2 nm + 1 is first vaporized on the substrate and then a layer of a composite layer is made of one or more of gold, silver, copper, aluminum and platinum with a thickness of 40 - 60 nm is then evaporated; and wherein an optical lens is further disposed on a reflection light path between the beam splitter and the image sensor.