CN110389121A - Fluorescence imaging system with multi-focus structured light illumination - Google Patents

Abstract

A multi-focus structured light illumination fluorescence imaging system, comprising: a light source; a first optical chip including an input end and a plurality of output ends, wherein the input end and the light source are connected through an optical fiber; a second optical chip including a plurality of input end and a plurality of output ends, wherein a plurality of input ends and a plurality of output ends of the first optical chip are connected one by one through optical fibers; a filter group is arranged on the optical path extension line of the output end of the second optical chip; a lens is set Between the second optical chip and the optical filter group; an objective lens is arranged on one side of the optical filter group, and the connection line between it and the optical filter group is perpendicular to the connection line between the second optical chip and the optical filter group; A stage is arranged between the objective lens and the optical filter group; a photodetector is arranged on the other side of the optical filter group, and the connection line between it and the optical filter group is perpendicular to the second optical chip and the The connection line of the optical filter group; an imaging lens, which is arranged between the photodetector and the optical filter group.

Description

Fluorescence imaging system with multi-focus structured light illumination

technical field

The invention relates to a fluorescent microscopic imaging system, in particular to a multi-focus structured light illumination fluorescent imaging system.

Background technique

At present, there are a variety of optical fluorescence microscopy imaging techniques that can break through the optical diffraction limit in the existing technology, including: stimulated emission depletion microscopy, light-activated localization microscopy, random optical reconstruction microscopy, and structured light illumination. Microscopic technology, etc., among which the structured light illumination adopted by structured illumination microscopic technology is divided into sinusoidal fringe, random pattern and multi-focus illumination.

In the existing technical solutions for multi-focus structured light illumination fluorescence imaging, spatial light modulators are mostly used to realize the projection and scanning of excitation light array lattices, which can accurately and quickly modulate the characteristics of the excitation light field by using spatial light modulators , form an array point excitation on the surface of the sample, and realize scanning sample imaging by translating the dot matrix, and realize fluorescence super-resolution imaging of the sample through the corresponding image reconstruction algorithm.

Although the realization of structured light illumination based on the spatial light modulator has the advantages of fast and accurate, the high cost of the device limits the wide application of structured light illumination super-resolution microscopy. The laser beam is incident on the spatial light modulator at a specific angle, which increases the volume of the system to a certain extent. At the same time, due to the diffraction effect of the spatial light modulator itself on light, its utilization rate of light energy is not high.

Therefore, based on the above problems in the prior art, it is urgent to develop an optical imaging system with high energy efficiency, low cost and compact structure.

Contents of the invention

(1) Technical problems to be solved

The present invention provides a multi-focus structured light illumination fluorescence imaging system to at least partly solve the shortcomings of high cost, low energy utilization rate and bulky spatial light modulator in the existing method.

(2) Technical solution

According to one aspect of the present invention, a multi-focus structured light illumination fluorescence imaging system is provided, comprising:

A light source, the light source is a laser light source for emitting light beams, the wavelength is 400-1600nm, and the light source can be a single-wavelength light source or an integrated light source with multiple wavelengths;

The first optical chip includes an input end and a plurality of output ends, wherein the input end is connected to the light source through an optical fiber, and is used to split the light beam to form multiple beams of outgoing light, and transmit light from the plurality of output ends respectively. output;

The second optical chip includes a plurality of input ends and a plurality of output ends, wherein the plurality of input ends and the plurality of output ends of the first optical chip are connected one by one through optical fibers, for connecting the first optical chip Each beam of outgoing light is divided into multiple beams and output from multiple output ends respectively, the output end face of the second optical chip is a plane, and the outgoing light is output in a rectangular lattice;

A filter group, arranged on the optical path extension line of the output end of the second optical chip, is used to reflect the outgoing light of the second optical chip to the object to be measured, and at the same time, the excitation light reflected by the object to be measured can be through the filter set to the photodetector;

A lens, arranged between the second optical chip and the filter group, for collecting the outgoing light output from the second optical chip;

An objective lens, arranged on one side of the optical filter group, the connection line between it and the optical filter group is perpendicular to the connection line between the second optical chip and the optical filter group, for projecting excitation light And collect the fluorescence emitted by the object to be measured;

A stage, arranged on the focal plane of the objective lens, for placing the object to be measured, the stage is a three-dimensional precision electric displacement platform;

A photodetector is arranged on the other side of the filter group, and the connection line between it and the filter group is perpendicular to the connection line between the second optical chip and the filter group. The detector can be configured as a scientific-grade CCD or CMOS camera, which is used to collect multi-frame fluorescence images of the object to be tested placed on the stage;

An imaging lens is arranged between the photodetector and the filter group, and is used for imaging the fluorescent signal of the object to be measured on the photodetector.

In a further solution, the first optical chip can be a silicon-based chip, a silicon nitride chip or a PLC (planar lightwave circuit, planar light waveguide) optical chip, such as a single 400-800nm visible light source for For single-photon fluorescence excitation, a silicon nitride chip with a working wavelength of visible light can be selected. If the light source is a single 800-1600nm infrared light for multi-photon fluorescence excitation, a silicon-based chip working at an infrared wavelength can be selected. If the light source is multiple Light of different wavelengths is used for multicolor fluorescence excitation, and a PLC chip that is not sensitive to wavelength can be selected.

In a further solution, the second optical chip is a stacked PLC optical chip, including a multilayer quartz substrate, each quartz substrate is provided with a plurality of splitting waveguides, and each layer of the stacked PLC optical chip All of them can divide one beam of input light into multiple beams of light output, and then realize the rectangular lattice output of outgoing light.

In a further solution, the filter set includes an excitation filter, a dichroic mirror and an emission filter.

(3) Beneficial effects

It can be seen from the above technical solutions that a multi-focus structured light illumination fluorescence imaging system provided by the present invention utilizes stacked PLC chips to realize arrayed dot matrix projection, which reduces the cost of building a multi-focus structured light illumination fluorescence imaging system, and at the same time The optical path is simplified; in addition, compared with the spatial light modulator, the loss of the optical diffraction of the grating-like structure of the PLC chip is low, and the energy utilization rate is high.

Description of drawings

FIG. 1 is a structural diagram of a multi-focus structured light illumination fluorescence imaging system according to an embodiment of the present invention.

FIG. 2 is a schematic diagram of a single-layer optical waveguide in the second optical chip of the system shown in FIG. 1 .

FIG. 3 is a schematic diagram of a second optical chip output section of the system shown in FIG. 1 .

[Description of Reference Signs]

1-light source; 2-first optical chip; 3-second optical chip; 4-lens;

5-filter group; 6-objective lens; 7-stage; 8-imaging lens; 9-photodetector;

301-quartz substrate; 302-split waveguide;

501 - excitation filter; 502 - dichroic mirror; 503 - emission filter.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

In the present invention, "disposed on" or "attached to" is used to include a direct contact relationship with a single or multiple components. Moreover, the ordinal numbers used in the specification and claims, such as "first", "second", "number one" or "number two", are used to modify the components claimed for protection, which do not themselves contain and represent the Parts have any previous ordinal numbers, nor do they imply the order of one part with another or in terms of manufacturing methods, and these ordinal numbers are used only to enable a part with a certain designation to be related to another part with the same designation Can make a clear distinction.

The present invention provides a fluorescent imaging system with multi-focus structured light illumination, which uses the first optical chip and the second optical chip to realize a dot matrix light source, and projects it on the object to be measured through a lens and an objective lens to excite the fluorescence of the object to be measured. At the same time, through the precise three-dimensional movement of the stage, the entire object to be measured is scanned with two-dimensional lattice light, and then the collection of multi-frame fluorescence images is realized by using the filter group, imaging lens and photodetector, and finally the corresponding image algorithm is used. Realize the fluorescence super-resolution imaging of the object to be measured.

Fig. 1 is a structural diagram of a multi-focus structured light illumination fluorescence imaging system according to an embodiment of the present invention, as shown in Fig. 1 , including:

A light source 1. The light source 1 is a laser light source with a wavelength of 400-1600nm. In this embodiment, the light source 1 can be a single-wavelength light source or an integrated light source with multiple wavelengths;

The first optical chip 2 includes an input end and a plurality of output ends, wherein the input end is connected to the light source 1 through an optical fiber, and is used to divide a single beam of incident light from the light source 1 into multiple beams, and separate them from the multiple beams. In this embodiment, the first optical chip 2 can be a silicon-based chip, a silicon nitride chip or a PLC (planar lightwave circuit, planar light waveguide) optical chip, such as the light source 1 is a single 400- 800nm visible light is used for single-photon fluorescence excitation, and silicon nitride chips with working wavelengths in visible light can be selected. For example, light source 1 is a single 800-1600nm infrared light for multi-photon fluorescence excitation, and silicon-based chips working at infrared wavelengths can be selected. , if the light source 1 is light of multiple different wavelengths for multicolor fluorescence excitation, a PLC chip that is not sensitive to wavelength can be selected;

The second optical chip 3 includes a plurality of input ends and a plurality of output ends, wherein the plurality of input ends and the plurality of output ends of the first optical chip 2 are connected one by one by optical fibers, and the output of the second optical chip 3 The end face is a plane, and the output light is a rectangular lattice output; in this embodiment, the second optical chip 3 is a stacked PLC optical chip, including a multilayer quartz substrate 301, and each quartz substrate 301 is provided with a plurality of splitting waveguides 302. Each layer of the second optical chip 3 can divide a single beam of incident light into multiple beams of outgoing light on average for output, and then realize outputting outgoing light in a rectangular lattice.

The optical filter group 5 is arranged on the optical path extension line of the output end of the second optical chip 3, and is used to refract the outgoing light of the second optical chip 3 onto the object to be measured on the stage 7. The sheet group 5 includes an excitation filter 501, a dichroic mirror 502 and an emission filter 503. Meanwhile, the excitation light reflected by the object to be measured can pass through the filter group 5 to the photodetector 9;

The lens 4 is arranged between the second optical chip 3 and the filter group 5, and is used to collect the outgoing light output from the second optical chip 3;

Objective lens 6, is arranged on the side of described optical filter group 5, and its connecting line with described optical filter group 5 is perpendicular to the connecting line of described second optical chip 3 and described optical filter group 5, uses Used to project excitation light and collect fluorescence emitted by the object to be measured;

The object stage 7 is arranged on the focusing surface of the objective lens 6, and is used to place the object to be measured. The object stage 7 is a three-dimensional precision electric displacement platform, which can perform precise three-dimensional movement, and is used to realize the second optical chip 3 Three-dimensional scanning of the object to be measured by the output rectangular lattice light;

The photodetector 9 is arranged on the other side of the filter group 5, and the connection line between it and the filter group 5 is perpendicular to the connection between the second optical chip 3 and the filter group 5 line, the photodetector 9 can be configured as a scientific research-grade CCD or CMOS camera, which is used to collect multi-frame fluorescence images of the object to be tested placed on the stage 7;

The imaging lens 8 is arranged between the photodetector 9 and the filter group 5, and is used for imaging the fluorescence signal of the object to be measured on the photodetector.

Please refer to FIG. 1 again. As shown in FIG. 1 , in this embodiment, the first optical chip 2 includes one input terminal and N output terminals (N≧16), which realizes 1*N uniform light splitting.

Fig. 2 is the schematic diagram of 3 single-layer optical waveguides in the second optical chip of the system shown in Fig. 1, as shown in Fig. 2, the second optical chip 3 includes N input ends and N*N output ends, consists of N layers Quartz substrates 301 are stacked. Each layer of quartz substrate 301 has N light-splitting waveguides 302, which can realize 1*N uniform light-splitting. The input end of each layer corresponds to the output end of the first optical chip 2 one by one, and is connected by optical fiber.

Fig. 3 is a schematic diagram of the output section of the second optical chip 3 of the system shown in Fig. 1. As shown in Fig. 3, the output section of the second optical chip 3 is a plane, which is perpendicular to the optical axis of the optical path, and each output point is pressed Rectangular lattice distribution.

Through the above statement, in view of the problems and shortcomings in the prior art, a multi-focus structured light illumination fluorescence imaging system provided by the embodiment of the present invention uses stacked PLC chips to realize arrayed dot matrix projection, which reduces the cost of building multi-focus The cost of the structured light illumination fluorescence imaging system is reduced, and the optical path is simplified at the same time; in addition, compared with the spatial light modulator, the loss of the optical diffraction of the grating-like structure of the PLC chip is lower, and it has a high energy utilization rate.

The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principles of the present invention, any modifications, equivalent replacements, improvements, etc., shall be included in the protection scope of the present invention.

Claims (9)

1. a kind of multifocal Structured Illumination fluoroscopic imaging systems, comprising:

One light source, for emitting light beam；

First optical chip, including an input terminal and multiple output ends, wherein the input terminal and the light source are connected by an optical fiber It connects, forms multi beam emergent light for the light beam to be carried out light splitting, and export respectively from multiple output ends；

Second optical chip, including multiple input terminals and multiple output ends, wherein the multiple input terminal and first optical chip Multiple output ends connected one by one by optical fiber, for every beam emergent light of first optical chip to be divided into multi beam, and respectively It is exported from multiple output ends；

One filter set is set on the optical path extended line of the second optical chip output end, is used for second optical chip Emergent light reflex to object under test；

One lens are set between second optical chip and filter set, for collecting from second optical chip output Emergent light；

One object lens are set to the side of the filter set, and the connecting line with the filter set is perpendicular to second light The connecting line of chip and the filter set, for project exciting light and acquire object under test transmitting fluorescence；

One objective table is set on the focusing surface of the object lens, for placing object under test；

One photodetector is set to the other side of the filter set, and the connecting line with the filter set is perpendicular to institute The connecting line for stating the second optical chip Yu the filter set, for acquiring the multiframe fluorescence for the object under test being placed on objective table Image；

One imaging len is set between the photodetector and the filter set, the fluorescence signal for object under test It is imaged on the photodetector.

2. system according to claim 1, wherein the light source is laser light source, wavelength 400-1600nm.

3. system according to claim 1, wherein the light source is the light source of single wavelength or the Integrated Light of multiple wavelength Source.

4. system according to claim 1, wherein first optical chip include silicon base chip, nitridation silicon chip or PLC optical chip.

5. system according to claim 1, wherein second optical chip is the PLC optical chip of lamination, including multilayer Quartz base plate is provided with multiple light splitting waveguides on every layer of quartz base plate.

6. system according to claim 5, wherein the output end face of second optical chip is plane, emergent light is square Shape dot matrix output.

7. system according to claim 1, wherein the objective table is three-dimensional precise electricity driving displacement platform.

8. system according to claim 1, wherein the filter set includes an exciter filter, a dichroscope and One transmitting optical filter.

9. system according to claim 1, wherein the photodetector is CCD or CMOS camera.