CN103335993A - Fluorescent dark field microscopy device and method based on waveguide constraint - Google Patents
Fluorescent dark field microscopy device and method based on waveguide constraint Download PDFInfo
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Abstract
The invention discloses a fluorescent dark field microscopy device and method based on waveguide constraint. The fluorescent dark field microscopy device comprises an illumination laser light source, a beam expansion lens, a near-end reflector, a carrier chip, a microobjective, a far-end reflector, a light filter, a collection lens and a CCD (Charge Coupled Device) image sensor; the method comprises the following steps that: the carrier chip which directly realizes the dark field function is prepared; lasers emitted by the illumination laser light source irradiate on a polymer film in the carrier chip through the near-end reflector after being subjected to beam expansion; fluorescence light emitted from fluorescent molecules in the light source forms a guiding mode in the carrier chip and is constrained at an interface of the polymer film and air, and thus, the bottom of a sample nearby the carrier chip is lightened by a near field and scattered; then, light is collected by the microobjective, after the illumination lasers are filtered by the far-end reflector and the light filter, the light is collected by the collection lens and forms an image at the CCD image sensor. The device and the method are based on the constraint of a fluorescent waveguide mode, the microscopy structure is simplified, and the dark field microscopy function is realized.
Description
Technical field
The present invention relates to the technical field of micro-imaging, particularly a kind of fluorescence details in a play not acted out on stage, but told through dialogues microscope equipment and method based on the waveguide constraint.
Background technology
Optical microscope is used by the association area personnel widely as the indispensable instrument in the micro-imaging field.It also is divided into light field microscope, dark field microscope, phase contrast microscope, videomicroscopy, fluorescent microscope, polarizing microscope, Laser Scanning Confocal Microscope, metaloscope etc. because purposes is different.Especially, at sample segment since its refractive index and surrounding environment relatively near under the situation, the observed result under the light field is not obvious, therefore often the method for use details in a play not acted out on stage, but told through dialogues micro-imaging is observed.Its principle generally be with some optical elements with lighting source with than oblique being mapped on the sample of wide-angle (greater than the corresponding angle of numerical aperture of collecting microcobjective), have only scattered light to be gathered into picture by object lens, can significantly characterize the edge contour of object, both see its pattern clearly, highlighted the contrast of sample again.
The method that realizes details in a play not acted out on stage, but told through dialogues is a lot, and with regard to prior art, general implementation method all is optical element such as axicon lens or diaphragm and the light beam that produces hollow is realized wide-angle incident.Its problem that mainly exists is: it is low that (1) sees through efficient.Because optical elements such as diaphragm make the far field excitation illumination light be difficult to all be utilized in the time of incident, cause energy loss.(2) cost performance is low.The details in a play not acted out on stage, but told through dialogues function of utilizing complicated technology such as optical elements sets to realize, its adding owing to a plurality of optical elements causes the lifting of price to be unfavorable for popularizing.(3) require harshness.In the common dark-field system, owing to want good elimination illumination light need very strict requirement be arranged to the collimation of light path, the expansion that this is unfavorable for imaging system is unfavorable for those skilled in the art's research.
Summary of the invention
Technology of the present invention is dealt with problems: the deficiency that overcomes existing dark field microscope imaging technique, a kind of fluorescence details in a play not acted out on stage, but told through dialogues microscope equipment and method based on the waveguide constraint proposed, realize convenient easily expansion, the details in a play not acted out on stage, but told through dialogues of economical and efficient is micro-, it is low to have solved traditional far field excitation efficient, light path complicated technology problem is a kind of low cost, practical micro-imaging technique.
For achieving the above object, technical scheme of the present invention is as follows: a kind of fluorescence details in a play not acted out on stage, but told through dialogues microscope equipment based on the waveguide constraint comprises: laser illuminator light source, extender lens, near-end catoptron, loading chip, microcobjective, far-end catoptron, optical filter, collecting lens and ccd image sensor; Be observed sample and place loading chip top; Described loading chip from bottom to top is made of successively the polymer film layer of glass-based bottom, metal film layer, doping fluorescent molecule, is used for the fluorescence that constraint is excited, and directly realizes the function of details in a play not acted out on stage, but told through dialogues; The laser that described laser illuminator light source sends expands by the near-end catoptron through extender lens and illuminates the loading chip, and excite the doping fluorescent molecule in its polymer film layer, the fluorescence that fluorescence molecule sends forms guided mode between upper strata air layer and lower metal thin layer, and be bound by polymeric film layer and air interface place, when sample places loading chip last, the sample bottom near the loading chip locate illuminated and scattering by fluorescence; Scattered light is directly collected by microcobjective, behind far-end catoptron and optical filter elimination laser illuminator, is collected the lens collection and images in ccd image sensor, realizes details in a play not acted out on stage, but told through dialogues observation.
A kind of fluorescence details in a play not acted out on stage, but told through dialogues microscopic method based on the waveguide constraint may further comprise the steps:
Sample preparation: prepare the loading chip with direct realization details in a play not acted out on stage, but told through dialogues function, and sample is put in loading chip top; The preparation of described loading chip is included as: evaporation layer of metal thin layer on substrate of glass, spin coating contains the PMMA(polymethylmethacrylate of fluorescence molecule again) and constitute polymer film layer, place sample after the oven dry up, make the loading chip;
Illumination: regulate the laser illuminator light source, the laser that the laser illuminator light source sends expands by the near-end catoptron through extender lens and illuminates the loading chip, and excite the doping fluorescent molecule in its polymer film layer, the fluorescence that fluorescence molecule sends forms guided mode between upper strata air layer and lower metal thin layer, and be bound by polymeric film layer and air interface place, when sample places loading chip last, the sample bottom near the loading chip locate illuminated and scattering by fluorescence;
Observation: fluorescence excitation goes filtration because the constraint of waveguide need not to add in addition optical element, collect scattered light by microcobjective, behind scattered light process far-end catoptron and the optical filter elimination laser illuminator, be collected the lens collection and image in ccd image sensor, realize details in a play not acted out on stage, but told through dialogues observation.
The principle of technical solution of the present invention is: excite lighting source illuminate the loading chip in the doping fluorescent molecule, the fluorescence that fluorescence molecule in the thin polymer film sends forms guided mode between upper strata air layer and lower metal layer, and is bound by polymer thin film and air interface place and can be transmitted to the far field.Only when sample placed loading chip last, sample was illuminated in the fluorescence near field, make its close loading chip in bottom locate be scattered, be gathered into picture by microcobjective then.
The advantage that the present invention compares with conventional art is:
(1) simple in structure: as to need not extra optical element and control the angle that excites lighting source, solved the complicated problem that is difficult for expansion of conventional details in a play not acted out on stage, but told through dialogues microscopic system light path;
(2) integration height, the loading chip plays loading simultaneously, and the effect of sample and shielding exciting light is directly illuminated in the fluorescence excitation near field;
(3) cost is low: the loading chip manufacturing is simple, favorable expandability, and cost is lower, thereby has realized the novel details in a play not acted out on stage, but told through dialogues microtechnic that is suitable for studying;
(4) good operability: the polymer layer of thickness in the loading chip, can realize the details in a play not acted out on stage, but told through dialogues of different-effect, thereby observation can change chip details in a play not acted out on stage, but told through dialogues the time at any time, to be fit to required sample.
(5) both to have protected the surface that it is difficult for above metal film layer oxidized for the PMMA layer of institute of the present invention spin coating, reduced the roughness of metal film layer again, makes its better fluorescence of being excited of constraint.
(6) the fluorescence details in a play not acted out on stage, but told through dialogues microscopic method of the waveguide among the present invention constraint, exciting light adopt can fluorescence excitation molecule R6G the 532nm laser instrument, the fluorescence that inspires is bound by among the waveguide, realizes the near field illumination.
When (7) the present invention inscribes the thickness of polymer film layer by change, can excite different guided modes.The constraint ability is also different, realizes the adjusting of the field of illumination, near field of sample in the details in a play not acted out on stage, but told through dialogues.
(8) evaporation is to suprabasil metal film among the present invention, and its THICKNESS CONTROL effectively makes fluorescence form guided mode in tens nanometers, has improved contrast.
Description of drawings
Fig. 1 is the fluorescence details in a play not acted out on stage, but told through dialogues microscope equipment structural representation that the present invention is based on the waveguide constraint;
Fig. 2 is details in a play not acted out on stage, but told through dialogues imaging synoptic diagram; Wherein a is for utilizing the details in a play not acted out on stage, but told through dialogues imaging synoptic diagram of the loading chip realization that silverskin is arranged, the details in a play not acted out on stage, but told through dialogues imaging synoptic diagram that b realizes for the loading chip that utilizes no silverskin;
Fig. 3 is the process flow diagram of the fluorescence details in a play not acted out on stage, but told through dialogues microscopic method based on waveguide constraint of the present invention.
Wherein, 1, the laser illuminator light source, 2, extender lens, 3, near-end catoptron, 4, glass-based bottom, 5, metal film layer, 6, the polymer film layer of doping fluorescent molecule, 7, microcobjective, 3, far-end catoptron, 9, optical filter, 10, collecting lens, 11, ccd image sensor.
Embodiment
As shown in Figure 1, the fluorescence details in a play not acted out on stage, but told through dialogues microscope equipment that the present invention is based on waveguide constraint comprises: laser illuminator light source 1, extender lens 2, near-end catoptron 3, loading chip, microcobjective 7, far-end catoptron 3, optical filter 9, collecting lens 10 and ccd image sensor 11.Be observed sample and place loading chip top; The loading chip from bottom to top is made of successively the polymer film layer 6 of glass-based bottom 4, metal film layer 5, doping fluorescent molecule, is used for the fluorescence that constraint is excited, and directly realizes the function of details in a play not acted out on stage, but told through dialogues; Wherein metal film layer 5 is made of one deck silver, and the polymer film layer 6 of doped with fluorescent dyes is by the PMMA(polymethylmethacrylate that contains fluorescence molecule) constitute; Fluorescence molecule is R6G; The wavelength of laser illuminator light source 1 is 532 nanometers.Metal film layer 5 thickness are 45 nanometers; The thickness of PMMA layer is 50 nanometers.Can excite guided mode, realize the details in a play not acted out on stage, but told through dialogues imaging function of high-contrast.
The laser that laser illuminator light source (1) sends expands by near-end catoptron 3 through extender lens 2 and illuminates the loading chip, and excite the doping fluorescent molecule in its polymer film layer 6, the fluorescence that fluorescence molecule sends forms guided mode between upper strata air layer and lower metal thin layer 5, and be bound by polymer film layer 6 and air interface place, when sample places loading chip last, the sample bottom near the loading chip locate illuminated and scattering by fluorescence; Scattered light is directly collected by microcobjective 7, behind far-end catoptron 3 and optical filter 9 elimination laser illuminators, is collected lens 10 collections and images in ccd image sensor 11, realizes details in a play not acted out on stage, but told through dialogues observation.
Shown in (a) among Fig. 2, the present invention has well realized the high-contrast details in a play not acted out on stage, but told through dialogues imaging of edge contour.Change along with the thickness of thin polymer film can excite the different guided wave mould in the thin polymer film, realizes the details in a play not acted out on stage, but told through dialogues function.Especially when the thickness of metal film layer 5 is zero, namely do not have metal film layer 5, still can excite the different guided wave mode in the thin polymer film this moment, realize the details in a play not acted out on stage, but told through dialogues imaging, shown in (b) among Fig. 2.
As shown in Figure 3, the performing step of the inventive method is:
The first step, sample preparation: prepare the loading chip with direct realization details in a play not acted out on stage, but told through dialogues function, and sample is put in loading chip top; The preparation of loading chip is included as: evaporation layer of metal thin layer 5 on substrate of glass 4, spin coating contains the PMMA(polymethylmethacrylate of fluorescence molecule again) and constitute polymer film layer 6, place sample after the oven dry up, make the loading chip;
Second, illumination: regulate laser illuminator light source 1, the laser that laser illuminator light source 1 sends expands by near-end catoptron 3 through extender lens 2 and illuminates the loading chip, and excite the doping fluorescent molecule in its polymer film layer 6, the fluorescence that fluorescence molecule sends forms guided mode between upper strata air layer and lower metal thin layer 5, and be bound by polymer film layer 6 and air interface place, when sample places loading chip last, the sample bottom near the loading chip locate illuminated and scattering by fluorescence;
The 3rd step, observation: fluorescence excitation goes filtration because the constraint of waveguide need not to add in addition optical element, collect scattered light by microcobjective 7, behind scattered light process far-end catoptron 3 and the optical filter 9 elimination laser illuminators, be collected lens 10 collections and image in ccd image sensor 11, realize details in a play not acted out on stage, but told through dialogues observation.
The part that the present invention does not elaborate belongs to techniques well known.
Claims (9)
1. the fluorescence details in a play not acted out on stage, but told through dialogues microscope equipment based on the waveguide constraint is characterized in that comprising: laser illuminator light source (1), extender lens (2), near-end catoptron (3), loading chip, microcobjective (7), far-end catoptron (3), optical filter (9), collecting lens (10) and ccd image sensor (11); Be observed sample and place loading chip top; Described loading chip from bottom to top is made of successively the polymer film layer (6) of glass-based bottom (4), metal film layer (5), doping fluorescent molecule, is used for the fluorescence that constraint is excited, and directly realizes the function of details in a play not acted out on stage, but told through dialogues; The laser that described laser illuminator light source (1) sends expands by near-end catoptron (3) through extender lens (2) and illuminates the loading chip, and excite the doping fluorescent molecule in its polymer film layer (6), the fluorescence that fluorescence molecule sends forms guided mode between upper strata air layer and lower metal thin layer (5), and be bound by polymer film layer (6) and air interface place, when sample places on the loading chip, sample bottom near the loading chip locate illuminated and scattering by fluorescence; Scattered light is directly collected by microcobjective (7), behind far-end catoptron (3) and optical filter (9) elimination laser illuminator, is collected lens (10) collection and images in ccd image sensor (11), realizes details in a play not acted out on stage, but told through dialogues observation.
2. a kind of fluorescence details in a play not acted out on stage, but told through dialogues microscope equipment based on waveguide constraint according to claim 1, it is characterized in that: described metal film layer (5) is made of one deck silver.
3. a kind of fluorescence details in a play not acted out on stage, but told through dialogues microscope equipment based on waveguide constraint according to claim 1, it is characterized in that: the polymer film layer of described doped with fluorescent dyes (6) is by the PMMA(polymethylmethacrylate that contains fluorescence molecule) constitute.
4. a kind of fluorescence details in a play not acted out on stage, but told through dialogues microscope equipment based on waveguide constraint according to claim 1, it is characterized in that: described fluorescence molecule is R6G.
5. a kind of fluorescence details in a play not acted out on stage, but told through dialogues microscope equipment based on waveguide constraint according to claim 1, it is characterized in that: the wavelength of described laser illuminator light source (1) is 532 nanometers.
6. a kind of fluorescence details in a play not acted out on stage, but told through dialogues microscope equipment based on waveguide constraint according to claim 1, it is characterized in that: the thickness range of described metal film layer (5) is that 0 nanometer is to 50 nanometers.
7. a kind of fluorescence details in a play not acted out on stage, but told through dialogues microscope equipment based on waveguide constraint according to claim 2 is characterized in that: the thickness of described PMMA layer can for 50 nanometers to 450 nanometers.
8. fluorescence details in a play not acted out on stage, but told through dialogues microscopic method based on waveguide constraint is characterized in that may further comprise the steps:
Sample preparation: prepare the loading chip with direct realization details in a play not acted out on stage, but told through dialogues function, and sample is put in loading chip top; The preparation of described loading chip is included as: go up evaporation layer of metal thin layer (5) in substrate of glass (4), spin coating contains the PMMA(polymethylmethacrylate of fluorescence molecule again) formation polymer film layer (6), place sample after the oven dry up, make the loading chip;
Illumination: regulate laser illuminator light source (1), the laser that laser illuminator light source (1) sends expands by near-end catoptron (3) through extender lens (2) and illuminates the loading chip, and excite the doping fluorescent molecule in its polymer film layer (6), the fluorescence that fluorescence molecule sends forms guided mode between upper strata air layer and lower metal thin layer (5), and be bound by polymer film layer (6) and air interface place, when sample places loading chip last, the sample bottom near the loading chip locate illuminated and scattering by fluorescence;
Observation: collect scattered light by microcobjective (7), behind scattered light process far-end catoptron (3) and optical filter (9) the elimination laser illuminator, be collected lens (10) collection and image in ccd image sensor (11), realize details in a play not acted out on stage, but told through dialogues observation.
9. the fluorescence details in a play not acted out on stage, but told through dialogues microscopic method based on waveguide constraint according to claim 8, it is characterized in that: when changing the thickness of described polymer film layer (6), can excite different guided modes, the constraint ability is also different, realizes the adjusting of the field of illumination, near field of sample in the details in a play not acted out on stage, but told through dialogues.
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| CN104567659A (en) * | 2014-12-22 | 2015-04-29 | 南京师范大学 | Vortex light lighting-based dark field digital holographic microscopy device and method |
| CN104567660A (en) * | 2014-12-22 | 2015-04-29 | 南京师范大学 | Dark field digital holographic microscopy device based on vortex round airy light illumination and method using device |
| CN104880453A (en) * | 2015-05-06 | 2015-09-02 | 华东理工大学 | Synchronous light-electricity sensing method for dark-field imaging-based solid nano channel |
| CN106580267A (en) * | 2017-01-24 | 2017-04-26 | 青岛大学附属医院 | Sidestream dark field imaging technique-based human body microvessel three-dimensional ultrastructure imaging system |
| CN108956571A (en) * | 2018-07-27 | 2018-12-07 | 南京理工大学 | It is a kind of that ccd target surface is imaged on based on compromising emanation and the calibration method and device of positioning surface angle error are installed |
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| CN113467066A (en) * | 2021-06-28 | 2021-10-01 | 中国科学技术大学 | Multifunctional microscopic imaging slide glass based on optical film |
| CN115097617A (en) * | 2022-07-15 | 2022-09-23 | 中国科学技术大学 | Total internal reflection fluorescence microscope system based on planar chip |
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| CN104567660A (en) * | 2014-12-22 | 2015-04-29 | 南京师范大学 | Dark field digital holographic microscopy device based on vortex round airy light illumination and method using device |
| CN104567659A (en) * | 2014-12-22 | 2015-04-29 | 南京师范大学 | Vortex light lighting-based dark field digital holographic microscopy device and method |
| CN104880453A (en) * | 2015-05-06 | 2015-09-02 | 华东理工大学 | Synchronous light-electricity sensing method for dark-field imaging-based solid nano channel |
| CN111413138A (en) * | 2015-12-23 | 2020-07-14 | 辰和科技药业股份有限公司 | Dual image based biometric imaging apparatus and techniques |
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| CN109073517A (en) * | 2016-04-27 | 2018-12-21 | 文塔纳医疗系统公司 | System and method for real-time volume control |
| CN106580267A (en) * | 2017-01-24 | 2017-04-26 | 青岛大学附属医院 | Sidestream dark field imaging technique-based human body microvessel three-dimensional ultrastructure imaging system |
| CN106580267B (en) * | 2017-01-24 | 2024-03-29 | 青岛大学附属医院 | Imaging system of human body microvascular three-dimensional ultrastructure based on sidestream dark field imaging technology |
| CN108956571A (en) * | 2018-07-27 | 2018-12-07 | 南京理工大学 | It is a kind of that ccd target surface is imaged on based on compromising emanation and the calibration method and device of positioning surface angle error are installed |
| CN108956571B (en) * | 2018-07-27 | 2023-12-15 | 南京理工大学 | Calibration method and device based on error of included angle between ccd target surface and installation positioning surface of leakage radiation imaging |
| CN110398479B (en) * | 2019-07-17 | 2022-04-08 | 南京理工大学 | Micro-spectrum measuring device and method based on optical chip substrate |
| CN110398479A (en) * | 2019-07-17 | 2019-11-01 | 南京理工大学 | A kind of microspectrum measuring device and method based on optical chip substrate |
| CN110609026B (en) * | 2019-10-31 | 2020-11-03 | 福州大学 | Laser power feedback control up-conversion fluorescence imaging device and imaging method thereof |
| CN110609026A (en) * | 2019-10-31 | 2019-12-24 | 福州大学 | Laser power feedback control up-conversion fluorescence imaging device and imaging method thereof |
| CN112557371A (en) * | 2020-12-08 | 2021-03-26 | 北京大学 | Nanoscopic multiphase interface transient observation device and method based on laser effect |
| CN112557371B (en) * | 2020-12-08 | 2021-11-02 | 北京大学 | Nanoscopic multiphase interface transient observation device and method based on laser effect |
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| CN115097617A (en) * | 2022-07-15 | 2022-09-23 | 中国科学技术大学 | Total internal reflection fluorescence microscope system based on planar chip |
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Application publication date: 20131002 |