CN101446406B - Illuminator in fiber-optical evanescent field - Google Patents
Illuminator in fiber-optical evanescent field Download PDFInfo
- Publication number
- CN101446406B CN101446406B CN2008102374140A CN200810237414A CN101446406B CN 101446406 B CN101446406 B CN 101446406B CN 2008102374140 A CN2008102374140 A CN 2008102374140A CN 200810237414 A CN200810237414 A CN 200810237414A CN 101446406 B CN101446406 B CN 101446406B
- Authority
- CN
- China
- Prior art keywords
- fiber
- optical
- plane
- illuminator
- optical fiber
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Microscoopes, Condenser (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention relates to an illuminator used in a fiber-optical evanescent field. Part of one end of the fiber optic is ground to be an inclined surface, and the inclined surface goes beyond the position of the fiber core 1 to 10 micron; when entering from the fiber core, the light is reflected to the end surface of the fiber optical in the position of the inclined surface, and a total internal reflection is generated on the end face of the fiber optical. By adopting the technology of combination of the optical fiber and the internal reflection imaging principle, the illuminator used in the evanescent field for biological illumination is fabricateed on a single optical fiber. Compared with the prior illuminator used in the evanescent field, the design of the invention has the advantages of simple structure, smaller volume, more flexible and stable light path. Therefore, a multifaceted illumination on the surface of the bioanalyte can be achieved, and a major breakthrough is made in the performence of the illuminator used in the evanescent field. A novel fluorescent micro-imaging system is convenneintly formed by adopting the technology of combination of the microscope, meanwhile, the illuminator is easily combined with other imaging technique and probing technique and has a wide application range.
Description
Technical field
The invention belongs to the fluorescent microscopic imaging technical field, particularly a kind of illuminator in fiber-optical evanescent field.
Background technology
The dynamic characteristic of a large amount of fact table clear-cells comes from the polymerization and the interaction of single protein molecule in the life science, just need the imaging technique of development ultrahigh resolution in order to study cyto-dynamics, thereby can on molecular scale, survey the details of cell activities.Under this demand, the thirties in 20th century, electron microscope grew up.It has caused the revolution of cell research, makes the biologist be able to from the inferior microscopic level understanding cell world.Enter the eighties, the non-optical class Scanning Probe Microscopy particularly appearance of AFM is advanced to the resolution ratio of imaging the precision of nanometer especially.But all there are difficulties such as system architecture complexity, imaging testing environment requirement harshness in these microscopies in varying degrees, important optical information (as polarization state, refractive index, spectrum etc.) especially can not be provided as optical microscopy and carry out the detection of non-invasi biological living, these have all limited their application in the high-resolution cell imaging greatly.Meanwhile, optical microscopy of new generation grows up.They become the focus that biologist, physicist and imaging scholars study once more with its high spatial resolution and temporal resolution, not damaged and feasibility that the unimolecule live body is surveyed.TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY is emerging in recent years a kind of optical image technology, the evanscent field that it utilizes total internal reflection the to produce sample that throws light on, thereby cause the fluorogen in the optics thin layer of thickness at hundred nano-scale to be excited, the signal to noise ratio of fluorescence imaging is very high.The imaging device of this method is simple, very easily combines with other imaging technique, Detection Techniques.At present successful realization 100nm even lower spatial resolution.
At present, the implementation of carrying out the fluorescent microscopic imaging of evanscent field illumination based on total internal reflection principle roughly has two kinds: lens type luminaire and object lens formula luminaire.Laser shines the cover glass surface after seeing through prism or object lens at a certain angle, forms total reflection, and evanescent wave excites the fluorescence molecule of biological sample, and the light that fluorescence molecule is launched is imaged onto on camera or the CCD through object lens, realizes the fluorescence records to biological sample.The microscope of two kinds of forms respectively has pluses and minuses, for lens type, the light that comes out from laser instrument incides on the prism with accurately selected incidence angle, on the surface of slide total reflection takes place, the evanscent field that produces excites the sample between two slides to launch fluorescence, implements simple relatively.Its shortcoming also clearly simultaneously: 1. prism dimensions is bigger, therefore the space of leaving between biological sample and the object lens is less, so it is very difficult such as other detection instruments such as AFM, NFMs that some are installed on this instrument, 2. because light path is comparatively complicated, make that link operation easier such as light path adjusting, sample replacing are very big.And for the object lens formula, then can overcome above shortcoming, laser focuses on object lens back focal plane and the edge incident of process object lens, the object lens emergent light is that directional light and oblique incidence are to cover glass, regulate the angle of laser incoming position and oblique incidence, can reach the total internal reflection requirement, thereby realize the evanscent field illumination.Evanscent field institute excited fluorescent still receives through object lens, filter the light of other wavelength except that fluorescence by dichroic mirror, be imaged on the camera or CCD at object lens rear, realization is to the fluorescence record of biological sample, and present most of biologists adopt the total internal reflection fluorescent microscope of object lens formula luminaire.Object lens formula luminaire equally also has significant disadvantages: 1. luminaire and microscopic examination system are total to light path, increased the complexity of light path, 2. because the restriction of form, lower surface that can only the observation of cell adherent growth, observation for the more significant cell surface of biological study especially side is then powerless, and 3. the introducing of large-numerical aperture micro objective has greatly increased system cost.
Summary of the invention
Purpose of the present invention at the deficiency of existing high-resolution fluorescent microscopic imaging technology, optical fiber technology is combined with total internal reflection principle, a kind of illuminator in fiber-optical evanescent field that is applicable to fluorescent microscopic imaging has been proposed, it is on 125 microns the simple optical fiber that the evanscent field luminaire is produced on diameter, make the evanscent field luminaire have simpler structure, littler size, light path is more flexible, stable, can realize multi-faceted illumination to the surface of biological sample, improve the performance of evanscent field luminaire greatly, and can combine the novel fluorescent microscopic imaging system of formation easily with microscopy.
Technical scheme of the present invention is:
A kind of illuminator in fiber-optical evanescent field is characterized in that: the part of optical fiber one end grinds to form the inclined-plane, and the inclined-plane crosses fiber cores position 1-10 micron, when light from fiber cores incident, be reflected onto the fiber end face place at the place, inclined-plane, and in the fiber end face experiences total internal reflection.
Described optical fiber can be single-mode fiber or multimode fibre.
Described fiber core can be in the optical fiber axle center, also can the disalignment.
Described inclined-plane forms by attrition process, can plating total reflection film on the inclined-plane also plated film not.
Described fiber end face can be perpendicular to the plane in axle center, also can be tapered plane.
The present invention has following characteristics: 1, the evanscent field luminaire is produced on diameter and is on 125 microns the simple optical fiber, make the evanscent field luminaire have simpler structure, littler size need not external optical system, and light path is more stable, operates more flexible.2, illuminator in fiber-optical evanescent field can be realized multi-faceted illumination to the surface of biological sample, has improved the performance of evanscent field luminaire greatly.3, illuminator in fiber-optical evanescent field adopts standard fiber to make, and is with low cost, and convenient with the light source coupling.4, illuminator in fiber-optical evanescent field constitutes novel fluorescent microscopic imaging system because physical size is little so can combine with other detection techniques easily.
Description of drawings
Fig. 1 is the structural representation of the evanscent field luminaire of axle center of the present invention optical fiber.
Fig. 2 plates the structural representation of full transmitting film for place, inclined-plane of the present invention.
Fig. 3 schemes with interface distance z for the light intensity I of evanscent field of the present invention.
Fig. 4 is light total reflection figure of the present invention.
Fig. 5 is the structural representation of the evanscent field luminaire of eccentric optical fiber of the present invention.
Fig. 6 constitutes the fluorescence microscopy system schematic for utilizing illuminator in fiber-optical evanescent field of the present invention.
The specific embodiment
Be described further below in conjunction with embodiment and accompanying drawing the present invention, but should do not limit protection scope of the present invention with this.
The present invention can realize by following mode:
Get one section optical fiber 1, utilize the fiber cut technology that fiber end face is cut into plane perpendicular to shaft axis of optic fibre.Utilize the optical fiber end grinding technology that fiber cores 2 positions are ground to form inclined-plane 3.If optical fiber 1 refractive index own is bigger, inclined-plane 3 angles can make the light in the fiber cores 2 that total reflection takes place herein, and guarantee to be radiated on the fiber end face also full emission to take place, and then inclined-plane 3 need not plated film herein.As shown in Figure 1.Otherwise for guaranteeing that total reflection takes place the light in the fiber cores herein, need utilize coating technique with inclined-plane plated film 4.As shown in Figure 2.
The evanscent field that fiber end face place total internal reflection produces can the irradiating biological sample, the fluorogen in the thick optics thin layer of hundred nano-scale is excited, and extraneous fluorogen is unaffected.
The present invention is based on following principle:
Total internal reflection is a kind of ubiquitous optical phenomena.Consider that a branch of planar light is reflected to the fiber end face place from fiber cores inclined-plane 3.Incident light reflects at the last branch of optical fiber surface, and another part then transmits optical fiber 1.Satisfy relational expression between incidence angle and the angle of transmission
n
1?sinθ
1＝n
2?sin?θ
2
Here n
1Be the refractive index of optical fiber, n
2Be the refractive index of the outer medium of optical fiber, θ
1Be incident angle, θ
2Be angle of transmission.When incidence angle increases, increase to critical angle θ
cThe time, angle of transmission at this moment is 90 °; When incidence angle continued to increase to greater than critical angle, light is transmission no longer, total reflection had just taken place, as shown in Figure 3.By the sne11 law as can be known
θ
2＝90°
By following formula as can be known, work as n
1＜n
2, total reflection just may take place.When light generation total reflection, because fluctuation effect, the energy of some light can pass contacting permeation and in the medium, be parallel to the interface and propagate outside optical fiber.This part light field is exactly so-called evanscent field.Consider a monochromic beam now, the cross section beam intensity is I
0, with angle θ incident greater than critical angle.The light intensity I of evanscent field with the pass of interface distance z is:
I＝I
0exp(-z/d)
λ is the incidence wave wavelength in the formula, and d is a transmission depth.D is generally less than 200nm, is about 100nm for visible light.
If optical fiber 1 is directly ground to form cone angle less than 90 °-θ
cInclined-plane 3, full emission can take place in 2 places in fiber cores, but there are some drawbacks in this way, as: 1. bevel angle is too small, and the cone zone is long, and optical fiber 1 can only be unfavorable for experimental implementation with very small angles near sample; 2. fiber cores 2 place's mode field diameters are usually below 10 microns, and the field of illumination is too small; 3. the light after the full emission has part power from the optical fiber head outgoing after optical fiber side reflects once more, these veiling glares make bias light strengthen, and have greatly reduced contrast.
Appropriate design optical fiber 1 end structure makes total reflection occur in fiber end face and then can effectively overcome above drawback.This be because: 1. utilize fiber end face as the field of illumination, effectively dwindled the physical size of luminaire, and the operation more flexible; 2. fiber end face has certain distance apart from fiber cores 2, and the divergence characterization of light beam itself can increase the field of illumination effectively; Light by end face reflection after secondary reflection again at optical fiber side opposite with illumination direction, thereby can not introduce veiling glare.
For making full emission occur in the fiber end face place, need process proper angle at fiber cores 2 places, thereby the light in the fiber cores 2 is reflexed to the fiber end face place, be example with fiber end face perpendicular to the optical fiber that optical fiber axle center, fiber cores 2 are positioned at the axle center.As shown in Figure 4.Light is reflexed to the fiber end face place by inclined-plane 3, and just in fiber end face generation total reflection, incident angle is θ
1, then inclined-plane, fiber cores place 3 with the angle of axis is
, medium is under the situation of water (refractive index 1.33) around, if optical fiber 1 refractive index own is higher, as n
1=1.6, the critical angle θ of total reflection then
c=56.23 °, make θ
1=58 °, α=29 ° then are because 90 °-α=61 are ° also greater than θ
c=56.23 °, on inclined-plane 3 total reflection can take place therefore, all light all can be reflected onto fiber end face, because θ
1＞θ
c, fiber end face can satisfy total reflection condition.If the refractive index of optical fiber own is not high, as n
1=1.46, the critical angle θ of total reflection then
c=65.64 °, make θ
1=66 °, α=33 ° then, because 90 °-α of incidence angle=57 ° less than θ
c=65.64 °, the transmission phenomenon can take place on the inclined-plane, all be reflected onto fiber end face for guaranteeing all light, need plate full transmitting film 4 at place, optical fiber inclined-plane.
Because during experiences total internal reflection, the typical transmission depth of evanscent field is generally about 100nm.Place if fiber end face is close to sample, then evanscent field also is about 100nm to the vertical irradiation degree of depth of sample.Have only this interior among a small circle fluorescence molecule to be excited, the fluorescence molecule beyond this scope is then unaffected fully.So TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY has the incomparable high signal to noise ratio of other formation method, the light injury and the photobleaching of cell are also very little.
Embodiment one: fiber core is located at the evanscent field luminaire in optical fiber axle center and makes.Illustrate that in conjunction with Fig. 2 making step is as follows:
1, the preliminary treatment of optical fiber: get one section optical fiber, utilize the about 30mm of coat of optical fiber wire stripper peeling optical fibre one end, fibre cladding is cleaned up with alcohol and ether mixed liquor.
2, cut in the middle of optical fiber drawing-down part with the optical fiber cutter, because optical fiber is mainly silica and makes, this action can cause the smooth fracture of optical fiber to come to be divided into two sections, and section is the plane.
3, utilize the optical fiber lapping technology that optical fiber end is ground tapered plane for α=29 °, grind the degree of depth and be advisable to surpass fiber cores position 1-10 micron.
4, the inclined-plane plating total reflection film that utilizes vacuum coating technology that step 3 is ground.
5, exciting light is introduced fiber cores, then produce the degree of depth at the fiber end face place and be the evanscent field about 100nm, place if fiber end face is close to sample, then evanscent field also is about 100nm to the vertical irradiation degree of depth of sample.Have only this interior among a small circle fluorescence molecule to be excited, the fluorescence molecule beyond this scope is then unaffected fully.
If the refractive index of optical fiber own is bigger, can be according to actual conditions cancellation step 4.
Embodiment two: the evanscent field luminaire of eccentric optical fiber is made.Illustrate that in conjunction with Fig. 5 making step is as follows:
1, the preliminary treatment of optical fiber: get one section optical fiber, utilize the about 30mm of coat of optical fiber wire stripper peeling optical fibre one end, fibre cladding is cleaned up with alcohol and ether mixed liquor.
2, cut in the middle of optical fiber drawing-down part with the optical fiber cutter, because optical fiber is mainly silica and makes, this action can cause the smooth fracture of optical fiber to come to be divided into two sections, and section is the plane.
3, utilize the optical fiber lapping technology that optical fiber end is ground truncated cone-shaped for α=29 °, also can be the truncated cone-shaped of other angles, satisfy the total reflection requirement and just become, grind the degree of depth just to be advisable near the fiber cores position.
4, utilize the optical fiber lapping technology on truncated cone-shaped, to continue optical fiber end is ground out the tapered plane of α=29 ° once more, also can be the tapered plane of other angles, satisfy the total reflection requirement and just become, grind the degree of depth and be advisable to surpass fiber cores position 1-10 micron.
5, the inclined-plane plating total reflection film that utilizes vacuum coating technology that step 4 is ground.
6, exciting light is introduced fiber cores, then produce the degree of depth at the fiber end face place and be the evanscent field about 100nm, place if fiber end face is close to sample, then evanscent field also is about 100nm to the vertical irradiation degree of depth of sample.Have only this interior among a small circle fluorescence molecule to be excited, the fluorescence molecule beyond this scope is then unaffected fully.
The evanscent field luminaire of making as shown in Figure 5, than the evanscent field luminaire of embodiment one made, the luminaire evanscent field illumination zone of eccentric optical fiber made will be bigger.
Embodiment three, utilize illuminator in fiber-optical evanescent field to realize the biological cell fluorescent microscopic imaging.Fig. 6 constitutes the fluorescence microscopy system schematic for utilizing illuminator in fiber-optical evanescent field.Light source uses wavelength X=488nm pump laser.After focusing on through lens 6, the light that light source 5 sends is coupled into optical fiber 7; illuminator in fiber-optical evanescent field with water pipe precaution after; but be installed on the mechanical adjustment frame 8 of a three-dimensional translating, one dimension rotation; can regulate insertion position, insertion depth and the insertion angle of optical fiber in sample cell 10 by this mechanical adjustment frame 8; sample cell places on the objective table 11 of an inverted biologic microscope, and image is imaged on the CCD14 by micro objective 13 through an optical filter 12 backs and gathers.
With microscopical objective table sample 9 is moved in the visual field, to regulate the illuminator in fiber-optical evanescent field that inserts angle then moves into the visual field and presses close to sample and treat the field of illumination, the evanscent field excited sample is sent fluorescence, and the nearly micro objective of fluorescence signal is collected, and is received and imaging by CCD.
Claims (4)
1. illuminator in fiber-optical evanescent field, it is characterized in that: the part of optical fiber one end grinds to form the inclined-plane, and fiber cores position 1-10 micron is crossed on the inclined-plane, when light from fiber cores incident, be reflected onto the fiber end face place at the place, inclined-plane, and in the fiber end face experiences total internal reflection, it is on 125 microns the simple optical fiber that the evanscent field luminaire is produced on diameter, the inclined-plane can make the light in the fiber cores that total reflection takes place at the place, inclined-plane, perhaps utilizes coating technique plating total reflection film on the inclined-plane.
2. a kind of illuminator in fiber-optical evanescent field according to claim 1 is characterized in that: described optical fiber is single-mode fiber or multimode fibre.
3. a kind of illuminator in fiber-optical evanescent field according to claim 1 is characterized in that: described fiber core is optical fiber axle center or disalignment.
4. a kind of illuminator in fiber-optical evanescent field according to claim 1 is characterized in that: described fiber end face is plane or tapered plane perpendicular to the axle center.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102374140A CN101446406B (en) | 2008-12-26 | 2008-12-26 | Illuminator in fiber-optical evanescent field |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102374140A CN101446406B (en) | 2008-12-26 | 2008-12-26 | Illuminator in fiber-optical evanescent field |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101446406A CN101446406A (en) | 2009-06-03 |
| CN101446406B true CN101446406B (en) | 2010-09-15 |
Family
ID=40742202
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008102374140A Expired - Fee Related CN101446406B (en) | 2008-12-26 | 2008-12-26 | Illuminator in fiber-optical evanescent field |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101446406B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101949849B (en) * | 2010-09-08 | 2011-09-21 | 华中科技大学 | Light-activated positioning microscopic imaging system based on optical fiber evanescent field illuminator |
| CN101949848B (en) * | 2010-09-08 | 2012-05-30 | 华中科技大学 | Photoactivation positioning microscopic imaging system based on micronano optical fiber evanescent field illuminator |
| CN103048272B (en) * | 2013-01-08 | 2014-12-10 | 浙江大学 | Frequency-shift super-resolution microimaging method and device based on evanescent field illumination |
| CN103901233B (en) * | 2014-04-11 | 2016-06-22 | 华中科技大学 | Optical fiber probe with polarization property and preparation method thereof |
-
2008
- 2008-12-26 CN CN2008102374140A patent/CN101446406B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN101446406A (en) | 2009-06-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102841083B (en) | Method and system of laser scanning phase-microscope imaging | |
| US7362500B2 (en) | Double-clad fiber scanning microscope | |
| CN102004307B (en) | System and method for realizing total internal reflection fluorescence microscopy by using concentric double conical surface lens | |
| CN106970461B (en) | Total internal reflection fluorescent microscopic imaging device based on ellipsoidal mirror | |
| CN103940796A (en) | Novel multi-angle and multi-mode quick switching circular optical illumination microscopic imaging system | |
| CN101446406B (en) | Illuminator in fiber-optical evanescent field | |
| CN103852458B (en) | A kind of microscopic method based on wide field stimulated emission difference and device | |
| CN202102170U (en) | System employing concentric double conical surface mirror for realizing total internal reflection fluorescence microscopy | |
| CN103105400A (en) | Method and device for detecting and classifying surface defects of large-aperture optical element | |
| CN107357044A (en) | A kind of step multimode fibre optical tweezer based on skew ray annular light field | |
| US20140218794A1 (en) | Confocal Fluorescence Microscope | |
| CN102818795A (en) | Biological fluorescence microscopic detection instrument | |
| CN102818794A (en) | Biological fluorescence microscopic detection instrument | |
| CN209661612U (en) | Three-dimensional line scans micro-optical probe | |
| CN101957308B (en) | Micro-nano optical fiber evanescent field illuminator | |
| CN108261179A (en) | Micro-optical is popped one's head in | |
| CN102419250B (en) | Active polymer plane waveguide propagation constant measuring instrument based on fluorescence imaging | |
| CN110664369B (en) | Self-adaptive confocal line scanning harmonic microscopic imaging method and device | |
| CN211014821U (en) | Microscope | |
| CN100464692C (en) | Method and apparatus for obtaining tissue micro tomography image and spectrum | |
| CN202403893U (en) | Active polymer planar waveguide propagation constant measuring instrument based on fluorescence imaging | |
| CN102226855B (en) | Three-dimensional super-resolution focusing method and device based on transparent medium pellet | |
| CN111060481A (en) | Nanometer microscopic imaging device based on coaxial double-waveguide optical fiber SPR (surface plasmon resonance) | |
| CN110568224A (en) | Composite near-field optical probe capable of simultaneously realizing high optical signal flux and high resolution and preparation method thereof | |
| CN203069524U (en) | Detection and classification device for surface defect of big-aperture optical element |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20100915 Termination date: 20131226 |