Use axicon lens to realize the method and the device of the micro-and fluorescence microscopy of details in a play not acted out on stage, but told through dialogues
Technical field
The present invention relates to a kind of method and device of realizing the micro-and fluorescence microscopy of details in a play not acted out on stage, but told through dialogues.
Background technology
Optical microscope is the important tool of research microworld.Different according to observation procedure and object of observation, optical microscope can be divided into kinds such as light field microscope, dark field microscope, fluorescent microscope.Most of research object can use the light field microscope to observe, but some samples are arranged because its refractive index and surrounding environment are very approaching, use common light field microscopic method be difficult to observe, at this moment use the micro-or fluorescence microscopy method of details in a play not acted out on stage, but told through dialogues then can address this problem.The micro-principle of details in a play not acted out on stage, but told through dialogues is to block the center section of lighting source, only allow the light of illuminating bundle marginal portion with certain angle oblique illumination sample, so, illuminating bundle does not directly enter microcobjective, and the scattered light that has only sample enters the object lens imaging, its effect just looks like the starry sky at night, presents bright sample structure under the dark background of sheet, and this microscopic method has greatly increased the contrast of image.As the means that a kind of very simple and effective microcosmic sample edge detects, the details in a play not acted out on stage, but told through dialogues microtechnic is widely used in every field.
The key distinction that details in a play not acted out on stage, but told through dialogues is micro-and light field is micro-is that their condenser design is different.What the micro-condenser of common light field produced is a taper focused beam illumination sample, and the micro-condenser of details in a play not acted out on stage, but told through dialogues then uses a circular light barrier to cover the center section of illuminating bundle, has formed a hollow taper focused beam.The summit of cone-shaped beam is focused sample surfaces, and light beam forms an inverted taper hollow beam after passing sample.Microcobjective is positioned at the hollow position of light beam, if the angle of divergence of illuminating bundle greater than the aperture angle of microcobjective, then illumination light will can directly not enter object lens, just can pass through the object lens imaging and have only by the light of sample scattering.The advantage of this dark field condenser is simple, has blocked most of illumination light but shortcoming is a light barrier, so transmitance is very low.Patent CN200410017069.1 has designed the micro-beam condensing unit of a kind of details in a play not acted out on stage, but told through dialogues, has the high advantage of illumination light utilization ratio, but need to use cone-shaped reflector and trapezoidal bucket reverberator and in complicated manufacture crafts such as lens center punchings, and this condenser can only be used for dark ground illumination.Patent CN200480020826.9 has designed a kind of more complicated condenser system, needs to use a series of diaphragms of working in coordination to realize, so the light transmission rate of total system is not high.
Fluorescence microscopy method is with special dyestuff testing sample to be handled earlier, uses the light of specific wavelength to remove to shine sample when observation then, and wherein dyestuff is subjected to exciting the fluorescence that can send another wavelength, the observing samples by this fluorescence imaging.Along with the appearance of various fluorescent dyes, use the different fluorescent dyes can dissimilar sample or the different members of mark sample interior of mark, thus meticulous formation that can observing samples.Fluorescent microscope has become the strong instrument of chemistry and biological sample imaging.But common fluorescent microscope will be at different wavelength of fluorescence, different narrow band pass filters is set before detector, stop that to select fluorescence is seen through the light of exciting light and other wavelength enters detector, this just makes will use different optical filters to different fluorescent dyes, and common fluorescent microscope does not have the micro-function of details in a play not acted out on stage, but told through dialogues simultaneously.
Summary of the invention
The present invention seeks to propose a kind of method and device of realizing the micro-and fluorescence microscopy of details in a play not acted out on stage, but told through dialogues, use axicon lens and one group of lens coupling as beam condensing unit, can realize simultaneously that details in a play not acted out on stage, but told through dialogues is micro-, fluorescence microscopy and light field be micro-, solved the not high and existing fluorescence microscopy of the micro-illumination light transmitance of existing details in a play not acted out on stage, but told through dialogues and will use different optical filters and can not have the micro-technical matters of details in a play not acted out on stage, but told through dialogues simultaneously different fluorescent dyes.
Technical solution of the present invention is:
A kind of method of using axicon lens to realize the micro-and fluorescence microscopy of details in a play not acted out on stage, but told through dialogues may further comprise the steps:
1] produces a parallel beam;
2] this parallel beam is dispersed the formation hollow beam by axicon lens 8;
3] angle of divergence of adjusting hollow beam makes the aperture angle of the angle of divergence of emergent ray greater than microcobjective 14;
4] sample 13 is placed on the center of hollow beam, with microcobjective 14 observation samples 13.
Above-mentioned parallel beam is illumination light or fluorescent exciting; Described fluorescent exciting light sources 4 adopts high-pressure sodium lamp or adopts the laser instrument of specific wavelength; The method of the angle of divergence of described adjusting hollow beam is: with hollow beam scioptics I9 successively and lens II11, make the aperture angle of the angle of divergence of lens II11 emergent ray greater than microcobjective 14; Should satisfy following relation between described axicon lens 8, lens I9, the lens II11:
Wherein: w
0The radius of-incident parallel beam; The base angle of γ-axicon lens 8; The refractive index of n-axicon lens 8 materials; u
1-incident parallel beam is by the angle of divergence behind the axicon lens 8; u
2-from the angle of divergence of lens II11 emergent ray; f
1The focal length of-lens I9; f
2The focal length of-lens II11; d
1Distance between-axicon lens 8 and the lens I9; d
2Distance between-lens I9 and the lens II11; The h-light beam incides the radius on the lens I9.
A kind of device that uses axicon lens to realize the micro-and fluorescence microscopy of details in a play not acted out on stage, but told through dialogues, comprise light-source system, be arranged on light-source system the place ahead optical system, be arranged on the sample placed in optical system the place ahead objective table 12, be arranged on the imaging system in sample the place ahead; Its special character is that described optical system comprises collimation lens 2, axicon lens 8, lens I9 and the lens II11 that is successively set on light-source system the place ahead; Described imaging system comprises microcobjective 14.
Above-mentioned light-source system comprises white-light illuminating light source 1 or fluorescent exciting light sources 4; Described fluorescent exciting light sources 4 is laser instruments.
Above-mentioned light-source system comprises fluorescent exciting light sources 4 and is arranged on the optical filter 5 in fluorescent exciting light sources 4 the place aheads; Described fluorescent exciting light sources 4 is high-pressure sodium lamps.
Above-mentioned light-source system comprises white-light illuminating light source 1 and fluorescent exciting light sources 4; Described fluorescent exciting light sources 4 is laser instruments; Described collimation lens comprises white light collimation lens 2 that is arranged on white-light illuminating light source 1 the place ahead and the collimation lens 6 that is arranged on fluorescent exciting light sources 4 the place aheads; Described optical system also comprises beam splitter 3; Described beam splitter 3 is used for the light beam that white-light illuminating light source 1 and fluorescent exciting light sources 4 send is incided axicon lens 8 simultaneously.
Above-mentioned light-source system comprises white-light illuminating light source 1 and fluorescent exciting light sources 4; Described fluorescent exciting light sources 4 is high-pressure sodium lamps; Described collimation lens comprises white light collimation lens 2 that is arranged on white-light illuminating light source 1 the place ahead and the collimation lens 6 that is arranged on fluorescent exciting light sources 4 the place aheads; The optical filter 5 that described optical system also comprises beam splitter 3 and is arranged on fluorescent exciting light sources 4 the place aheads; Described beam splitter 3 is used for the light beam that white-light illuminating light source 1 and fluorescent exciting light sources 4 send is incided axicon lens 8 simultaneously; Described optical filter 5 is used for selecting suitable excitation wavelength from the light that fluorescent exciting light sources 4 sends.
Above-mentioned imaging system also comprises the ccd video camera 16 that is arranged on microcobjective 14 the place aheads.
Above-mentioned optical system also comprise the polarizer 7 that is arranged on axicon lens 8 rears and be arranged on microcobjective 14 and ccd video camera 16 between analyzer 15.
Above-mentioned imaging system also comprises monitor screen or the computing machine 17 that links to each other with ccd video camera 16.
Advantage of the present invention is:
1, system's transmitance height.The present invention adopts the combination of axicon lens and one group of lens as beam condensing unit, and system's transmitance is very high, near 100%.
2, can realize the micro-and micro-switching of details in a play not acted out on stage, but told through dialogues of light field easily.By adjusting the interval between the lens combination in the illumination path, can change the dispersion angle of illuminating ray, thereby realize the conversion between light field and the details in a play not acted out on stage, but told through dialogues.
3, the present invention uses axicon lens to realize the function of the micro-and fluorescence microscopy of details in a play not acted out on stage, but told through dialogues simultaneously, can change mutually between bright field illumination and the dark ground illumination, and save the fluorescence narrow band pass filter is set before detector.
Description of drawings
Fig. 1 is the principle schematic that the present invention uses the axicon lens beam split;
Fig. 2 is that the present invention uses axicon lens and lens combination to realize the schematic diagram of details in a play not acted out on stage, but told through dialogues and the micro-illumination of light field;
Fig. 3 is apparatus of the present invention structural representation and index path;
Wherein: 1-white-light illuminating light source, 2-collimation lens, 3-beam splitter, 4-fluorescent exciting light sources (high-pressure sodium lamp or laser instrument), 5-optical filter, 6-collimation lens, the 7-polarizer, 8-axicon lens, 9-lens I, the 10-completely reflecting mirror, 11-lens II, 12-objective table, the 13-sample, 14-microcobjective, 15-analyzer, the 16-CCD video camera, the 17-computing machine;
Fig. 4 is the Al that apparatus of the present invention are taken
2O
3The light field of particulate and details in a play not acted out on stage, but told through dialogues microphoto;
Wherein: Fig. 4 a is Al
2O
3The light field micro-image of particle; Fig. 4 b is Al
2O
3The details in a play not acted out on stage, but told through dialogues micro-image of particle;
Fig. 5 is the fluorescence micrograph of the ZnS:Cu particle of apparatus of the present invention shooting.
Embodiment
A kind of method of using axicon lens to realize the micro-and fluorescence microscopy of details in a play not acted out on stage, but told through dialogues may further comprise the steps:
1] produce a parallel beam, this parallel beam is illumination light or fluorescent exciting, and wherein fluorescent exciting light sources adopts the laser instrument of high-pressure sodium lamp or employing specific wavelength;
2] this parallel beam is passed through axicon lens, disperse the formation hollow beam;
3], make the aperture angle of the angle of divergence of lens II emergent ray greater than microcobjective with hollow beam scioptics I successively and lens II;
Wherein should satisfy following relation between axicon lens, lens I, the lens II:
Wherein: w
0The radius of-incident parallel beam; The base angle of γ-axicon lens 8; The refractive index of n-axicon lens 8 materials; u
1-incident parallel beam is by the angle of divergence behind the axicon lens 8; u
2-from the angle of divergence of lens II11 emergent ray; f
1The focal length of-lens I9; f
2The focal length of-lens II11; d
1Distance between-axicon lens 8 and the lens I9; d
2Distance between-lens I9 and the lens II11; The h-light beam incides the radius on the lens I9;
4] sample is placed on the center of hollow beam, uses the microcobjective observation sample.
As shown in Figure 1, radius is w
0Parallel beam by axicon lens after because refraction can diverge to hollow beam, when the base angle of axicon lens γ hour (less than 10 degree), the angle of divergence is approximately:
u=(n-1)γ (1)
Wherein n is the refractive index of axicon lens material.
In dark ground illumination, the angle of divergence of illuminating bundle must not enter object lens to guarantee illumination light, and have only scattered light to enter object lens greater than the aperture angle of microcobjective.But in order to guarantee the transmitance of illumination light, the base angle γ of axicon lens can not be too big, and light can be reflected back otherwise have greatly.So, use single axicon lens can not obtain enough big beam divergence angle u.
In order to enlarge the angle of divergence of illuminating bundle, we have placed lens I and lens II behind axicon lens, as shown in Figure 2.According to the geometric relationship among Fig. 2, can obtain following formula:
The angle of divergence u of last emergent ray
2Expression.By selecting the suitable focal length of lens (f
1, f
2) and regulate distance (d between each element
1, d
2) can change u
2Work as u
2During greater than the aperture angle of microcobjective, illumination (or exciting) light can not enter microcobjective, has only scattered light (or fluorescence) can enter object lens, thereby can realize details in a play not acted out on stage, but told through dialogues micro-(or fluorescence microscopy).
Work as u
2During less than the aperture angle of object lens, illuminating ray can directly enter object lens, thereby can realize the micro-illumination of light field.
Referring to Fig. 3, a kind of device that uses axicon lens to realize the micro-and fluorescence microscopy of details in a play not acted out on stage, but told through dialogues, comprise light-source system, be arranged on light-source system the place ahead optical system, be arranged on the sample placed in optical system the place ahead objective table, be arranged on the imaging system in sample the place ahead; Light-source system comprises white-light illuminating light source and fluorescent exciting light sources; Fluorescent exciting light sources is laser instrument or high-pressure sodium lamp; Optical system comprises that the white light collimation lens that is arranged on white-light illuminating light source the place ahead, the collimation lens that is arranged on fluorescent exciting light sources the place ahead, the light beam that white-light illuminating light source and fluorescent exciting light sources can be sent incide beam splitter, axicon lens, lens I and lens II on the axicon lens simultaneously; Imaging system comprises microcobjective, is arranged on the ccd video camera in microcobjective the place ahead, the monitor screen that links to each other with ccd video camera or computing machine; Objective table is automatically controlled objective table or manual objective table, and ccd video camera is simulation ccd video camera or digital ccd video camera.
If use high-pressure sodium lamp as fluorescent exciting light sources, then need before fluorescent exciting light sources, place an optical filter, optical filter is used for selecting suitable fluorescence exciting wavelength; If use laser instrument as fluorescent exciting light sources, then do not need optical filter.
In order to regulate the brightness that scattered light enters ccd video camera, a polarizer can be set between beam splitter and axicon lens, an analyzer is set between microcobjective and ccd video camera simultaneously, regulate the polarization direction of the preceding analyzer of ccd video camera, make its polarization direction vertical, can reach the purpose that improves the fluorescence microscope images signal to noise ratio (S/N ratio) like this with the polarizer.
In order to reduce the volume of apparatus of the present invention, a completely reflecting mirror can be set before lens II, reduce the device height of microcobjective below.
The course of work of apparatus of the present invention:
1, the micro-and light field micromanipulation of details in a play not acted out on stage, but told through dialogues
Under details in a play not acted out on stage, but told through dialogues and the micro-working method of light field, turn off fluorescent exciting light sources.The white-light illuminating light source becomes hollow cone-shaped beam irradiation sample through the polarizer, axicon lens, lens I, completely reflecting mirror, lens II after the white light collimation lens becomes parallel beam.By selecting the suitable focal length of lens (f
1, f
2) and regulate distance (d between each element
1, d
2) can change the angle u of outgoing beam and optical axis
2Work as u
2During greater than the aperture angle of microcobjective, illumination light can not enter microcobjective, has only scattering luminous energy to enter object lens, realizes that details in a play not acted out on stage, but told through dialogues is micro-; Work as u
2During less than the aperture angle of object lens, illuminating ray can directly enter object lens, thereby can realize that light field is micro-.Regulate the polarization direction of the preceding analyzer of ccd video camera, can change the brightness of visual field and the signal to noise ratio (S/N ratio) of image.Among the embodiment, base angle γ=5 of axicon lens °, the focal length of lens I and lens II is respectively f
1=150mm and f
2=18mm, the spacing d of axicon lens 8 and lens I
1=100mm, the spacing d of lens I and lens II
2=300mm, incident light radius w
0=10mm.Under this parameter, can obtain u
2=43.8 °, this angle can realize the micro-illumination of details in a play not acted out on stage, but told through dialogues greater than the aperture angle (aperture angle as the 25X/NA0.4 object lens is 23.6 °, and the aperture angle of 40X/NA0.65 object lens is 40.5 °) of most of microcobjective.Under this configuration,, can change d simultaneously by regulating the position of lens I between axicon lens 8 and lens II
1And d
2, in order to change the angle of divergence u of illumination light emergent ray
2, work as u
2During less than the aperture angle of object lens 14, illumination light can directly enter object lens, thereby can realize the micro-illumination of light field.Fig. 4 is that apparatus of the present invention are to Al
2O
3The contrast of the micro-and details in a play not acted out on stage, but told through dialogues micro-image of particulate light field, Fig. 4 (a) is the light field microphoto, the detail section of particle surface is not obvious; Fig. 4 (b) is the details in a play not acted out on stage, but told through dialogues microphoto, can clearly observe the rough detail section of particle surface, and contrast and stereoscopic sensation are more intense.
2, fluorescence microscopy operation
Under the fluorescence microscopy working method, turn off the white-light illuminating light source.Fluorescent exciting light sources can use high-pressure sodium lamp or wavelength specific laser.Excitation source (if use laser instrument as fluorescent exciting light sources, does not then need optical filter) after the mating plate after filtration, again through focusing on the sample behind collimating mirror, beam splitter, the polarizer, axicon lens, lens I, completely reflecting mirror, the lens II.By selecting the suitable focal length of lens (f
1, f
2) and regulate distance (d between each element
1, d
2) can change the angle u of fluorescent exciting outgoing beam and optical axis
2Work as u
2During greater than the aperture angle of microcobjective, do not excite and can enter microcobjective, the fluorescence that has only sample to send can enter the object lens imaging, reaches the purpose of fluorescence microscopy.Regulate the polarization direction of the preceding analyzer of ccd video camera, make its polarization direction with the polarizer vertical, the scattered light that can weaken exciting light like this enters ccd video camera, reaches the purpose that improves the fluorescence microscope images signal to noise ratio (S/N ratio).Among the embodiment, the semiconductor laser that uses wavelength 405nm removes optical filter as fluorescent exciting light sources, base angle γ=5 of axicon lens °, and the focal length of lens I and lens II is respectively f
1=150mm and f
2=18mm, the spacing d of axicon lens and lens I
1=100mm, the spacing d of lens I and lens II
2=300mm.Fig. 5 is the fluorescence microscope images of the ZnS:Cu particle of apparatus of the present invention shooting, and the ZnS:Cu particle sends green fluorescence under 405nm laser excitation, and shooting effect is fine.